Our findings elucidate the novel downstream pathways of CSPGs and suggest potential synergy of blocking their two PTP receptors.Receptor protein tyrosine phosphatase σ (PTPσ) and its subfamily member LAR act as transmembrane receptors that mediate growth inhibition of chondroitin sulfate proteoglycans (CSPGs). Inhibition of either receptor increases axon growth into and beyond scar tissues after CNS injury. However, it is unclear why neurons express two similar CSPG receptors, nor whether they use the same or different intracellular pathways. We have now studied the signaling pathways of these two receptors using N2A cells and primary neurons derived from knockout mice. We demonstrate that both receptors share certain signaling pathways , but also use distinct signals to mediate CSPG actions. Activation of PTPσ by CSPGs selectively inactivated CRMP2, APC, S6 kinase and CREB. By contrast LAR activation inactivated PKCζ, cofilin and LKB1. For the first time, we propose a model of the signaling pathways downstream of these two CSPG receptors. We also demonstrate that deleting both receptors exhibits additive enhancement of axon growth in adult neuronal cultures  After CNS injuries, a family of extracellular matrix (ECM) molecules, the chondroitin sulfate proteoglycans (CSPGs), are highly upregulated by reactive scars and potently inhibit axon growth into and beyond the lesion area1246in vitro910121315161718The presence of sulfated GAG chains is particularly important because removing GAGs or preventing GAG sulfation neutralizes most of the suppression of axon growth by CSPGs in vitro.Deficiency of PTPσ or LAR in adult mice increased regrowth of various projection tracts after spinal cord injury (SCI), including sensory, corticospinal and serotonergic axons, into the caudal spinal cord151716This study supports a novel model of signaling pathways downstream of the two CSPG receptors and potential synergistic actions of blocking both simultaneously.RhoA is a prominent intracellular signal that regulates cytoskeletal dynamics, transcription and cell growth, and is thought to mediate functions of multiple axon growth inhibitors, including myelin- associated inhibitors21232628Treatment with CSPGs for 20 min significantly increased active RhoA levels in N2A cells transfected with either PTPσ or LAR, compared with those in control cells . BecauseTo validate the findings from N2A cells in primary neuronal cultures, we evaluated whether PTPσ or LAR deletion alters activities of intracellular signals, including active RhoA, in cultured cerebellar granule neurons (CGNs) following incubation in CSPGs. We could easily collect enough samples for multiple Western blot analyses using CGN cultures derived from postnatal 7–10 day mice. Following 24 hours of growth, CGNs were treated with CSPGs (1.5 μg/ml) for 20 minutes and the levels of active RhoA in the supernatants were detected by precipitation with rhotekin binding domain beads and then by Western blotting. Application of CSPGs at 1.5 μg/ml significantly enhanced levels of active RhoA in CGNs derived from PTPσ+/+ mice . HoweverAs a downstream mediator of RhoA action, Rock can phosphorylate and activate LIM kinase (LIM-K), which in turn phosphorylates and inactivates cofilin3032The intracellular PI3K/Akt pathway is essential for regulating neuronal growth during development and contributes to suppression of axon growth by CSPGs16212236Deletion of PTEN, a negative regulator of PI3K/Akt signaling, stimulated neuronal growth and survival following axotomy by activating mTOR38CRMP2 is critical for regulating axon formation and outgrowth during development by binding tubulin heterodimers and promoting microtubule assembly4243454647The tumor suppressor APC accumulates at the tip of the growth cone during axon elongation and its phosphorylation by GSK-3β suppresses axon growth by reducing its ability to bind microtubules1621Extracellular regulated kinases (Erks) are expressed in multiple cell types including neurons, and are essential for regulating protein synthesis/degradation and maintaining levels of proteins required for growth in response to many extracellular axon growth and guidance cues, including neurotrophins. Although it has not been determined whether Erk signaling mediates CSPG function, nerve growth factor (NGF) promoted growth of conditioning DRG neurons cultured on CSPGsThe 90 kDa ribosomal S6 kinases (p90RSKs), also known as MAPK-activated protein kinase 1, are Ser/Thr kinases characterized by two non-identical functional kinase domains and a carboxy-terminal docking site for Erks51Activation of Erk can enhance CREB phosphorylation at serine 133, thus increasing its activity as an upstream signal53The serine/threonine kinase 11 (known as LKB1) and its downstream signaling target SAD/MARK regulate neuronal polarization and axon elongation during development5758As a member of the atypical protein kinase C subfamily, PKCζ phosphorylates LKB1 at Ser-428/431 and mediates activation of AMPK in endothelial cells60in vitro by counting the number of neurites that cross a gradient of CSPGs1516in vitro.Our experimental results with either N2A cells or primary neurons support that LAR and PTPσ share certain signaling pathways, but also employ distinct signals to covey CSPG effects on neurons. It was thus important to determine whether targeting both PTP receptors would have additive actions in overcoming CSPG-mediated inhibition of axon growth. CSPG-spot assay is a reliable method for measuring axon growth To compare downstream signals of the two RPTPs, we conducted parallel experiments, measuring alterations of multiple signaling proteins in the lysates of PTPσ- or LAR-overexpressing N2A cells. These neuroblastoma-derived cultures possess the functional properties of neurons27656621223435Based on the results of the present study and previous reports, we propose a simplified model of convergent and divergent pathways downstream of the two PTP CSPG receptors . The int323472PTPσ and LAR do not employ identical signaling to mediate CSPG actions, although both use the Akt/GSK-3β signaling pathway . ActivatAs a downstream signal for many neurotrophic factors747659Moreover, activation of PTPσ, but not LAR, by CSPGs inactivated CREB by reducing its phosphorylation, suggesting that CSPG-PTPσ interactions inactivate Erk and subsequently reduce the activity of CREB, an important transcription factor for controlling neuronal growth5354As a downstream signal of Erk, LKB1 appears to be critical for regulating axon elongation during developmentWhy PTPσ and LAR employ both convergent and divergent downstream signaling pathways remains unclear. It may relate to diversity in the ligands and substrates of RPTPs, indirect links along the different intracellular pathways and multiple interactions among the signals. Although CSPGs and Although HSPGs appear to be the substrates of PTPσ and LAR1516846889091To explain how PTPσ and LAR differ in their CSPG-signaling functions, we cannot exclude possible subtle roles of other signaling proteins that did not appear significantly altered by CSPG actions in the current experiments. For example, given the trend of their slight alterations , 4E-BP1 94We detected a crucial role of atypical PKCζ, but not the conventional PKC, in regulating CSPG-LAR interactions . In cont17In this study, we examined 15 signaling proteins that potentially act as the downstream pathways mediating the interactions between CSPGs and two PTP receptors  and usedIn identifying the signaling pathways of the two CSPG receptors, we primarily employed one-way ANOVA to determine whether CSPG stimulation induces statistically significant differences at several time points. The overall alterations of the signaling proteins in N2A cells and CGN cultures matched well. Because the properties of N2A cells may not be identical to those of primary CGNs, the extents and latencies of the signaling changes might not be the same. For example, p-S6 showed a greater degree of reduction at more delayed time points in CGNs than in PTPσ-transfected N2A cells . HoweverOn the basis of the present results and previous studies, we summarize convergent and divergent pathways that mediate the axon growth-inhibitory effects of CSPG-RPTP interactions in in vitro to a greater degree than deletion of either receptor alone  or transcription factors (such as KLFs)389899Transgenic or pharmacological inhibition of either PTPσ or LAR induces significant regrowth of several projection fiber tracts after SCI, including serotonergic, corticospinal and sensory fibers, and improves functional recovery16181516or alone . MoreoveAll experimental protocols were approved by the Institutional Animal Care & Use Committee at Temple University and the methods were carried out in accordance with the relevant guidelines and regulations.Antibodies against the following proteins were used: mouse mAb phospho-Akt , rabbit mAb phospho-S6 ribosomal protein (Ser235/236), rabbit mAb phospho-Erk1/2 (p44/42), rabbit pAb phospho-CRMP2 (Thr514), rabbit pAb phospho-cofilin (Ser3), rabbit mAb phospho-4E- BP1 , rabbit mAb phospho-CREB , rabbit mAb phospho-PKA C , rabbit pAb phospho-90 kDa ribosomal S6 kinase , rabbit pAb phospho-PKCζ/λ (Thr410/403), rabbit mAb phospho-PKC (pan) , rabbit mAb phospho-LKB1  , rabbit pAb phospho-MAP1B , rabbit pAb phospho-APC , mouse anti-actin clone C4  and mouse mAb against RhoA . The major proteins employed include rhotekin binding domain beads , a mixture of purified CSPGs , laminin (Sigma) and several protease inhibitors . A plasmid with the human LAR sequence was provided by Dr. Morgan Sheng and amplified in our lab16N2A, a mouse neural crest-derived cell line, possesses neuronal properties656667102PTPσ and LAR knockout mice were provided by Drs. Michel TremblaySupernatant samples from N2A or CGN cultures (in 35 mm dishes) were prepared as above. After quantification of total proteins in lysates, using Bio-Rad DC protein assay reagents, a sample containing the same amount of proteins from each dish was incubated with rhotekin binding domain coupled beads (45 μg/sample) for 50 min at 4 °C. GTP-bound RhoA and total RhoA in cell lysates were detected by Western blot using a mouse monoclonal antibody against RhoA® scanner and Odyssey® imaging software. For the Western blotting assays, at least three separate experiments were performed and the representative blots are shown in the figures.Expression changes of each studied signaling protein were determined with Western blots in N2A cells and CGNs after CSPG treatment. The supernatants of cell lysates containing the same amount of total proteins were loaded onto Tris-Glycine gels and transferred to nitrocellulose membranes. The membranes were blocked with 5% blotting grade milk (BIO-RAD), blotted with primary antibodies described above, and then incubated with appropriate secondary antibodies conjugated to IRDye 800CW (LI-COR) or IRDye 680RD (LI-COR). To determine the levels of loading control proteins, membranes were probed with anti-total signaling proteins and/or actin using separate membranes with the same amount of total loading proteins. Visualization and quantification were carried out with the LI-COR OdysseyDRGs were harvested from various genotypes of mice . Quantification data for each signaling protein were collected from 3–5 separate experiments.SigmaPlot software was used for statistical analysis. Data in graphs are shown as means ± SEM. The comparisons between multiple groups were analyzed with a repeated measures ANOVA. The experiments comparing a single determination of means between two independent groups, including the active RhoA and neurite growth assay, were analyzed with Student’s How to cite this article: Ohtake, Y. et al. Two PTP receptors mediate CSPG inhibition by convergent and divergent signaling pathways in neurons. Sci. Rep.6, 37152; doi: 10.1038/srep37152 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


AOX genes in mandarin (Citrus clementina) and sweet orange (Citrus sinensis) at nucleotide and protein levels, including promoter analysis, phylogenetic analysis and C. sinensis gene expression. This study also aimed to do the homology modeling of one AOX isoform (CcAOXd). Moreover, the molecular docking of the CcAOXd protein with the ubiquinone (UQ) was performed. Four AOX genes were identified in each citrus species. These genes have an open reading frame (ORF) ranging from 852 bp to 1150 bp and a number of exons ranging from 4 to 9. The 1500 bp-upstream region of each AOX gene contained regulatory cis-elements related to internal and external response factors. CsAOX genes showed a differential expression in citrus tissues. All AOX proteins were predicted to be located in mitochondria. They contained the conserved motifs LET, NERMHL, LEEEA and RADE-H as well as several putative post-translational modification sites. The CcAOXd protein was modeled by homology to the AOX of Trypanosona brucei (45% of identity). The 3-D structure of CcAOXd showed the presence of two hydrophobic helices that could be involved in the anchoring of the protein in the inner mitochondrial membrane. The active site of the protein is located in a hydrophobic environment deep inside the AOX structure and contains a diiron center. The molecular docking of CcAOXd with UQ showed that the binding site is a recessed pocket formed by the helices and submerged in the membrane. These data are important for future functional studies of citrus AOX genes and/or proteins, as well as for biotechnological approaches leading to AOX inhibition using UQ homologs.The alternative oxidase (AOX) protein is present in plants, fungi, protozoa and some invertebrates. It is involved in the mitochondrial respiratory chain, providing an alternative route for the transport of electrons, leading to the reduction of oxygen to form water. The present study aimed to characterize the family of  The term oxidase refers to any enzyme that catalyzes the oxidation–reduction reaction involving molecular oxygen as an electron acceptor. In these reactions, the oxygen is reduced to water or to hydrogen peroxide. The alternative oxidase (AOX) protein is present in plants, fungi, protozoa and some invertebrates, but it has not been found in mammals. It is located on the matrix side of the inner mitochondrial membrane and is involved in the mitochondrial respiratory chain, providing an alternative route for the passage of electrons. The main electron transport route in eukaryotes passes through the complex IV (known as cyanide-sensitive cytochrome oxidase) of the respiratory chain, but in some organisms the electron transport route goes through the AOX protein . Both routes lead to the transportation of electrons and the reduction of oxygen to form water , 2. HoweAOX genes  that are conserved in the AOX proteins of different plant species and assumed to be involved in the redox regulation of AOX activity were identified in the CcAOXd structure (C68 (I) and C118 (II); 68 (I) and C118 (II) also play a role in the post-translational regulation of most angiosperm AOX proteins [221 that is highly conserved across other AOX proteins [124, E163, E214 and E265) and 2 histidine (H166 and H268) residues, all of which are highly conserved among AOX proteins  while the CsAOXb was lowly expressed (<1 RPKM) (CsAOXa gene was highly expressed in the fruit (17.5 RPKM) but also showed significant expression levels in callus, flower and leaf  and significant expression levels in fruit and flower  and also close values of expression in flower and leaf  [AOX genes were associated to gametophyte development [AOX genes related to climacteric fruit ripening presented elements responsive to ethylene in their promoter sequences [CsAOXa and CSAOXd genes did not present any elements responsive to ethylene in their promoter sequences; this could be related to the fact that citrus are non-climacteric fruits, or may suggest an involvement of these CsAOX genes in fruit formation more than in fruit ripening  . The CsAthaliana , 54. Theo CsAOXc –in chillor mango –58. Some fruits) , 57, whiAOX gene family in C. clementina and C. sinensis. Four AOX genes were identified in each species; the C. clementina genes were orthologues of the C. sinensis genes. Phylogenetic analysis of the AOX citrus and A. thaliana sequences showed that the CcAOXb and CsAOXb were closed to the AtAOX1D sequence while CcAOXd and CsAOXd were closed to AtAOX2. According to the cis-element present in the citrus AOX promoters, the gene expression may be regulated by several external or internal factors. Expression of CsAOX genes revealed that CsAOXc was highly expressed in callus while CsAOXa and CsAOXd were highly expressed in fruits. Other regulation levels were also predicted, such as alternative splicing and post-translational modifications. The corresponding proteins were predicted to be directed to the mitochondria, and the analysis of the 3-D structure of one the C. clementina AOX isoforms showed the presence of two hydrophobic helices that may be involved in the anchoring of the protein in the inner mitochondrial membrane. The active site of the protein is located in a hydrophobic environment deep inside the AOX structure and contains a diiron center. The molecular docking of CcAOXd with UQ showed that the binding site is a recessed pocket formed by the helices and submerged into the membrane. These data are important for future functional studies of citrus AOX genes and/or proteins, as well as for biotechnological approaches leading to AOX inhibition using UQ homologs.To the best of our knowledge, this is the first characterization of the S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S4 Fig(DOCX)Click here for additional data file.S5 Fig(DOCX)Click here for additional data file.S6 Fig(DOCX)Click here for additional data file.S7 Fig(DOCX)Click here for additional data file.


Mitochondrial alternative respiratory-chain enzymes are phylogenetically widespread, and buffer stresses affecting oxidative phosphorylation in species that possess them. However, they have been lost in the evolutionary lineages leading to vertebrates and arthropods, raising the question as to what survival or reproductive disadvantages they confer. Recent interest in using them in therapy lends a biomedical dimension to this question.Ciona intestinalis alternative oxidase, AOX, on the reproductive success of Drosophila melanogaster males. Sperm-competition assays were performed between flies carrying three copies of a ubiquitously expressed AOX construct, driven by the α-tubulin promoter, and wild-type males of the same genetic background.Here, we examined the impact of the expression of In sperm-competition assays, AOX conferred a substantial disadvantage, associated with decreased production of mature sperm. Sperm differentiation appeared to proceed until the last stages, but was spatially deranged, with spermatozoids retained in the testis instead of being released to the seminal vesicle. High AOX expression was detected in the outermost cell-layer of the testis sheath, which we hypothesize may disrupt a signal required for sperm maturation.Drosophila thus has effects that are deleterious to male reproductive function. Our results imply that AOX therapy must be developed with caution.AOX expression in The online version of this article (doi:10.1186/s12861-017-0151-3) contains supplementary material, which is available to authorized users. In the animal kingdom, the alternative mitochondrial respiratory chain is widely represented, as illustrated by the broad phylogenetic distribution of the gene for the alternative oxidase (AOX) , 2. AOX Ciona intestinalis (Ascidiacea) was expressed in cultured human cells, it promoted resistance to inhibitors of complexes III and IV [Drosophila melanogaster, transgenic expression of C. intestinalis AOX had no apparent effect on viability, reproduction and health of the organism [dj-1β Parkinson’s disease model [C. intestinalis AOX has also been successfully expressed in the mouse without any significant effect on major physiological parameters [In fact, only marine invertebrates appear to still possess genes for alternative enzymes –3. NonetI and IV  and compI and IV . In the organism . It conforganism , mitigatse model  and amelse model , defectise model , or exprse model . C. interameters , 11. It rameters , 11.These benefits of AOX expression raise the possibility of its eventual use for treatment of human patients with complexes III and/or IV deficiencies, as a by-pass therapy . HoweverD. melanogaster males expressing AOX in competition assays with AOX-nonexpressors. AOX produced a clear impairment to reproductive function that was associated with decreased production of mature sperm cells. This suggests that possible gene therapy applications of AOX must be developed with caution.We set out to investigate the paradox between the beneficial by-pass and the possible maladaptive consequences of AOX expression in higher metazoans, by directly testing its effects on male reproductive function. Sperm are highly dependent on biological energy. Therefore, we reasoned that it may be a relevant target in which to test for subtle but functionally meaningful detriments of AOX expression. We challenged Drosophila, as in many species, females typically mate with a succession of males, storing sperm inside the female body to fertilize oocytes as they mature [In y mature . Sperm cy mature , 15; thiy mature . Newly ay mature .D. melanogaster males expressing AOX, we performed sperm-competition assays between flies carrying three copies of the α-tubulin-AOX construct, as described previously . In the ‘defensive’ approach, 3Xtub-AOX males were first allowed to mate with virgin w1118 females, which were then mated with w1118 males in the absence of 3Xtub-AOX males. Progeny from the competing males can be distinguished by eye-colour: those from transgenic males have red eyes, whilst those from w1118 males have white eyes. Controls consistently show that the eye-colour marker, as such, has no influence over the outcome . This result is consistent with previously published data using diverse genetic backgrounds [P1’ which measures the proportion of progeny sired by the first male to mate in sperm-competition assays [tub-AOX males sired statistically fewer offspring than some of the AOX-nonexpressor controls . In fact, even after 10 days since w1118 males were removed from the mating vials, eggs fertilized with the original male sperm were still preferentially laid over the ones fertilized with 3Xtub-AOX sperm . Control experiments performed with red-eyed daughterless-GAL4 (daGAL4)  and tubulin-GeneSwitch (tubGS) and also UAS-empty2nd and UAS-empty3rd , whereiUAS-AOX using the daGAL4 driver [tubAOX , whilst at the same time their SVs remain immature. In addition, we observed an accumulation of whitish material at the proximal end of the testes of 10-day-old 3Xtub-AOX males , which we investigated further (see below). The mature spermatozoids produced by these males, although few in number, did not appear to have any motility defects, as judged by visual inspection.Successful sperm competition in ve tract , 23–25. l mating . Therefoles Fig. , inset, tubGS) system, in which the same α-tubulin promoter as used in the tub-AOX constructs controls the expression of a modified GAL4 that is inducible by mifepristone (RU486). This allows time-regulated expression of a GAL4-dependent AOX transgene in exactly the same tissues as in the tubAOX lines. By transferring males to food-vials containing the inducing drug on the day of eclosion, any possible developmental disturbance is avoided, whilst accurate controls can be implemented, notably flies with transgene, but lacking driver and/or drug , and again only under inducing conditions . To confirm that this is an α-tubulin promoter-specific phenotype, we dissected the reproductive organs of 10-day-old males expressing UAS-AOX driven by daGAL4. The SVs had a normal morphology , but no obvious alterations were observed between AOX-expressing and control males, based on visual inspection of >70 dissected flies of each genotype. Although AOX was not expressed in the accessory glands . Howeveone Fig. , was belα-tubulin promoter . Some cells highly expressing ATP5A, a complex V subunit traditionally used as a mitochondrial marker, appeared negative for AOX , which is a marker for the pigment cells [At higher resolution, it was clear that not all cells in the ensheathing tissues of the testes and SVs express AOX at a high level when driven by the  connect . This isnt cells , 32. ParD. melanogaster testis is also the region where the individualization process starts. This process is essential for the final stage of elongated spermatid differentiation [tubGS, but did observe an elevated number of WBs . In control males, few of these individualized GFP-positive cells were found in this region of the testes , as expected, given that they should move to the SVs after individualization. The failure of the individualized, elongated spermatids to move into the SV and the associated disturbance in the spatial distribution of ICs could account for the accumulation of the whitish material seen by light microscopy, and is the simplest explanation for the defect seen in sperm-competition assays, although the reason why they are retained in the testis is not obvious morphologically.To investigate the nature of the whitish material found at the proximal end of the testis of AOX-expressing males, we used a transgenic construct that expresses GFP-tagged Don Juan protein (DJ-GFP) in the tail of elongated spermatids and mature spermatozoids , 36, enaC. intestinalis AOX in Drosophila did not affect viability or fertility under non-competitive lab conditions. Here we demonstrate that transgenic AOX expression in Drosophila under the α-tubulin promoter does cause a clear detriment to male reproductive success, using a stringent sperm-competition assay. This was accompanied by a decreased and disorganized production of mature spermatozoids. Although some sperm cells appear to complete the process of morphological differentiation, following individualization they appear to remain lodged in the proximal region of the testis, in the outer sheath of which AOX is highly expressed.In previous studies , 7 we reα-tubulin promoter or via the tubGS driver, is present at high abundance in the pigment cells of the testis sheath and SV, and is also found at very low levels in some somatic cyst cells. However, it is below the level of detection in the germline. The α-tubulin promoter has been reported to drive germline expression of transgenes up to the late spermatocyte phase [AOX, when expressed directly under the te phase . Clearlyte phase . Surpriste phase . On the tub-AOX males . A calctachment . Drosophrmations , whereasAn interference with cell signaling may also involve ROS. AOX has been reported to decrease mitochondrial ROS production even under conditions of normal oxidative phosphorylation activity , 42. In Clearly, much remains to be learned about the physiological effects of AOX expression. Our findings indicate the importance of studying these effects and their mechanisms in detail, before implementing AOX in gene therapy. Some of its properties may need to be modified, before the enzyme can be safely deployed.Drosophila. Transgenic flies ubiquitously expressing AOX were earlier found to exhibit normal development, reproduction and lifespan.AOX offers a potential therapeutic strategy for buffering pathological stresses in mitochondria. Whilst it is, indeed, able to compensate for a number of pathological insults, it has remarkably little, if any effect on normal physiology of model organisms, including In the present study we took this analysis to a deeper level, by applying a more stringent test of reproductive capacity, the well-established sperm-competition assay. Despite being fertile, AOX-expressing males showed a specific detriment in this test, which correlates with total transgene expression dose. Morphologically, we found this to be correlated with spatially deranged spermatogenesis and failure to release mature sperm into the storage organ  in normal quantities, although the defect is inferred to arise in the somatic pigment cells of the testis, not the germline. This is the first demonstration of a developmental defect caused by AOX expression in a metazoan, and needs also to be taken into account when considering therapeutic uses of AOX in humans.w1118, CyO and TM3,Sb balancers, UAS-StingerGFP, daGAL4, tubGS, and DJ-GFP were obtained from stock centres. The lines UAS-AOXF6 [tub-AOX35, tub-AOX112, tub-AOX7, 2Xtub-AOX, 3Xtub-AOX [UAS-AOXwt8.1, UAS-AOXmut, UAS-empty2nd, and UAS-empty3rd [w1118 background for six-to-ten generations, and were maintained in standard diet at 25 °C [tubGS line.Standard lines mpty3rd  were desw1118 females (white eyes), aged between 3 and 7 days, were initially allowed to mate individually with 5–8 day-old (virgin) transgenic males  for 3 days. The males were discarded and the females transferred to new vials and allowed to mate individually with 5–8 day-old w1118 males (white eyes), for a further 3 days, after which the second males were also discarded. The females were again transferred to new vials, allowed to lay eggs for 3 days, then transferred finally for further egg laying over 5 days. In the offensive paradigm, approximately 50 virgin w1118 females (white eyes) were first crossed with w1118 males (white eyes), and then with transgenic males , following the same mating and egg laying scheme of the defensive paradigm. Reproductive success was measured by counting the number of red- and white-eyed progeny, which represent, respectively, the progeny of transgenic and w1118 males. The small number of vials derived from any single female that contained progeny exclusively of one eye colour were considered to represent only a single mating and were excluded from the analysis. Data were plotted as mean number of progeny per female of each eye colour, in the four successive mating/egg laying vial sets, ± standard deviation.Sperm-competition assays were conducted using two approaches. In the defensive paradigm, approximately 50 virgin 2 prior to transferring to a dissection board containing phosphate-buffered saline (PBS). Using thin dissection forceps, the internal organs were removed from the abdomen by dislocating the external genitalia, followed by manual isolation of the reproductive organs, which were imaged immediately using the Nikon SMZ 745 T system under white light. The images were analyzed and testis and SV thickness was measured using NIS Elements D4.20 software .Reproductive organs were dissected by anesthetizing males of the indicated age and genotype with COFor immunofluorescence imaging, dissected organs were fixed in 4% paraformaldehyde for 20 min at room temperature, and then transferred to PBS prior to permeabilization. The organs were rinsed twice with PBS containing 0.1% Triton X-100 and 0.1% BSA, then permeabilized and blocked with PBS containing 1% Triton X-100 and 1% BSA for 30 min at room temperature, followed by overnight incubation at 4 °C with the primary antibody diluted in PBS containing 0.3% Triton X-100 and 0.5% BSA. The samples were then rinsed twice and washed three times (30 min each) with PBS containing 0.1% Triton X-100 and 0.1% BSA at room temperature, followed by overnight incubation at 4 °C with the secondary antibody diluted in PBS containing 0.3% Triton X-100 and 0.5% BSA. Washes were performed as described above, and samples were rinsed with PBS and then Milli-Q water, prior to transferring to optically clear 35 mm glass bottom dishes  containing ProLong Gold Antifade Mounting medium with DAPI . Primary antibodies used were rabbit polyclonal anti-AOX , mouse gmax for 10 min at 4 °C, and supernatant protein concentration was measured by the Bradford method. Crude mitochondrial preparations were obtained by homogenizing 50–100 adult males in ice-cold isolation buffer  followed by centrifugation at 200 gmax for 3 min at 4 °C. The supernatant was transferred to a new tube and centrifuged at 9000 gmax for 10 min at 4 °C. The pellet was resuspended in 50 μl of isolation buffer, and protein concentrations were determined using the Bradford method.Protein extracts from whole adult males (20–30) and from dissected testes and SVs of 50–100 males were prepared by gentle grinding of tissues in 50–200 μl PBS containing 1.5% Triton X-100 and Complete, Mini, EDTA-free Protease Inhibitor Cocktail . The suspension was centrifuged at 16,000 100 μg of total protein extracts and 40 μg of mitochondrial preparations were mixed with 5× Laemmli buffer , denatured at 95 °C for 5 min and resolved by SDS-PAGE on 4–20% or Any kD Criterion Precast Gels (Bio-Rad) at 120 V for approximately 1.5 h. Proteins were transferred to nitrocellulose membranes using the iBlot Gel Transfer system  for 7 min at room temperature. Membranes were blocked in TBST  containing 5% dried nonfat milk for 2 h at room temperature or overnight at 4 °C. Primary antibodies  were incubated for at least 1 h at room temperature, followed by three washes (10 min each) with TBST. Secondary antibodies  were incubated for at least 2 h at room temperature, and washed as described. Membranes were incubated in Luminol substrate detection system Immun-Star HRP , and chemiluminescence signals were detected on Fuji Medical X-ray films . Band densitometry was performed with ImageJ software .P1’ and P2’ [Measurements of organ thickness were performed as schematized in Additional file  and P2’  were tes


Human T-lymphotropic virus type 1 (HTLV-1) has been discovered in 1980 and has been linked to tropical spastic paraparesis (HAM/TSP) in 1985 in Martinique. There is no data on HAM/TSP incidence trends. We report, in the present work, the temporal trends incidence of HAM/TSP in Martinique over 25 years.P trends were assessed from simple Poisson regression models. Number of HTLV-1 infection among first-time blood donors was retrospectively collected from the central computer data system of the Martinique blood bank. The HTLV-1 seroprevalence into this population has been calculated for four 5-year periods between 1996 and 2015.Martinique is a Caribbean French West Indies island deserved by a unique Neurology Department involved in HAM/TSP diagnosis and management. A registry has been set up since 1986 and patients diagnosed for a HAM/TSP were prospectively registered. Only patients with a definite HAM/TSP onset between 1986 and 2010 were included in the present study. The 25-year study time was stratified in five-year periods. Crude incidence rates with 95% confidence interval (95%CI) were calculated using Poisson distribution for each period. Age-standardized rates were calculated using the direct method and the Martinique population census of 1990 as reference. Standardized incidence rate ratios with 95% CIs and Overall, 153 patients were identified . Crude HAM/TSP incidence rates per 100,000 per 5 years (95%CI) in 1986–1990, 1991–1995, 1996–2000, 2001–2005 and 2006–2010 periods were 10.01 (6.78–13.28), 13.02 (9.34–16.70), 11.54 (8.13–14.95), 4.27 (2.24–6.28) and 2.03 (0.62–3.43). Age-standardized 5-year incidence rates significantly decreased by 69% and 87% in 2001–2005 and 2006–2010 study periods. Patients characteristics did not differ regarding 1986–2000 and 2001–2010 onset periods. Between 1996–2000 and 2011–2015 study periods, the HTLV-1 seroprevalence significantly decreased by 63%.Martinique faces a sudden and rapid decline of HAM/TSP incidence from 2001 in comparison to 1986–2000 periods. Reduction of HTLV-1 seroprevalence, that may result from transmission prevention strategy, could account for HAM/TSP incidence decrease. Human T-lymphotropic virus type 1 (HTLV-1) was discovered in 1980 and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) was described five years later in 1985. HAM/TSP is a progressive disabling disorder characterized by spastic paraparesis with bladder and bowel dysfunction that constitutes a significant public health problem in endemic areas. Up to date, there is no efficiency treatment of HAM/TSP and prevention of HTLV-1 transmission is critical to limit the disease spreading throughout communities. In the present 25-year-study time, we report a significant decrease of HAM/TSP incidence estimated more than 70% in early 2000 compared to 1986–2000 period in Martinique a French West Indies Island. We found a trend to a significant older age at onset after 2000  that may reflect an age cohort effect and that could be indicative of a rapid decrease in HTLV-1 seroprevalence. We showed a significant decline in HTLV-1 infection among first-time blood donors between 1996–2000 and 2011–2015 study periods. Thus, probable HTLV-1 seroprevalence decrease secondary to HTLV-1 antibodies screening in blood donors and pregnant women and to iterative information campaigns could partly account for HAM/TSP incidence decline. This study emphasizes the importance of prevention strategies to control HAM/TSP development in HTLV-1 endemic areas. Human T-lymphotropic virus type 1 (HTLV-1) is associated with many diseases including HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). It is estimated that about 10–20 million people are infected with HTLV-1 throughout the world [Whereas HTLV-1 seroprevalence is unknown for 80% of world population , data arThe risk of developing HAM/TSP in HTLV-1 infected individuals has been assessed and deeply varies between studies and ethnic groups. In southern Japan the lifetime risk is 0.25%  while thWe report in the present study the incidence of HAM/TSP and its temporal trends, in the population of Martinique, French West Indies, over a 25-year period from 1986 to 2010. Temporal trends in HTLV-1 infection prevalence among first-time blood donors between 1996 and 2015 have also been calculated.Martinique is one of the most highly developed islands in the Caribbean, classified high (41st) in terms of global human development at the world level. The population steadily increased between 1982  and 2010 . The great majority (95%) of the population is Afro-Caribbean.Our Neurology department, located at the University Hospital of Martinique, constitutes the only neurological facility for patients in Martinique. It is highly involved in HAM/TSP diagnosis and management since the original description of the association between HTLV-1 and HAM/TSP . The uniTo reduce HTLV-1 transmission among Martinique population, prevention programs were implemented in the early nineties. Blood-donor and organ-donor screening was systematically introduced. Blood and organ donations are invariably rejected if HTLV-1 is detected. Similarly, antenatal screening was implemented to prevent mother-to-child transmissions of HTLV-1 by breastfeeding avoidance. Information campaigns were regularly performed encouraging the use of condom to prevent sexually transmitted infection including HIV and HTLV-1.The present study is based on a retrospective analysis on data prospectively collected between 1986 and 2015 in a dedicated registry. Patients with a HAM/TSP onset between 1986 and 2010 were considered.A registry has been set up since January 1986 and was still in progress in June 2015. HAM/TSP characteristics were collected carefully and continuously. Physicians involved in HTLV-1 diseases including neurologists, rehabilitation physicians, ophthalmologists, hematologists and dermatologists reported patients included in the registry. On a yearly basis, a cross-analysis of virology and clinical database was performed.A standardized case report form has been elaborated and collected the demographic data, the medical history, the clinical symptoms, the initial symptoms (gait impairment or urinary disturbances), the onset-to-diagnosis delay and the CSF analysis. The year of first symptoms, such as stiffness or weakness in the legs or urinary disturbances, defined the year of HAM/TSP onset.6 cells stored at -80°C after preparation from blood sample PBMCs. Forward and reverse primers for HTLV-I DNA quantitation: SK110 . Internal HTLV-I TaqMan probe: 5′CTTTACTGACAAACCCGACCTACCCATGGA-3′; Located between position 4829 and 4858 of the HTLV-I genome, and carried a 5′ reporter dye FAM (6-carboxy fluorescein) and a 3′ quencher dye TAMRA (6-carboxy tetramethyl rhodamine). Forward and reverse primers for human albumin DNA quantification: Alb-S (5′-GCTGTCATCTCTTGTGGGCTGT-3′) and Alb-AS (5′-AAACTCATGGGAGCTGCT GGTT-3′). Alb TaqMan probe (5′-FAM-CCTGTCATGCCCACACAAATCTC TCC-TAMRA-3′) were used as described previously [6 PBMCs.Peripheral blood mononuclear cells (PBMCs) were isolated from EDTA-enhanced blood by density gradient centrifugation. Real Time TaqMan PCR was performed on DNA extracted from dry pellets of 10eviously . ResultsFor the purpose of the present study, each chart was reviewed and HAM/TSP diagnosis was defined according to an updated staged approach to the World Health Organization criteria . Only deIn Martinique, all blood donations are performed in the center for blood transfusion (Etablissement Français du Sang). Demographic characteristics of all first-time blood donors tested positive for HTLV-1 were retrospectively collected from the central computer data system of the blood bank. Only donors tested positive with ELISA assay and confirmed with Western blot assay were retained. Data were available for the 20-year period from January 1, 1996 and December 31 2015. Characteristics of all donors were not available for the present study.The present research was conducted according to the principles of the declaration of Helsinki. In 1995, a hospital ethic committee  was constituted and approved the HAM/TSP registry. Between 1986 and 1994, only oral informed consents were obtained whereas all patients included in the registry after 1994 have given their written informed consent. After standardized information, the oral consent was obtained and collected in the patient’s chart. The hospital ethic committee allowed retrospectively oral consents for 1986–1994 period. For child inclusion participant, a parent has provided informed consent on the child’s behalf. Analysis was performed on anonymized data to ensure confidentiality.Due to the small number of events per year, the 25-year study time, from January 1, 1986 to December 31, 2010, was stratified in five-year periods for HAM/TSP incidence analysis. Similarly, the 20-year study time, from January 1, 1996 to December 31, 2015, for HTLV-1 seroprevalence in first-time blood donors was stratified in four 5-year periods.www.insee.fr). By convention, the population of the last year of each defined period was considered for calculation. Crude incidence rates with 95% confidence intervals (95%CIs) were calculated using Poisson distribution for each period. Age-standardized rates were calculated using the direct method and the Martinique population census of 1990 as reference. The Martinique population census of 1990 constituted the denominator to calculate HAM/TSP incidence for the first study period 1986–1990. Then populations of 1995, 2000, 2005 and 2010 were used for 1991–1995, 1996–2000, 2001–2005 and 2006–2010 study periods. Standardized incidence rate ratios with 95% CIs and P trends were assessed from simple Poisson regression models. P for trend was used to either support or reject a linear trend in HAM/TSP standardized incidence rates from the first to the last study period.The numerator for calculation of incidence rate was the number of HAM/TSP patients with a disease onset in the defining period. The denominator was based on data provided by French National Institute for Statistical and Economic Studies  was used as reference to calculate the odds ratio of seroprevalence with 95%CI in the four study periods.t test or Mann-Whitney test were used to examine differences in nominal and continuous values. All analyses were performed in SAS 9.3  and MS Excel software.The χ2 test or Fisher-exact test, as appropriate, and Student Between January 1986 and June 2015, 326 HAM/TSP patients were included in our registry. We excluded 84 patients who met the diagnosis of probable HAM/TSP according to WHO criteria. Among the 242 definite HAM/TSP patients, 89 were excluded regarding their year of onset before 1986 (83) or after 2010 (6). Eventually, 153 definite HAM/TSP patients experienced a disease onset over the 25-year study period. The mean age at onset was 53 ± 13 years and females predominated (79.7%). Patients experienced gait impairment as first symptoms in 86% and the median onset-diagnosis delay was 3 years. Patient’s characteristics are summarized in The 5-year crude incidence rates (95%CI) per 100,000 in 1986–1990, 1991–1995, 1996–2000, 2001–2005 and 2006–2010 study periods are reported in Regarding HAM/TSP incidence rate decline from 2001, we compared patient’s characteristics between the two groups whose disease onset was in 1986–2000 and 2001–2010 periods.Sex ratio, initial symptoms, onset-to-diagnosis delay and history of blood transfusion before 1990 were comparable between the two groups. We found a trend to significance for an older age at onset in 2001–2010 incident cases.Level of HTLV-1 in PBMC was available for 123 out of 153 patients. The mean level of HTLV-1 proviral load did not differed between the two study periods.Over 25 years, 1986–2010, our study reports a significant decrease of new definite HAM/TSP cases. Whereas the crude 5-year incidence rates remained stable between 1986 and 2000, ranging from 10.1 to 12.2/100,000, the rates rapidly decline between 2001 and 2010, ranging from 3.2 and 2.9/100,000. The age-standardized 5-year incidence rates exhibit a dramatic decrease by 70% from 2001 and reaching 87% for 2006–2010 periods.In accordance with previous studies, the age at onset of HAM/TSP patients was around 50 years old –18. We fSchistosoma proteins may reduce inflammatory process associated with HTLV-1 infection through interferon-gamma level decrease [Strongyloides is associated with formation of new HTLV-1-infected T-cell clones and with oligoclonal proliferation of certain HTLV-1 clones, increasing thereby the risk of HTLV-1 disease expression. On the other hand, whereas HTLV-1 and Strongyloides infect 43% and 35% of tested indigenous in Central Australia [In 1989, HTLV-1 seroprevalence was appraised at 2.2% in the general population of Martinique , while idecrease , anotherdecrease . Gillet decrease  suggest ustralia , the HAMustralia . StudiesWhereas meta-analyses show no clear evidence that structural interventions at community level, to increase condom use, prevent the transmission of HIV and other sexually transmitted infections , no reliDespite the large study period of 25 years, the number of new HAM/TSP cases remains limited with low statistical power particularly in comparing the characteristics of the two groups of patients with a disease onset before and after 2001. Between 1986 and 2015, technological improvement of imaging and laboratory investigations may have influenced HAM/TSP diagnosis accuracy. However, HAM/TSP diagnosis criteria did not change during the study time and imaging methods that are crucial to rule out alternative myelopathy and especially spinal cord compression, were available through the whole study time. A reduction of HAM/TSP patients referred to our network by the primary health setting over the study period might be raised. However, only slight changes occurred to the global health care network for HTLV-1 infected patients. Moreover, we assume that improvement of medical management of HAM/TSP patients in Neurology and Rehabilitation centers may have prompted general practitioners and primary care centers to refer more systematically patients to our network. We showed that HTLV-1 seroprevalence have decreased over 20 years among first-time blood donors, but the absence of demographic characteristics of this population limits our findings interpretation.In summary, Martinique faces a rapid and sustained decline of HAM/TSP incidence from 2001 estimated by 70% in comparison to 1986–2000 periods. Implementation of HTLV-1 transmission prevention strategy may result in seroprevalence reduction and in HAM/TSP incidence decrease observed 15 years later. The monitoring of new HAM/TSP cases has to be continued and HTLV-1 seroprevalence in blood donors and pregnant women must be sequentially assessed.S1 Checklist(DOC)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


Individuals with grapheme-color synesthesia experience idiosyncratic colors when viewing achromatic letters or digits. Despite large individual differences in grapheme-color association, synesthetes tend to associate graphemes sharing a perceptual feature with similar synesthetic colors. Sound has been suggested as one such feature. In the present study, we investigated whether graphemes of which representative phonemes have similar phonetic features tend to be associated with analogous synesthetic colors. We tested five Korean multilingual synesthetes on a color-matching task using graphemes from Korean, English, and Japanese orthography. We then compared the similarity of synesthetic colors induced by those characters sharing a phonetic feature. Results showed that graphemes associated with the same phonetic feature tend to induce synesthetic color in both within- and cross-script analyses. Moreover, this tendency was consistent for graphemes that are not transliterable into each other as well as graphemes that are. These results suggest that it is the perceptual—i.e., phonetic—properties associated with graphemes, not just conceptual associations such as transliteration, that determine synesthetic color. Individuals with grapheme-color synesthesia experience “colors” when viewing achromatic letters or digits. The association in each pair of the inducing grapheme and the induced color is idiosyncratic. In addition, the synesthetic color induced by a grapheme is not the same among different synesthetes. Therefore, the large individual differences in grapheme-color associations have long been considered a key characteristic in synesthesia. More recently, however, it has been recognized that there may be potential regularities of synesthetic grapheme-color association hidden behind the large individual variances . In contrast, Asano and Yokosawa  induced similar synesthetic color. These results imply that the sound quality is indeed what determines synesthetic color, which is not confined to a specific language.Some previous works have taken cross-linguistic approaches to consider the possibility that inherent differences between languages affect synesthetic grapheme-color association  is overlapped to the color of Hangul “” (Korean grapheme pronounced as /h/)”. If transliterability between graphemes of different languages were the source of the previous results, it weakens the implication of the results that graphemes sharing a phonetic quality tend to induce similar synesthetic color.While these results extended the findings of Asano and Yokosawa , 2012 anThis poses a serious concern because the investigation into the non-random association between a certain aspect of inducers and concurrents in synesthesia (graphemes and colors respectively in this paper) bears implications for cross-modal association between features in human languages and thoughts in general ' are transliterated with each other, whereas “t” and “” (Korean consonant pronounced similar to /r/ or /l/)' are not. Another benefit of employing the rules is that they offer a more objective set of criteria to pair and compare graphemes from different languages, since the rules are universal classification which can be applied to every language.Introducing the articulatory rules has several advantages. First, as mentioned earlier, it can rule out the confounding factor of conceptual association, since each category includes graphemes that are not transliterated with each other despite the shared phonetic quality, as well as those that are. For example, in the alveolar category, the acoustic qualities of graphemes “t” and “”), to examine whether they are still associated with similar synesthetic colors. This final analysis was expected to distinguish conceptual association via transliteration from perceptual similarity. Detailed methods and the results of those three analyses are described below.We tested five Korean multilingual synesthetes on a color-matching task using graphemes from Korean (hangul), English , and Japanese orthography (hiragana and katakana) as inducing stimuli. We analyzed the matched colors in three ways. First, in the within-script analysis, we examined the similarity of the synesthetic colors induced by those graphemes whose representative sounds are classified into the same category within each script. Second, in the cross-script analysis, we investigated whether the influence of the phonetic features on synesthetic color also exists between graphemes across different scripts. Finally, we took a closer look at those graphemes categorized as the same but which cannot be transliterated into each other . All participants use Korean as their first language and had studied English as their second language for about 10 years at the time of the study. They experience synesthetic colors for hangul, Latin graphemes, and Japanese syllabaries (hiragana and katakana); three of them experience colors on both hiragana and katakana, whereas two participants experience colors only on hiragana. All of them showed high consistency over multiple color matching results, and identified as synesthetes whose consistency scores are below 135  were used as stimuli for synesthetic color matching task. Based on the previous work showing the similarity of synesthetic colors of graphemes and syllabaries sharing initial phonemes  controlled by an Intel PC using Matlab 7.0.4  analysis was applied to each participant separately in order to correct potential type-I error in multiple comparisons . The results from the CVI analyses on within-script pairs reassured us that characters belonging to the same phonetic category tended to take on similar synesthetic colors. Figure t(238) = −5.826, KBL t(276) = −19.633, p < 0.001, HWP t(106) = −3.851, p < 0.01, KML t(115) = −9.767, YMK t(90) = −6.313, p < 0.001, FDR corrected for all synesthetes]. In the velar category, CVIs of three synesthetes significantly differed from the baseline CVI . When it comes to nasal category, two synesthetes showed statistically significant differences . These results imply that graphemes belonging to the same phonetic category tend to induce similar synesthetic colors. Moreover, such results do not seem to be solely driven by the script of their native language (hangul) but also within the scripts of the second (L2) or the third languages (L3), when the results are broken down into different scripts  = −4.358, p < 0.05; Velar: t(4) = −3.210, p < 0.05; Supplementary Figure Such a tendency is more noticeable in certain categories than in others. In the alveolar category of the place of articulation, quite a few of the reported synesthetic colors experienced by the five synesthetes were similar. Moreover, the alveolar category comprised multiple graphemes in all four scripts tested  = −5.881, KBL t(625) = −23.269, HWP t(270) = −4.30, KML t(114) = −6.967, YMK t(118) = −4.574, p < 0.001, FDR corrected]. Three synesthetes showed statistically significant difference both in the velar and in the nasal conditions . These results imply that the influence of phonetic feature of inducing graphemes on the induced synesthetic colors is not limited within a script. Results also showed that the relationship between phonetic feature and synesthetic color is not limited between scripts of the same language  either  = −3.283, p < 0.05; Supplementary Figure We again focused on the alveolar, velar and nasal categories as in the within-script CVI analyses. Specifically, all five synesthetes showed statistically significant difference in the alveolar category . In the velar category, two synesthetes had smaller mean CVI both in pairs of graphemes that are transliterable and in those that are not and one synesthete had smaller mean CVI only in pairs of graphemes that are not transliterable . In the nasal category, two synesthetes showed smaller mean CVI only in pairs of transliterable graphemes but two synesthetes showed smaller mean CVI both in pairs of graphemes that are transliterable and in those that are not . When we conducted the group-level analysis, the mean CVI of the alveolar category was significantly different from the mean of baseline both in pairs of graphemes that are transliterable and in those that are not [Alveolar T: t(4) = −3.212, NT: t(4) = −3.331, p < 0.05, FDR corrected; Supplementary Figure Results from the within- and cross-script CVI analyses showed that graphemes  of the same phonetic feature tend to induce similar synesthetic colors, which leads us to conclude that phonetic feature is an important determining factor of synesthetic color. However, it is still possible that conceptual association between graphemes within the same phonetic category such as transliterability, not the acoustic feature itself, might base the finding. Therefore, we divided graphemes within each phonetic category into two subgroups: one includes those that are conceptually associated and transliterable based on readily recognizable sound similarity, and the other contains those of which phonetic similarity is not readily recognizable and thus not transliterable. For example, “t” and “By exploiting such articulatory rules as the place and the manner of articulation, the current results show that graphemes associated with a shared phonetic feature tend to induce similar synesthetic colors. This was the case not just within a script, but also across multiple scripts of different languages. It is one step further from the previous findings  funded by the Ministry of Science, Information and Communications Technologies (ICT) and Future Planning.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Sc) in the brain. PrPSc accumulation in the endoplasmic reticulum (ER) result in a dysregulated calcium (Ca2+) homeostasis and subsequent initiation of unfolded protein response (UPR) leading to neuronal dysfunction and apoptosis. The molecular mechanisms for the transition between adaptation to ER stress and ER stress-induced apoptosis are still unclear. Mitogen-activated protein kinases (MAPKs) are serine/threonine protein kinases that rule the signaling of many extracellular stimuli from plasma membrane to the nucleus. However the identification of numerous points of cross talk between the UPR and MAPK signaling pathways may contribute to our understanding of the consequences of ER stress in prion diseases. Indeed the MAPK signaling network is known to regulate cell cycle progression and cell survival or death responses following a variety of stresses including misfolded protein response stress. In this article, we review the UPR signaling in prion diseases and discuss the triad of MAPK signaling pathways. We also describe the role played by MAPK signaling cascades in Alzheimer’s (AD) and Parkinson’s disease (PD). We will also overview the mechanisms of cell death and the role of MAPK signaling in prion disease progression and highlight potential avenues for therapeutic intervention.Prion diseases are neurodegenerative pathologies characterized by the accumulation of a protease-resistant form of the cellular prion protein named prion protein scrapie (PrP Sc  are a group of fatal neurodegenerative disorders affecting both humans and animals. Major animals TSEs include bovine spongiform encephalopathies (BSE) in cattle, scrapie in sheep and goats and chronic wasting disease in elk and deer. Human TSEs include Creutzfeldt-Jakob disease (CJD), Gerstman-Sträussler-Scheinker syndrome (GSS), Fatal Familial Insomnia (FFI) and kuru  is a multifunctional organelle responsible for folding and processing of secretory and transmembrane proteins. Balanced protein load and folding capacity are two important factors for correct protein folding and processing in the ER. Under pathological conditions this balance between folding load and capacity is disrupted and misfolded/unfolded proteins are accumulated that subsequently initiates ER stress  and the p38 kinases. The widely accepted perception that exists about these MAPKs is that JNK/SAPK (stress-activated protein kinase) and p38 MAPK help in promoting cell death in face of chronic stress stimuli, while ERK1/2 opposes cell death signaling cascades , Parkinson’s disease (PD) and prion diseases  and prion diseases have harmful effects on humans and animals due to lack of effective therapeutic strategies and presymptomatic diagnostic tools. Thus there is a need of novel therapeutic intervention strategies to control these diseases. Here in this review article, we will focus on the role of UPR and MAPK signaling cascades in neurodegenerative diseases with special focus on prion diseases.c to PrPSc is the hallmark of all TSEs . The general decline in several synaptic proteins levels during infection was proposed to be a key trigger for neurodegeneration  reversing the translational repression of the synaptic proteins is a valid therapeutic strategy for prion disease.The main purpose of the PERK pathway signaling cascade is to relieve the ER stress by reducing the amount of proteins entering the ER  and the p38 family of kinases (p38 MAPKs). They are activated through three-tier kinase signaling mechanism. As mentioned above, the MAPK subgroups are activated upon phosphorylation via the dual threonine/tyrosine motif in their activation loop: ERK1/2 with glutamine (Glu), proline (Pro) and glycine (Gly) embedded between threonine/tyrosine motif (Thr-Glu-Tyr), JNK (Thr-Pro-Tyr), and p38 (Thr-Gly-Tyr). There are a variety of selected MAP kinase kinases (MKKs) which phosphorylate these threonine/tyrosine motifs. The MKKs are in turn activated via the phosphorylation of a number of MAP kinase kinase kinases (MKKKs). The intricate mechanisms of biological outcomes of MAPK signaling are dependent on several factors including the sub-cellular localization of the MAPK signaling (determined by the amount of misfolded proteins and scaffolding proteins). In addition, the magnitude and duration of MAPK pathways activation and the effect of other signaling pathways such as UPR on MAPK pathways determines cell fate  through the regulation of the BCL2 family members  treatment Coffey, . There aThe p38 MAPKs activation has been observed in response to a variety of inflammatory cellular stresses such as hyperosmolarity, arsenite or taxol and pro-inflammatory cytokines released in response to activated microglia due to the accumulation of misfolded proteins  misfolds and transform to prion protein scrapie (PrPSc). There is an increase in the β- sheet content and a corresponding decrease in α-helices . But there are several cases where ERK1/2 can be apoptotic . The mechanism of MAPK signaling requires further molecular research to determine whether MAPK-dependent signaling can promote cell proliferation, survival or cell death. Scientists are agreed on the analogy with the other investigations that the response to ER stress could prove crucial in reflecting the differences in the duration of signaling, magnitude of signaling, relationship with other signaling pathways and to some extent cell type-specific expression of MAPK cascades. Although there are still several challenges in understanding the role of MAPK signaling and ER stress in prion diseases there are cases where there are indications that therapeutics to manipulate both UPR and MAPK signaling could be advantageous. To date studies on prion disease in vivo models or in vitro models none has focused on inhibiting UPR and MAPK pathways simultaneously. Moreno et al.’s  reversed caspase mediated cell death in prion disease cell model -ASK signaling or due to the Caet al.’s  work on et al.’s , 2013. OIt is clear from previous investigations that useful therapeutic investigation for all the major misfolding diseases such as AD, PD, HD and TSEs should be focused on targeting either the misfolded proteins directly or indirectly by targeting the subsequent downstream signaling pathways. These pathways are initiated in response to the ER stress due to accumulation of the pathogenic proteins. UPR and MAPK pathways need further molecular research to exactly pinpoint the signaling cascades that are activated in response to the accumulation of prion protein in TSEs. In addition, focusing on the UPR and MAPK pathways simultaneously might be a good idea as all the previous research conducted on prion diseases was focused solely either on UPR inhibition or MAPK inhibition individually. Modern molecular level pharmacological research has contributed significantly towards achieving the goal of inhibiting MAPK pathways. Currently all available inhibitors in the market can only target any one of the arms of MAPK pathways. By contrast to other arms of MAPK pathways, many p38 MAPK inhibitors are available such as PH-797804, BIRB 796, VX-702, SB 239063, SB202190, SCIO 469, and BMS 582949. ERK1/2 pathway inhibitors available are PD98059, BVD-523 and U0126. JNK pathway inhibitor commonly used in research is SP600125. On the other hand there are very few inhibitors available to target the UPR signaling cascades. That means simultaneous targeting of UPR and MAPK pathways via inhibitors may be difficult. But Modern scientific tools such as gene manipulation techniques  can be adopted to target UPR and MAPK pathways simultaneously in protein misfolding neurodegenerative diseases to achieve the goal of stress free environment within the nervous cell.SZAS wrote the manuscript, DZ gave the idea, TH helped in figures and LY critically reviewed the manuscript before final submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Caenorhabditis elegans for suppressors of activated Gq. One of the suppressors is an allele of sek-1, which encodes a mitogen-activated protein kinase kinase (MAPKK) in the p38 MAPK pathway. Here, we show that sek-1 mutants have a slow locomotion rate and that sek-1 acts in acetylcholine neurons to modulate both locomotion rate and Gq signaling. Furthermore, we find that sek-1 acts in mature neurons to modulate locomotion. Using genetic and behavioral approaches, we demonstrate that other components of the p38 MAPK pathway also play a positive role in modulating locomotion and Gq signaling. Finally, we find that mutants in the SEK-1 p38 MAPK pathway partially suppress an activated mutant of the sodium leak channel, NCA-1/NALCN, a downstream target of Gq signaling. Our results suggest that the SEK-1 p38 pathway may modulate the output of Gq signaling through NCA-1(unc-77).Gq is a heterotrimeric G protein that is widely expressed in neurons and regulates neuronal activity. To identify pathways regulating neuronal Gq signaling, we performed a forward genetic screen in  Gq is a widely expressed heterotrimeric G protein that regulates a variety of biological processes, ranging from neurotransmission to cardiovascular pathophysiology . In addiCaenorhabditiselegans, which has a single Gαq homolog (EGL-30) and conservation of the other components of the Gq signaling pathway  (UNC-73 (ortholog of Trio RhoGEF) (UNC-73 activates RHO-1 (ortholog of RhoA), which has been shown to enhance neurotransmitter release through both diacylglycerol kinase (DGK-1)–dependent and –independent pathways  (egl-30(tg26) mutant animals exhibit hyperactive locomotion and a “loopy” posture, in which worms have exaggerated, deep body bends and loop onto themselves (sek-1. SEK-1 is a mitogen-activated protein kinase kinase (MAPKK), the C. elegans ortholog of mammalian MKK3/6 in the p38 MAPK pathway  . egl-30( pathway . HoweverSEK-1 is activated by the MAPKKK NSY-1 (ortholog of ASK1) and activates the p38 MAPKs PMK-1 and PMK-2   . Here, wsek-1(yak42) mutant was isolated from an ENU mutagenesis suppressor screen of the activated Gq mutant, egl-30(tg26) (sek-1(yak42) was outcrossed away from egl-30(tg26) before further analysis. Double mutant strains were constructed using standard methods  suppression phenotype. yak42 was initially mapped to the X chromosome using strains EG1000 and EG1020, which carry visible marker mutations. These experiments showed that yak42 was linked to lon-2, but it was at least several map units (cM) away. yak42 was further mapped to ∼1 cM away from the red fluorescence insertion marker oxTi668, which is located at +0.19 cM on the X chromosome.egl-30(tg26); yak42 was used for whole-genome sequencing to identify candidate yak42 mutations. XZ1233 was constructed by crossing a yak42 strain outcrossed two times, back to egl-30(tg26). Thus, in XZ1233, yak42 has been outcrossed three times from its original isolate. DNA was isolated from XZ1233 and purified according to the Hobert Laboratory protocol (http://hobertlab.org/whole-genome-sequencing/). Ion torrent sequencing was performed at the University of Utah DNA Sequencing Core Facility. The resulting data contained 10,063,209 reads of a mean read length of 144 bases, resulting in ∼14× average coverage of the C. elegans genome. The sequencing data were uploaded to the Galaxy web platform, and we used the public server at usegalaxy.org to analyze the data , looking for regions that lacked sequence coverage. We found a 3713-bp deletion that was subsequently confirmed to be the yak42 causal mutation, affecting the gene sek-1 located at −1.14 cM.Strain XZ1233 Escherichia coliOP50 stock culture, spread with sterile glass beads to cover the entire plate. Bacterial lawns were grown at room temperature (22.5–24.5°) for 24 hr, and then stored at 4° until needed. All locomotion assays were performed on first-day adults at room temperature (22.5–24.5°). L4 stage larvae were picked the day before the assay and the experimenter was blind to the genotypes of the strains assayed. For experiments on strains carrying extrachromosomal arrays, the sek-1(km4) control worms were animals from the same plate that had lost the array.Locomotion assay plates were made by seeding 10 cm nematode growth medium plates with 150 μl of an egl-8, unc-73, and rund-1 mutants, worms were allowed a minimal recovery period  prior to counting body bends.Body bend assays were performed as described p::sek-1(cDNA)::tbb-2utr::gpd-2 operon::GFP::H2B:cye-1utr . Plasmids and primers used are found in Table S2 in C. elegans strains with extrachromosomal arrays were generated by standard methods (sek-1(yak42) worms. To achieve a power of 0.95, it was calculated that 17 animals should be assayed per experiment. Data were analyzed to check for a normal distribution , and then subjected to the appropriate analysis using GraphPad Prism 5. For data sets with three or more groups, if the data were normal, they were analyzed with a one-way ANOVA; if they were not, they were analyzed with a Kruskal–Wallis test. Post hoc tests were used to compare data sets within an experiment. Reported P-values are corrected. Table S3 in File S1 contains the statistical tests for each experiment. * P < 0.05, ** P < 0.01, *** P < 0.001.At the beginning of the project, a power study was conducted on pilot body bend assays using wild-type and File S1, and are available from the Caenorhabditis Genetics Center or upon request. The authors state that all data necessary for confirming the conclusions presented in the article are represented fully within the article and Supplemental Material.Strains and plasmids are shown in Tables S1 and S2 in egl-30(tg26) (egl-30(tg26) worms are hyperactive and have a loopy posture, characterized by an exaggerated waveform . Whole-genome sequencing revealed that yak42 carries a large deletion of the sek-1 gene from upstream of the start codon into exon 4 , a previously published sek-1 deletion allele, for the Gq suppression phenotype  . egl-30 double mutants with either sek-1(yak42) or sek-1(km4) are not loopy (File S1). sek-1(yak42) was outcrossed from egl-30(tg26) and assayed for locomotion defects. Both the sek-1(yak42) and sek-1(km4) mutants are coordinated but move more slowly than the wild type (sek-1(ag1) point mutation (File S1). To test whether the egl-30(tg26) suppression phenotype might be an indirect effect of the slow locomotion of a sek-1 mutant, we built an egl-30(tg26) double mutant with a mutation in unc-82, a gene required for normal muscle structure. unc-82 mutants are coordinated but move slowly, similar to sek-1 mutants (egl-30(tg26) unc-82(e1220) double mutant moves more slowly than an egl-30(tg26) mutant , it is still loopy  allele causes an R243Q missense mutation in the Gα switch III region that has been shown to reduce both the intrinsic GTPase activity of the G protein and render it insensitive to GTPase-activation by a regulator of G protein signaling (RGS) protein, thus leading to increased G protein activation (egl-30(tg26) by sek-1 is specific for this egl-30 allele, we built a double mutant between sek-1(km4) and the weaker activating mutation egl-30(js126). egl-30(js126) causes a V180M missense mutation in the Gα switch I region immediately adjacent to one of the key residues required for GTPase catalysis  mutant also suppresses the hyperactivity and loopy waveform of egl-30(js126)  , demonstEGL-30/Gαq is negatively regulated by GOA-1, the worm Gαo/i ortholog, and the RGS protein EAT-16  suppresses the hyperactivity and loopy waveform of goa-1(sa734) . However, although sek-1(km4) suppresses the hyperactivity of eat-16(tm775), it did not significantly suppress the loopy waveform . One possible downstream effector of GOA-1 is the DAG kinase DGK-1, which inhibits DAG-dependent functions such as synaptic vesicle release (dgk-1(sy428) animals are hyperactive, but the sek-1dgk-1 double mutant is uncoordinated and looks like neither sek-1 nor dgk-1 mutants, confounding the interpretation of how sek-1 genetically interacts with dgk-1.egl-30 is widely expressed and acts in neurons to modulate locomotion  or in acetylcholine neurons (unc-17 promoter) was sufficient to rescue the sek-1 mutant slow locomotion phenotype, but expression in GABA neurons (unc-47 promoter) was not sufficient (sek-1 acts in acetylcholine neurons to modulate locomotion rate.To identify the cell type responsible for the fficient . These rsek-1 acts in neurons to suppress egl-30(tg26). Expression of sek-1 under pan-neuronal and acetylcholine neuron promoters reversed the sek-1 suppression of egl-30(tg26). Specifically, egl-30(tg26) sek-1 double mutants expressing wild-type sek-1 in all neurons or acetylcholine neurons resembled the egl-30(tg26) single mutant  and motorneuron (unc-17β promoter) acetylcholine neuron subclasses , suggesting that the slow locomotion of sek-1 mutants is due to a loss of sek-1 in acetylcholine motorneurons. However, expression of sek-1 in either the head acetylcholine neurons or motorneurons partially reversed the sek-1 suppression of egl-30(tg26) hyperactivity , suggesting that the hyperactivity of activated Gq mutants may result from excessive Gq signaling in both head acetylcholine neurons and acetylcholine motorneurons; sek-1 may act in Gq signaling in both neuronal cell types. By contrast, expression of sek-1 in head acetylcholine neurons but not motorneurons reversed the sek-1 suppression of the egl-30(tg26) loopy waveform , suggesting that the loopy posture of activated Gq mutants may result from excessive Gq signaling in head acetylcholine neurons, and sek-1 may act in those neurons to control body posture.To narrow down the site of sek-1 acts in the development of the AWC asymmetric neurons, we asked whether sek-1 also has a developmental role in modulating locomotion by testing whether adult-specific sek-1 expression (driven by a heat-shock promoter) is sufficient to rescue the sek-1 mutant. We found that sek-1 expression in adults rescues the sek-1 slow locomotion phenotype , SEK-1 (MAPKK), and PMK-1 or PMK-2 (MAPKs) (tir-1(tm3036) and nsy-1(ok593) mutant animals have slow locomotion on their own, and also suppress the hyperactivity and loopy waveform of egl-30(tg26)  and a pmk-2pmk-1 double mutant. Although we found that the pmk-2 and pmk-3 single mutants were slightly slow on their own, only the pmk-2pmk-1 double mutant phenocopied sek-1 and suppressed both the hyperactivity and loopy waveform of egl-30(tg26) (pmk-2 and pmk-1 act redundantly downstream of sek-1 to suppress egl-30(tg26). These data suggest that the p38 MAPK pathway modulates locomotion rate in C. elegans and acts genetically downstream of egl-30. (MAPKs) . We test30(tg26) . We also30(tg26) . Thus, pC. elegans. Specifically, the JNK pathway members jkk-1 (JNK MAPKK) and jnk-1 (JNK MAPK) have been shown to act in GABA neurons to modulate locomotion . Moreover, neither jkk-1 nor jnk-1 suppressed the loopy phenotype of egl-30(tg26) . Thus, the JNK and p38 MAPK pathways modulate locomotion independently, and the JNK pathway is not involved in Gq signaling.The JNK MAPK pathway, related to the p38 MAPK family, also modulates locomotion in comotion . We founPMK-1 is the transcription factor ATF-7 (atf-7(qd22qd130) loss-of-function mutant and the atf-7(qd22) gain-of-function mutant moved slowly compared to wild-type animals . However, atf-7(qd22qd130) did not suppress the loopy waveform of egl-30(tg26) , suggesting that atf-7 is not a target of this pathway, or else it acts redundantly with other downstream p38 MAPK targets. We also tested gap-2, the closest C. elegans homolog of ASK1-interacting protein (AIP1), which activates ASK1 (the ortholog of C. elegansNSY-1) in mammalian systems . Finally, we tested VHP-1, a phosphatase for p38 and JNK MAPKs that inhibits p38 MAPK signaling (vhp-1(sa366) mutant also showed no locomotion defect .We also tested the involvement of possible p38 MAPK pathway effectors. One of the targets of egl-30(tg26) animals are loopy and hyperactive, so we tested whether increased activation of the TIR-1/p38 MAPK signaling module causes similar phenotypes. The tir-1(ky648) allele leads to a gain-of-function phenotype in the AWC neuron specification (File S1).egl-30(tg26) identified mutants that fall into three different categories: mutants in the canonical Gq pathway, such as the PLC egl-8 , unc-73(ox317), and rund-1(tm3622) have slow locomotion , have a loopy posture reminiscent of egl-30(tg26) (rho-1(gf) sek-1(km4) double mutants had a loopy body posture like rho-1(gf) animals, and an even slower locomotion rate (sek-1 and rho-1(gf) mutants have additive locomotion phenotypes. However, both sek-1(km4) and sek-1(yak42) weakly suppress the slow growth rate of the rho-1(gf) mutant (data not shown). Because sek-1 does not enhance unc-73 mutants and suppresses some aspects of the rho-1(gf) mutant, sek-1 may modulate output of the Rho pathway, although it probably is not a direct transducer of Rho signaling.We next tested whether 30(tg26) . rho-1. The nca-1(gf) animals are coiled and uncoordinated; thus, it is difficult to measure their locomotion rate by the body bend assay because they do not reliably propagate sinusoidal waves down the entire length of their body. Instead, we used a radial locomotion assay in which we measured the distance animals moved from the center of a plate. nca-1(gf) double mutants with either sek-1(km4) or nsy-1(ok593) uncoiled a bit, but still exhibited uncoordinated locomotion (nca-1(gf), they propagated body waves to their posterior half even more poorly than the nca-1(gf) mutant. However, both sek-1 and nsy-1 partially suppressed the loopy waveform of the nca-1(gf) mutant (sek-1 and nsy-1 weakly suppressed the nca-1(gf) locomotion defect  . Together, these data suggest that mutations in the SEK-1 p38 MAPK pathway suppress some aspects of the nca-1(gf) mutant.We tested whether comotion . In fact) mutant , and in n defect . Additiosek-1 acts in acetylcholine neurons to modulate wild-type and egl-30(tg26) locomotion, we tested whether sek-1 also acts in these neurons to suppress nca-1(gf). Expression of sek-1 in all neurons or in acetylcholine neurons of nca-1(gf) sek-1(km4) animals restored the nca-1(gf) loopy phenotype (sek-1 in GABA neurons did not affect the loopy posture of the nca-1(gf) sek-1 double mutant (sek-1 acts in acetylcholine neurons to modulate the body posture of nca-1(gf) as well. However, in radial locomotion assays, expression of sek-1 in any of these neuron classes did not significantly alter the movement of the nca-1(gf) sek-1 double mutant , although the weak suppression of nca-1(gf) by sek-1 in this assay makes it difficult to interpret these negative results. To further narrow down the site of action of sek-1 for its NCA suppression phenotypes, we expressed it in subclasses of acetylcholine neurons. Surprisingly, expression of sek-1 in acetylcholine motorneurons but not head acetylcholine neurons was sufficient to restore the loopy posture of the nca-1(gf) mutant  mutants may result from excessive NCA-1 activity in acetylcholine motorneurons. Additionally, expression of sek-1 in either the head acetylcholine neurons or the motorneurons restored the nca-1(gf) small body size phenotype . We make the tentative conclusion that sek-1 acts in acetylcholine neurons to modulate nca-1(gf) body posture and size, but we are not able to conclusively narrow down its site of action further, possibly due to the uncoordinated phenotype of nca-1(gf) and the weaker suppression of nca-1(gf) by sek-1.Given that henotype . By conte mutant . These d) mutant , the oppSEK-1 and the p38 MAPK pathway as a positive modulator of locomotion rate and Gq signaling. The physiological importance of this pathway is clear under conditions of elevated Gq signaling but is less obvious during normal wild-type locomotion, consistent with the observation that sek-1 mutations have a relatively weak effect on synaptic transmission in a wild-type background (SEK-1 p38 MAPK pathway may be more important for modulation of Gq signaling and synaptic strength than for synaptic transmission per se.The p38 MAPK pathway has been best characterized as a pathway activated by a variety of cellular stresses and inflammatory cytokines , but it SEK-1, we identified other p38 pathway components that modulate Gq signaling. Specifically, we found that tir-1, nsy-1, and pmk-1pmk-2 mutants exhibit locomotion defects identical to sek-1 and suppress activated Gq, suggesting that they act in a single p38 pathway to modulate signaling downstream of Gq. These results indicate a redundant function for PMK-1 and PMK-2 in modulating locomotion rate and Gq signaling. PMK-1 and PMK-2 also act redundantly for some other neuronal roles of the p38 pathway, such as the development of the asymmetric AWC neurons and to regulate induction of serotonin biosynthesis in the ADF neurons in response to pathogenic bacteria (PMK-1 acts alone in the intestine to regulate innate immunity, and in interneurons to regulate trafficking of the GLR-1 glutamate receptor (In addition to receptor .SEK-1 p38 MAPK pathway that modulate locomotion? There are several known downstream effectors of p38 MAPK signaling in C. elegans, including the transcription factor ATF-7 (ATF-7 is not required for the p38 MAPK–dependent modulation of Gq signaling. The p38 MAPK pathway may activate molecules other than transcription factors, or may activate multiple downstream effectors.What are the downstream effectors of the SEK-1 p38 pathway modulate the output of Gq signaling? One of the pathways that transduces signals from Gq includes the RhoGEF Trio/UNC-73, the small GTPase Rho, and the cation channel NALCN/NCA-1 (SEK-1 p38 MAPK pathway strongly suppress the loopy waveform of the activated Gq mutant, suggesting that the SEK-1 pathway might modulate Gq signal output through the Rho-Nca branch. Consistent with this, we found that mutations in the SEK-1 p38 MAPK pathway partially suppress an activated NCA-1 mutant. Given the precedence for direct phosphorylation of sodium channels by p38 to regulate channel properties (PMK-1 and PMK-2 phosphorylate NCA-1 to regulate its expression, localization, or activity.How does the sek-1 acts in acetylcholine neurons to modulate the locomotion rate in both wild-type and activated Gq mutants. sek-1 also acts in acetylcholine neurons to modulate the loopy waveform of both activated Gq and activated nca-1 mutants, and the size of activated nca-1 mutants. However, our data obtained from attempting to narrow down the site of action of sek-1 suggest that it may act in both head acetylcholine neurons and acetylcholine motorneurons, and that the waveform is probably controlled by at least partially distinct neurons from those that control locomotion rate. Further work will be required to identify the specific neurons where Gq, NCA-1, and the SEK-1 pathway act to modulate locomotion rate and waveform, and determine whether they all act together in the same cell.Consistent with the observation that Gq acts in acetylcholine neurons to stimulate synaptic transmission (www.g3journal.org/lookup/suppl/doi:10.1534/g3.117.043273/-/DC1.Supplemental material is available online at Click here for additional data file.


Almost 40 years ago, the first pathogenic human retrovirus was discovered and shown to cause a T-cell leukemia in humans  . GVN represents a coalition of virologists on a global scale, and its HTLV-1 task-force includes major HTLV-1 researchers from all-over the world . The three wrote the manuscript together.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Although human T-lymphotropic virus (HTLV) is transmitted via the same routes as human immunodeficiency virus (HIV), its worldwide seroprevalence differs drastically because HTLV is transmitted mainly via infected cells rather than free virus. The sharing of needles and other equipment places people who inject drugs (PWID) at particularly high-risk for such blood-borne diseases.To validate the methodology used to process and analyze the dried blood spots (DBS) utilized in the study, dried serum spots (DSS) with dilutions of sera from known HTLV infected individuals were analyzed by ELISA and Western blot. DBS collected between 2011 and 2015 from 2,077 PWID in eight German cities recruited by respondent-driven sampling were tested for HTLV-specific antibodies.The validation demonstrated that the use of DSS allowed identification of samples with even low titers of HTLV-specific antibodies, although a confirmatory Western blot with an additional venous blood sample would often be required. Despite numerous HIV and HCV positive individuals being identified within the study population, none tested positive for HTLV.While the HIV and HCV prevalences in German PWID are comparable to those in other European countries, the very low prevalence of HTLV reflects the situation in the general population. Human T-lymphotropic virus (HTLV) was the first exogenous human retrovirus discovered , and altThere are three well-studied routes of human-to-human transmission for both HTLV-1 and HTLV-2: sexual contact, breastfeeding and blood products containing HTLV infected lymphocytes , 12. DesThe storage and shipping of samples for subsequent detection of antibodies in epidemiological surveys can be simplified if dried blood spots (DBS) or dried serum spots (DSS) on filter paper are used. Numerous studies investigating various pathogens have shown DBS to be reliable for antibody detection and comparable to venous blood samples. DBS were successfully evaluated soon after the start of the HIV/AIDS pandemic and the method has since proven to be very useful for HIV-specific antibody screening , 26. DBSThe first aim of the study presented here was to validate, in our hands, the use of DBS for HTLV testing using a small set of sera from known HTLV-positive individuals. The second was to determine the prevalence of HTLV in a German cohort of people who inject drugs (and therefore at high risk for blood-borne infections) using 2,077 DBS from PWID in eight German cities that had been collected using respondent-driven sampling as part of a multicenter sero-behavioural survey for HIV and hepatitis B and C .As part of a recent study using respondent-driven sampling, 2,077 DBS from PWID in eight German cities had been collected, with the number of DBS in the respective cities ranging from 130 (Leipzig) to 337 (Berlin). The capillary blood samples, collected by a trained person, were spotted with ~30μl per spot on filter cards . The filter cards were dried for at least three hours or overnight at room temperature, then placed in plastic bags and sent to our laboratory. Antibodies were eluted in PBS containing 0.05% Tween-20 and 3% FCS by incubation overnight at 4°C, resulting in a 1:15 dilution with respect to the original blood volume. Eluates were stored at -20°C until testing for the presence of HTLV-specific antibodies according to a modified algorithm proposed by the HTLV European Research Network . First, To validate HTLV testing from DBS, 14 randomly selected sera from individuals previously tested positive for HTLV antibodies were used. All subjects were asymptomatic carriers (AC) to reflect the expected situation within a group of non-hospitalized PWID. Characterized HTLV-1 and HTLV-2 sera for the Dried Blood Spot validation were additionally provided by the Communicable Diseases Research Tissue Bank of Imperial College Healthcare NHS Trust, London, UK. The proviral loads of these HTLV infected subjects ranged between 0.01% and 35%. Thirty μl of the undiluted sera as well as of three serial 10-fold dilutions in negative human sera were spotted onto two replicate filter papers. These DSS were then processed in the same way as the DBS, eluting the antibodies from the filters to give an additional 1:15 dilution before testing in ELISA. A confirmation Western blot of the highest dilution of each sample still reactive in ELISA was carried out as described above.Details of the study protocol have been published elsewhere , 41. BriThe Federal Commissioner for Data Protection and Freedom of Information approved the study protocol on 29/11/2012 (III-401/008#0035). Ethics approval was received on 19/11/2012 from the ethics committee at the medical university of Charité, Berlin (EA4/036/11) . All aduTo validate the use of DBS for HTLV screening, we tested sera from six known HTLV-1 infected individuals and eight HTLV-2 infected individuals with the diagnostic ELISA routinely used in our laboratory. In addition to the negative control sera provided with the assay, one serum from a healthy donor was included as control. To account for the probable different magnitudes of reactivity in individuals progressing to disease and asymptomatic carriers, serial dilutions up to 1:1000 were also tested. All HTLV-1 and HTLV-2 positive sera tested positive at the 1:100 dilution. Three HTLV-1 positive sera  and three HTLV-2 positive sera  were still positive at the 1:1000 dilution .The same sera (and dilutions thereof) were then used to create DSS to simulate the conditions and processing used for the study samples. As expected, the inherent 1:15 dilution resulting from elution of the sera from the filters reduced detectability . All DSSAll sera were then subjected to diagnostic Western blot to test this assay's suitability for confirmation . AccordiAfter spotting on filter paper and eluting, only two HTLV-1 positive sera  remained unambiguously positive , Table 1Taken together, all DSS eluates of the validation panel were found to be positive in ELISA, but testing eluates of six DSS from HTLV-1 infected asymptomatic carriers resulted in only two cases that could be confirmed by Western blot. In contrast, testing eight DSS from HTLV-2 infected asymptomatic carriers resulted in five confirmed HTLV-2 infections. A summary of the validation, with details of the ELISA and Western blot results, is given in Initial screening of the DBS eluates was performed by ELISA as described above. Of the 2,077 eluates from DBS tested, six were initially found to be reactive . Three wHIV and HTLV are the only known exogenous human retroviruses, and of the four types of HTLV, only HTLV-1 and HTLV-2 are of epidemiological relevance, both being distributed worldwide, albeit usually in different population groups. Both viruses can spread easily within people who inject drugs (PWID), with especially HTLV-2 being found at high rates in PWID within the United States and Europe .Here we report the results of screening for anti-HTLV-1/-2 antibodies in 2,077 samples from German PWID collected during 2011–2014 as part of a multicenter sero-behavioural survey using respondent-driven sampling . The freOur study was performed with DBS material prepared from capillary blood. The use of DBS samples for HTLV seroprevalence studies is not without precedence. In one Spanish study, DBS from 484 PWID were analyzed and 27 cases of HTLV-2 infection were detected , using bThe prevalence of HTLV in PWID is quite different across European countries. In the Irish city of Dublin, a prevalence of 14.6% HTLV-2 seropositivity in a sample of 103 PWID infected with HIV-1 was reported, but no HTLV-1. In this study, sera were tested by ELISA and infections were confirmed by Western blot and/or PCR  using thIt is currently assumed that at least two different introductions of HTLV-2 from the US into PWID populations in Europe took place, based on the observation that the HTLV-2a subtype is found predominantly in Northern and Eastern Europe whereas the HTLV-2b subtype is mainly found in Western and Southern Europe , 42. TheIn conclusion, this analysis of 2,077 DBS samples from German PWID recruited by respondent-driven sampling in eight German cities between 2011 and 2014 found no evidence for cases of HTLV infection. These results are in line with previous serological surveys carried out in Germany but are somewhat different from the situation in some other European countries where low HTLV seroprevalences have been reported, particularly in PWIDs.


Neurodevelopmental biology, coupled with the application of advanced histological, imaging, molecular, cellular, biochemical, and genetic approaches, has provided new insights into these intricate genetic, cellular, and molecular events. During telencephalic development, specific neural progenitor cells (NPCs) proliferate, differentiate into numerous cell types, migrate to their apposite positions, and form an integrated circuitry. Critical disturbance to this dynamic process via genetic and environmental risk can cause neurological disorders and disability. The phosphatidylinositol-3-OH kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) signaling cascade contributes to mediate various cellular processes, including cell proliferation and growth, and nutrient uptake. In light of its critical function, dysregulation of this node has been regarded as a root cause of several neurodevelopmental diseases, such as megalencephaly (“big brain”), microcephaly , autism spectrum disorders, intellectual disability, schizophrenia, and epilepsy. In this review, particular emphasis will be given to the PI3K-Akt-mTOR signaling pathway and their paramount importance in neurodevelopment of the cerebral neocortex, because of its critical roles in complex cognition, emotional regulation, language, and behaviors. The mammalian neocortex is a complicated, tightly orchestrated, and six-layered construction that contains various neuronal subtypes and glias. The diverse neurons and glias of the central nervous system (CNS) are produced by a small, heterogeneous population of neural progenitor cells (NPCs) that undergo transcriptional changes to sequentially specific distinct cell fates, guided by temporal cell extrinsic and intrinsic cues. Astrocytes and oligodendrocytes (OLs), the major glial sub-lineages in the CNS, play key roles in telencephalic development and homeostatic maintenance with increasing brain complexity, controlling various aspects of neurodevelopment and diseases  is a member of the serine/threonine protein kinase AGC family and has three isoforms Akt1/Pkbα, Akt2/Pkbβ, and Akt3/Pkbγ. Akt, as a central node, is a positive regulator of several signaling pathways including cell proliferation, growth, survival, and metabolism across many species  in human genome have been reported in a variety of developmental aberrations of the brain, including agenesis of the corpus callosum (ACC) and microcephaly. Several clinical studies demonstrate that haploinsufficiency of AKT3 in this region causes microcephaly and ACC  exhibits widespread p-Akt localization in the developing cortex, with remarkable enrichment in neural progenitor cells in the ventricular zone, suggesting Akt’s primary role in brain development  and HME. De novo germline 1q43q44 (encompassing the AKT3 gene) trisomy has been reported in megalencephaly  tetrasomy and a gain of function mutation in AKT3 , have been reported in HME   leads to a dynamic trajectory of brain overgrowth and altered scaling of neural cells, with an elevation of beta-catenin signaling (Chen et al. Pten+/− mice, which provide a new perspective that Pten and beta-catenin signaling act in conjunction to control neural cell number and normal brain growth trajectory (Chen et al. Loss of Together, the emerging consensus is that elevation of the PI3K-AKT-mTOR signaling pathway leads to enhanced proliferation of progenitors, neuronal hypertrophy, and excessive dendritic branching, whereas suppression exhibits the opposite consequences (Costa-Mattioli & Monteggia Germline or widespread somatic mutations of PI3K-AKT-mTOR signaling networks may elicit overt brain architecture defects, whereas subtle, somatic, or cell type-specific mutations may lead to localized and restricted abnormalities. The severity and medical characteristics of neurodevelopmental diseases may be determined partially by the stage at which the mosaicism occurs relative to the distinct period of neurogenesis and gliogenesis, and which types of neural cells are affected. Therefore, more detailed researches are needed to decipher the cell type-specific effects of mosaic mutation and to determine which type of pathologies is attributed to specialized neural malfunction (Cai et al.