Mitochondrial dysfunction has been implicated in the aetiology of many complex diseases, as well as the ageing process. Much of the research on mitochondrial dysfunction has focused on how mitochondrial damage may potentiate pathological phenotypes. The purpose of this review is to draw attention to the less well-studied mechanisms by which the cell adapts to mitochondrial perturbations. This involves communication of stress to the cell and successful induction of quality control responses, which include mitophagy, unfolded protein response, upregulation of antioxidant and DNA repair enzymes, morphological changes, and if all else fails apoptosis. The mitochondrion is an inherently stressful environment and we speculate that dysregulation of stress signaling or an inability to switch on these adaptations during times of mitochondrial stress may underpin mitochondrial dysfunction and hence amount to pathological states over time. Approximately 1.45 billion years ago, gram negative bacteria were engulfed by primitive eukaryotic cells giving rise to the mitochondrion . Howeverβ-cell failure [Being the site of programmed cell death and energy metabolism, the cells survival is ultimately dependent on precise coordination between mitochondria and the rest of the cell. Consequently there are a number of mitochondrial stress signals that are communicated to the rest of the cell that stimulate cellular adaptions, which support this organelle-host symbiotic relationship. This is an emerging area in mitochondrial biology that has not been well studied to date. We propose that inability for the cell to perceive and respond to mitochondrial stress may be a platform for mitochondrial dysfunction . Mitocho failure , and ins failure , as well failure . As suchMuch of the previous literature linking mitochondrial dysfunction with disease has focused on the linear view that mitochondrial stress causes damage, which subsequently causes disease. However, mitochondria respond to various stresses by inducing a complex array of cellular responses and adaptations to reduce the impact of subsequent stressors. The purpose of this review is to summarise the literature surrounding cellular adaptations and quality control processes to cope with mitochondrial stress, which may be potential pharmacological targets.2+ fluxes, permeability transition pore opening, and perhaps secretion of proteins/peptides  is the process whereby electrons gained during substrate oxidation are transported along the electron transport chain (ETC), coupled with the pumping of protons across the innermitochondrial membrane and the generation of a proton gradient, which is used as the driving force for ATP production at complex V (ATP synthase) in conjunction with oxygen consumption at complex IV. Defective oxphos reduces bioenergetic capacity and is a sign of cellular stress. Perturbations in oxphos can lead to reduced ATP production, changes in redox status, innermitochondrial membrane depolarization, and excessive reactive oxygen species (ROS) generation.ATP Levels. Oxphos is a multistep process that effectively results in ATP production from fuel substrates. ATP is a major output of oxphos function  protein expression in myocardial tissue [In addition to regulating mitochondrial substrate oxidation, AMPK has other effects on mitochondrial parameters. Metformin treatment activates AMPK activity in conjunction with inducing PGC1l tissue . A gain l tissue  which iml tissue , highligl tissue . These lNAD+ Levels. In addition to ATP production, mitochondrial oxphos involves electrons being stripped out of fuel substrates and carried on NAD+, making NAD+ a signal of mitochondrial oxphos function  polymerase 1 (PARP1) which is important for maintenance and repair of genomic DNA [NADomic DNA  and alsoomic DNA . These d+/NADH ratio are the sirtuin (SIRT) family of NAD+-dependent deacetylases. Through alterations in posttranslational modifications  on lysine residues, SIRT proteins influence a number of metabolic processes and cellular functions.Another important group of enzymes regulated by changes in the NAD+ and via deacetylation of the master regulator of mitochondrial biogenesis, PGC1α, and has been reported to increase mitochondrial bioenergetic capacity [SIRT1 is known to regulate mitochondrial function in an anterograde, or nucleus to mitochondria fashion, signaling in response to redox status . SIRT1 icapacity , 23. In capacity . SIRT3 icapacity , 26. Altcapacity , as wellcapacity , a transIn addition to changes in ATP and nutrient intermediates, alterations in oxphos can induce a stress response via changes in membrane potential. More specifically, the innermitochondrial membrane becomes polarized during oxphos and loss of the membrane potential or depolarization can act as a signal , promotiMitophagy. Mitochondrial depolarization can induce lysozomal degradation of mitochondria in a process called mitophagy. A major pathway mediating mitophagy has been identified which involves the PD associated proteins PTEN-induced putative kinase 1 (PINK1) and parkin. Parkin is an E3 ubiquitin ligase which translocates to mitochondria upon membrane depolarization in a PINK1-dependent process [ process . PINK1 a process . Mitophaβ-cell dysfunction [in vivo models in this area means that it is unclear if these cellular effects are physiologically relevant or whether it is cause or consequence of disease. Another feature of these studies that makes it difficult to discern the relevance of reduced autophagic clearance of mitochondria in the aetiology of chronic disease is that defective mitophagy is usually defined as reduced parkin translocation in response to the uncoupling agents CCCP/FCCP under nonphysiological circumstances [in vivo in mammalian disease models would greatly facilitate insight into the role of defective mitophagy in disease development.Defective mitophagy coincides with cell models of neurological diseases including AD  and PD , as wellfunction , cardiomfunction , and hepfunction . These fmstances , 37. Litmstances , suggestIn all, mitophagy is a quality control process that enables adaptation to oxphos stress and may be protective against complex disease development. However, it is not currently understood how this process is switched on during physiologically normal mitochondrial membrane depolarization.Permeability Transition Changes. Membrane depolarization can also initiate cell death by opening of the MPTP. This pore is a protein complex of VDAC, cyclophilin D (CypD), and adenine nucleotide translocase that can open and close and allows small substances to cross the mitochondrial membrane in response to membrane depolarization [rization . Openingrization . Increasrization  and convrization .+, a signal of low energy status by SIRT3 deacetylation of CypD [Mitochondrial perturbations can lead to MPTP resistance as a cellular adaption. MPTP opening can be inhibited by NAD of CypD . Inhibit of CypD .The phospholipid composition of the mitochondrial membrane also affects PMTP opening and now there is emerging evidence indicating that fatty acids and ceramides play a role in regulating the activity of MPTP which links lipids with mitochondrial dysfunction. Omega 3 fatty acid incorporation into the mitochondrial membrane delays MPTP opening . CeramidMembrane depolarization may result in either mitophagy or apoptosis depending on the extent of damage, which improves cellular or whole body functioning by cleansing damaged organelles or cells, respectively.Mitochondria are a major site of ROS production , as byprOxidative damage has been linked with a number of complex diseases, including neurological disorders , 51, carMuch of the literature surrounding ROS induced damage has incubated cultured cells in supraphysiological concentrations of exogenous hydrogen peroxide or has used high doses of pharmacological inhibitors of complex I and III for short periods of time –62. ThesC. elegans in response to low-grade arsenic exposure [C. elegans and improve functioning in mammalian cell culture, in vivo mammalian studies are lacking.Extending on the idea that oxidative stress is involved in preconditioning phenomena, there is evidence that mild mitochondrial ROS stressors may actually improve lifespan, through a process termed “mitochondrial hormesis” (mitohormesis). This theory describes the link between low-grade mitochondrial stressors and enhanced cellular function . Mitohorexposure  and low-exposure . Low-graexposure , and lowexposure , implicacis-acting enhancer sequence that controls expression of a variety of antioxidant enzymes. A ROS-mediated signaling cascade that results in activation of this enhancer has been identified, which provides evidence for cellular adaptation in response to ROS signals, where nuclear respiratory factor 2 (NRF2) transcription factor activates the ARE enhancer to switch on an antioxidant gene program [There are other examples where ROS induces cellular adaptations to clear subsequent ROS insults or improve mitochondrial functioning, which highlights an important negative feedback role for these molecules. One major mechanism that switches on antioxidant defences in the cell is the activation of antioxidant response element (ARE). ARE is a  program . Oxidati program . In addi program , which i program  catalase program  and PrxI program  which haα (PGC1α) and PGC1β promoter activity and expression, and in addition to stimulating mitochondrial biogenesis, these transcriptional coactivators also stimulate the expression of multiple antioxidant defences [In vivo overexpression of pgc1β in rat skeletal muscle resulted in increased expression of antioxidant enzymes and an overall reduction in oxidative damage [ROS not only induce antioxidant responses in a negative feedback type mechanism, but also can stimulate cellular adaptations that improve mitochondrial capacity in general. ROS increases mitochondrial biogenesis and mitochondrial DNA content in fibroblasts , 78. Hyddefences , 80. In e damage , highlige damage , which ie damage  and hencin vitro. Increased understanding of the mechanisms that cells use to respond to mitochondrial oxidative stress in vivo is less thoroughly investigated to date and may be relevant in understanding the pathogenesis of complex disease, particularly as a reduced capacity to mount and antioxidant response is linked with a number of disease states [Overall, there has been a large focus in the literature on the effects of exogenous ROS e states , 58, 59,e states .Mitochondria are dynamic organelles that constantly undergo morphological changes in the opposing processes of fusion and fission. In fusion, multiple mitochondria form one elongated mitochondrion, whereas, in fission, a mitochondrion generates small, fragmented mitochondria. Mitochondrial fission and division occurs when Drp-1 is recruited from the cytosol to divide the mitochondrion . Fusion Changes in mitochondrial morphology are known to occur in response to mitochondrial stressors and may be a cellular adaptation to regulate cell survival. The pro-survival protein Pim-1 and apoptotic protein PUMA have opposing roles in Drp-1 localisation to the mitochondria , suggestDysregulation of mitochondrial quality control processes has been implicated in pathological states and ageing. Down-regulation of Drp1 and mitochondrial elongation can be caused by a pharmacological DNA damage inducer  and cellOverall the evidence in the literature indicates that the inherent plasticity in mitochondrial morphology provides the capacity for changes in this organelle in response to the prevailing environmental conditions, which provides a platform for rapidly changing bioenergetics and apoptotic state of the mitochondrion in response to specific stress signals.Mitochondrial DNA (mtDNA) is double stranded and organised into a circular structure which encodes 37 genes, of which 13 are subunits of the ETC, 2 are rRNAs, and 22 are tRNAs . mtDNA imtDNA repair enzymes play an important role in responding to different stresses with increased activity of these enzymes that are protecting against oxidative stress induced apoptosis , palmitaInterestingly, many of the cellular adaptations to oxphos stress also lead to increased expression of mtDNA repair enzyme 8-oxoguanine glycosylase (OGG1), the major mitochondrial DNA repair enzyme. For example, the NRF2, part of the ROS induced antioxidant adaptation, can bind to the OGG1 promoter region to induce OGG1 expression , reinforThus, while mtDNA does not have the same level of protection and repair as nDNA, there are a number of feedback mechanisms in place to respond to mitochondrial genotoxic stress.β-amyloid specifically in the mitochondrial fraction has been associated with severe mitochondrial dysfunction and cell death [It is well known that aberrant protein folding and subsequent formation of toxic oligomeric intermediates, such as amyloids, are implicated in chronic diseases of ageing. Given that mitochondrial dysfunction is also widely implicated in complex diseases of ageing and mitochondria are vulnerable to proteotoxic stress, it is possible that mitochondrial specific protein misfolding is a component of chronic disease. Consistent with this idea, accumulation of ll death .The cell has a quality control pathway to adapt to mitochondrial proteotoxic stress called the mitochondrial unfolded protein response (mtUPR) . mtUPR wC. elegans, by Haynes et al. which facilitated some further characterisation of steps involved in this pathway [The topic was approached in a simpler model organism,  pathway . mtUPR a pathway , suggest pathway , a bZip  pathway . Nuclear pathway .Although HAF-1 is homologous to mammalian ATP binding cassette proteins, mitochondrially located ATP binding cassette proteins have roles in heme transport, whereas ATP binding cassette proteins with peptide efflux roles are located elsewhere in the cell , so thus+ concentrations have been implicated in longevity, notably through the activation of SIRT proteins or related homologues. Pharmacological increases in NAD+, increased hsp-6 reporter activity, and lifespan in C. elegans independently of the C. elegans SIRT1 homologue [+ activity. Aged wild type mice display higher Hsp60 protein expression in midbrain than young mice, but this age-dependent effect is diminished in DJ-1 knockout mice [α-synuclein toxicity [mtUPR is clearly a control process providing cellular adaptation to mitochondrial proteotoxic stress and there is some evidence suggesting a role for the activation of this pathway in chronic disease and ageing. Increased NADomologue  reveal mout mice  providintoxicity , which rtoxicity . Interestoxicity . These fC. elegans induces mtUPR induction in the gut, which provides evidence for cellular adaptations to mitochondrial stress in a noncell autonomous manner [Transcriptomic analysis of ATFS-1-dependent genes demonstrates signaling genes and transcriptional regulators in addition to mitochondrial quality control genes , suggests manner . Indeed s manner  and belis manner . It has s manner . An exams manner . Overalls manner ; howeverMitochondria should not simply be thought of as isolated organelles that generate energy. They have a complex relationship with the rest of the cell requiring back and forth coordination of two genomes and constant communication about the bioenergetic status of the cell. Dysregulation of mitochondrial communication processes and reduced cellular capacity to activate mitochondrial quality control processes in times of stress may therefore be important part of the molecular basis for the mitochondrial component of complex disease.


Moreover, fluoxetine inhibits the differentiation of hypothalamic neuroprogenitor cells, as demonstrated by decreased number of mature neurons (Neu-N+ cells) and increased number of undifferentiated cells (SOX-2+ cells). Additionally, fluoxetine-induced proliferation and maintenance of hypothalamic neuroprogenitor cells leads to changes in the mRNA levels of appetite regulator neuropeptides, including Neuropeptide Y (NPY) and Cocaine-and-Amphetamine-Regulated-Transcript (CART). This study provides the first evidence that SSRIs affect the development of hypothalamic neuroprogenitor cells in vitro with consequent alterations on appetite neuropeptides.A significant number of children undergo maternal exposure to antidepressants and they often present low birth weight. Therefore, it is important to understand how selective serotonin reuptake inhibitors (SSRIs) affect the development of the hypothalamus, the key center for metabolism regulation. In this study we investigated the proliferative actions of fluoxetine in fetal hypothalamic neuroprogenitor cells and demonstrate that fluoxetine induces the proliferation of these cells, as shown by increased neurospheres size and number of proliferative cells (Ki-67 Food intake and body weight are centrally regulated by the hypothalamus, where arcuate nucleus (ARC) neurons sense and integrate peripheral signals of nutrition to downstream circuits Adult hypothalamic neurogenesis occurs at low rates in rodents but it is essential for body weight and feeding regulation Proliferation of hypothalamic neuroprogenitor cells during the perinatal period is influenced by maternal nutrition and hormones availability in mice Selective serotonin reuptake inhibitors (SSRIs) are antidepressant drugs also known for their neurogenic effect in perinatal hippocampal and cerebellar neuroprogenitor cells in vitro model previously described by our group Therefore, in this study we investigated whether the SSRI fluoxetine alters the proliferation and differentiation of rat embryonic hypothalamic neuroprogenitor cells. Moreover, using an All experimental procedures were performed in accordance with the European Union Directive 86/609/EEC for the care and use of laboratory animals. In addition, animals were housed in a licensed animal facility  and the CNC animal experimentation board approved the utilization of animals for this project. Moreover, people working with animals have received appropriate education (FELASA course) as required by the Portuguese authorities.Hypothalamic neuroprogenitor cells were isolated and cultured as floating neurospheres as previously described After 6–7 days in culture, neurospheres were dissociated and plated in Poly-D-Lysine coated culture plates as previously described Fluoxetine, Cytosine Arabinoside (AraC) and tyrosine kinases (Trk) receptor inhibitor K252a were purchased from Sigma-Aldrich.A recent rodent study showed that about 80% of plasma fluoxetine is transferred to the pup after maternal injection, with high levels of fluoxetine being detected in the pup brain Hypothalamic neurospheres were cultured in the presence of fluoxetine (1 µM); treatment with fluoxetine started 3 days after isolation of hypothalamic neuroprogenitor cells. Control hypothalamic neurospheres were cultured in the absence of fluoxetine.To investigate the proliferative effects of fluoxetine and contribution of neurotrophins to the fluoxetine-induced proliferation, control and fluoxetine P3 hypothalamic neurospheres were incubated with the proliferation inhibitor AraC (10 µM) or the Trk receptor inhibitor K252a (200 nM) during the last 24 hours before RNA collection or plating.Hypothalamic neuroprogenitor cultures were allowed to differentiate in the presence of fluoxetine (1 µM). Treatment with fluoxetine started 2 days after plating of hypothalamic neurospheres. Incubation with Trk receptor inhibitor K252a (200 nM) occurred during the last 24 hours before RNA collection or fixation. Control hypothalamic neurospheres differentiated in the absence of fluoxetine.Twenty-four hours after incubation with the inhibitors, P3 hypothalamic neurospheres were plated in Poly-D-Lysine coated 12-well culture plates and kept in culture for 1 day with fresh medium (DMEM-F12/Glutamax supplemented with growth factors) before fixation.P3 hypothalamic neurospheres and differentiated hypothalamic neuroprogenitor cultures were fixed with 4% paraformaldehyde (Sigma) and permeabilized with 1% Triton X-100 (Sigma) followed by one hour blocking with 3% BSA (Sigma) and incubation with the primary antibody overnight at 4°C. Thereafter, the incubation with the secondary antibody was performed for one hour at room temperature. Nuclei were stained with 4′6-diamidino-2-phenylindoline, DAPI .The primary antibodies used were: mouse anti-Ki-67 , mouse anti-Neu-N  and mouse anti-SOX-2 . The secondary antibodies used were: goat anti-mouse Alexa Fluor 594 and goat anti-mouse Alexa Fluor 488 .To investigate the effect of fluoxetine in the proliferation and differentiation of hypothalamic neuroprogenitor cells, the percentage of Ki-67, Neu-N and SOX-2 positive cells was calculated in P3 hypothalamic neurospheres and differentiated hypothalamic neuroprogenitor cultures. Fluorescence images were recorded in the monolayer formed at the neurospheres periphery (Ki-67) or in the center of the neurospheres (Neu-N and SOX-2) using a confocal microscope . The percentage of Ki-67, Neu-N and SOX-2 positive cells was calculated in ten independent microscopic fields and normalized to total number of nuclei with DAPI staining (approximately 80–120 nuclei per microscopic field). Data are shown as percentage of control. The procedure was performed for four independent culture preparations.The size of control and fluoxetine-treated neurospheres was evaluated immediately before the next passage, using the Axiovision software (Zeiss) to determine their diameter. The neurospheres diameters oscillated between 50 and 250 µm and this interval was divided into four subintervals , to obtain the size distribution of hypothalamic neurospheres. Size distribution was determined as percentage of total number of neurospheres evaluated for each treatment. The procedure was performed for four independent culture preparations.Hypothalamic neurospheres were collected by centrifugation and immediately frozen in dry ice as described before 1A and serotonin pre-synaptic transporter (SERT) were previously described by others Each PCR reaction contained 50 ng of template cDNA, 0.2 µl of Phusion Polymerase (Finnzymes), 200 µM dNTPs (ThermoScientific) and 0.5 µM of reverse and forward primers, to a final volume of 20 µl. Primers sequences for tryptophan hidroxylase type 2 (TPH2), serotonin receptor type 5-HTQuantitative PCR was performed in an iQ5 thermocycler (Bio-Rad) using 96-well microtitre plates and the QuantiTect SYBR Green PCR Master Mix (QIAGEN) as described before Relative mRNA quantification was performed using the ΔCt method for genes with the same amplification efficiency. The mRNA quantification of different neuropeptides was performed in the same culture preparations. For the experiments using inhibitors the mRNA quantification is shown as percentage of control. The procedure was performed for five independent culture preparations.All the experiments were replicated in four to five independent culture preparations. The values in the figures are expressed as mean ± SEM. For the experiments involving fluoxetine and inhibitors treatment, data were analyzed by one-way ANOVA with post hoc test to determine significant differences between groups. For the experiments involving neuropeptides quantification during passages and neurospheres percentage for size intervals, data were analyzed by two-way ANOVA with post hoc test to determine significant differences between control and fluoxetine groups.1A  The neurobiological actions of neurotrophins are mediated by a family a tyrosine kinase receptors: TrkA, TrkB and TrkC receptors. Therefore, to investigate the involvement of neurotrophins in the proliferation of hypothalamic neuroprogenitor cells induced by fluoxetine we used K252a, a Trk receptors inhibitor. K252a is an alkaloid extensively used as Trk receptors antagonist, In our study, we quantified the number of Ki-67 positive cells after incubation with cell proliferation inhibitor AraC or Trk receptors inhibitor K252a, during the last 24 hours before cell fixation. The elevated number of proliferative cells was reversed to control levels by incubation with AraC or K252a in fluoxetine treated cultures . Both inAdditionally, incubation of P3 neurospheres with AraC did not change significantly the expression of BDNF mRNA in both conditions . HoweverOverall, these results show that fluoxetine promotes the proliferation of hypothalamic neuroprogenitor cells, via a mechanism involving neurotrophins.Proliferation changes during hypothalamic neurodevelopment can lead to an impaired balance between neurons expressing orexigenic and anorexigenic neuropeptides Concerning the orexigenic neuropeptides, fluoxetine significantly increased the expression of NPY in hypothalamic neurospheres, with a 1.5-fold increase in mRNA levels at P2, and a 2-fold increase at P3 . To inveAdditionally, fluoxetine led to an earlier rise on AgRP levels, with a 5-fold increase in the mRNA levels occurring at P1 . In contAll together, these data show that fluoxetine modifies the expression levels of neuropeptides important for the regulation of feeding and that this effect is dependent of the proliferative effect of fluoxetine on hypothalamic neuroprogenitor cells.+ cells) and progenitor cells (SOX-2+ cells) + cells) was robustly increased in the cultures incubated with fluoxetine during differentiation , pre-synaptic transporter and 5-HTin vitro model, particularly the possibility that the mechanisms observed for the cell culture preparations will not be recapitulated in the living organism. Nevertheless, the neurospheres culture model has been extensively used by researchers for studying proliferation of well-known neurogenic regions like the lateral ventricles and hippocampus In this study we have investigated the neurogenic actions of fluoxetine in hypothalamic neuroprogenitor cells cultured as neurospheres. This model presents the limitations of an Fluoxetine promotes the proliferation and survival of new neurons in the adult hippocampus Furthermore, the involvement of BDNF is suggested by the data showing an upregulation of BDNF mRNA levels in the neurospheres treated with fluoxetine at P3, the passage with maximal proliferative effect (as shown with neurospheres size). Noteworthy, previous reports showing that the response of neuroprogenitor cells to fluoxetine is mediated by an upregulation of BDNF levels in the hippocampus Moreover, BDNF is an important molecule to the cellular physiology of the hypothalamus since it is involved in the neuronal development and maturation of this brain region In opposition, the inhibition of neuronal differentiation produced by fluoxetine does not seem to be mediated by neurotrophins: the mRNA level of BDNF is similar in hypothalamic neuroprogenitor cells differentiated in the presence or absence of fluoxetine and Trk receptors inhibitor does not reverse the high number of SOX-2 positive cells observed in fluoxetine-treated cell cultures. However, the incubation with K252a partially reversed the low number of mature neurons quantified in cultures differentiated in the presence of this antidepressant. Other mediators of fluoxetine neurogenic activity, such as the recently described glial cell-derived neurotrophic factor (GDNF), cyclin-dependent kinase inhibitor p21 and Wnt3a In this study, we showed that fluoxetine alters the expression levels of neuropeptides important for the regulation of appetite, including upregulation of orexigenic neuropeptides NPY and AgRP, during the proliferation of hypothalamic neuroprogenitor cells, and down regulation of abundant neuropeptides NPY and CART, during the differentiation of these cells. Moreover, our results suggest that the alterations of neuropeptides levels promoted by fluoxetine are mediated by its effects in the proliferation and maintenance of neuroprogenitor cells.The expression of NPY (and other neuropeptides) in embryonic mitotic cells, and in particular, in hypothalamic neuroprogenitor/neuroprecursor cells was previously demonstrated in neurospheres cultures Therefore, changes of proliferation and neuropeptides content in hypothalamic neuroprogenitor cells induced by fluoxetine may influence the neuronal differentiation process. This hypothesis is in accordance to a previous study showing that the neurogenesis of orexigenic precursors can be altered by stimuli reaching the hypothalamus and lead to increased density of orexigenic neurons The involvement of neurotrophins in the effects of fluoxetine further supports the possibility that fluoxetine modifies the expression of neuropeptides by promoting the propagation of hypothalamic neuroprogenitor cells. First, the most significant increase of NPY mRNA occurs simultaneously with the BDNF upregulation and the most robust increase in cellular proliferation. Second, the levels of NPY mRNA return to control after incubation with Trk receptors inhibitor, as it occurred with proliferation inhibitor. Similarly to the data reported here, BDNF upregulates the levels of NPY in cortical cell cultures and hippocampal slices Surprisingly, previous studies reporting the effects of fluoxetine and BDNF in the adult hypothalamus show opposite evidences from the ones reported in this work. For example, administration of fluoxetine and other serotonergic drugs decreases the content and release of NPY These evidences lead us to hypothesize that the SSRI fluoxetine has divergent actions in mature and neuroprogenitor hypothalamic cells, with the neurogenic activity being more evident on this last type of cells . Our hypothesis is further supported by previous reports showing that the cognitive outcomes of serotonin transporter blockade during development are sometimes dramatically different from the effects during adulthood in vitro model described here. This hypothesis reinforces the need of understanding the consequences of exposing embryonic and adult hypothalamic neuroprogenitor cells to antidepressants. In this context, the in vitro model used for the present study can represent a valuable model to screen possible neurogenic effects of other drugs in hypothalamic neuroprogenitor cells.Moreover, we can speculate that due to the reduced number of neuroprogenitor cells in the adult hypothalamus The importance of adult hypothalamic neurogenesis and plasticity to the metabolic response to dietary challenges was recently demonstrated in vitro study shows that SSRIs antidepressants have the potential to change the proliferation/differentiation of neuroprogenitor cells from the embryonic hypothalamus. Although future in vivo studies are needed, based on the presented results, we can speculate that antidepressant drugs may be yet another contributor to impaired neurodevelopment of this key metabolism center by increasing the pool of neuroprogenitor cells and inhibiting the differentiation of these cells.Our Figure S1Trk receptors inhibitor K252a upregulates the levels of BDNF in P3 hypothalamic neurospheres. Incubation with K252a for 24 hours results in a compensatory upregulation of the mRNA levels of neurotrophic factor BDNF. Incubation with proliferation inhibitor AraC for 24 hours does not modify the mRNA of BDNF. One-Way ANOVA; ns, p>0.05; *, p<0.05 compared to control. P, passage.(TIFF)Click here for additional data file.


AbstractBrachymelecta Linsley and Sinomelecta Baker (Apinae: Melectini) are redescribed, each represented by a single species which has not been reencountered since capture of the type series ca. 1878 and 1900, respectively. Both genera are the only melectines to possess two submarginal cells in the forewing but are otherwise wholly dissimilar. Brachymelecta mucida (Cresson), a species known only from the male holotype collected in “Nevada”, is newly described and figured, including the first account of the hidden sterna and genitalia. Sinomelecta oreina Baker is similarly described and figured based on the holotype male and paratype female, apparently collected from the eastern Tibetan Plateau. Both genera are valid and from the available data do not appear to represent merely autapomorphic forms of Melecta Latreille. Indeed, the terminalia of Sinomelecta oreina are in some respects more similar to those of species of Thyreus Panzer.The enigmatic, cleptoparasitic bee genera  Melectini. Each is known from only one or two specimens collected over a century ago and preserved without adequate locality data. One or both may now be extinct. PageBreakThe two genera do not appear to be close relatives, although they share a striking common character: both have only two submarginal cells in the forewing although all other Melectini have three. The genera involved are Brachymelecta Linsley and Sinomelecta Baker; each is discussed and described below. Since each genus contains only a single species, the descriptions combine specific characters with probable generic characters, i.e., characters that differentiate other genera of Melectini. A very unusual feature of Sinomelecta is that the male, like the female, has 12 antennomeres. In nearly all bees males have 13 antennomeres. There is no reason to believe that Brachymelecta shares this character but since the antennae of the sole specimen of that genus are broken, new material will be necessary if the antennomeres of that taxon are to be counted.The objective of this paper is to fully describe two genera of the anthophorine tribe Brachymelecta is preserved in the Academy of Natural Sciences of Philadelphia. Described in 1879 as “Melecta? mucida”, its source was given as “Nevada (Morrison)” ], mostly from the western United States but some from the southeast (Georgia). In spite of extensive collecting in the western United States by persons interested in bees, no other specimens have been obtained. Brachymelecta might be extinct, or may exist in or near Nevada, less likely elsewhere. If not extinct, it is presumably exceedingly rare. Like other Melectini, it would be a cleptoparasite in nests of other bees. The only equally small North American melectine is Zacosmia Ashmead which also occurs in the western United States and is a cleptoparasite of Anthophora (Heliophila).Given the rarity of this material, the few references to them in the literature, and thereby their general unfamiliarity to many, it is worthwhile to summarize the historical details for each genus. The only known specimen of rrison)”  althoughrrison)” . The labSinomelecta, one of each sex, were in the collection of Donald B. Baker (1922–2004) for many years and are today preserved in the Division of Entomology of the University of Kansas Natural History Museum. They were described by Baker as Sinomelecta oreina in 1997 , while the holotype male and paratype female of Sinomelecta oreina are in the Division of Entomology, University of Kansas Natural History Museum, Lawrence (SEMC). Through the kindness of Jason Weitraub (ANSP) we were permitted to dissect the metasomal apex of the Brachymelecta mucida holotype and to clean the genitalia for imaging. Despite being over 134 years old , the genital capsule and hidden sterna were easily removed from between TVII and SVI and after a gentle bath in a weak potassium hydroxide solution, most of the surrounding connective tissue proved no challenge to detach from the sclerites. The terminalia of Sinomelecta oreina had been dissected and partially cleaned by the late Donald B. Baker at some point prior to his published account of the species . Metasomal T1 to T4 densely covered with pale brown, appressed, plumose setae; T1 with midlength of horizontal surface subequal to that of vertical (anterior) surface and considerably shorter than midlength of exposed part of T2.(Cresson)http://species-id.net/wiki/Brachymelecta_mucidaMelecta?mucida Cresson, 1879a: 205 [♂]. [Melecta]?mucida Cresson; Melecta mucida Cresson; Brachymelecta mucida (Cresson); Brachymelecta mucida (Cresson); ♂ , labeledvide supra).As for the genus : Body length 9 mm, forewing length 8 mm. Head width 2.7 mm; head length  2.1 mm. Intertegular distance 2.0 mm; distance between outer margins of tegulae 3.0 mm.PageBreak with outer surface coarsely nodulose; outer apical margins of tibiae protuberant but without conspicuous spines; mesotibial spur and outer metatibial spur about as long as tibial diameter; inner metatibial spur longer than tibial diameter; arolia well developed; pretarsal claws cleft, outer ramus slender, sharply pointed, inner ramus flattened, expanded, much shorter than outer ramus, apex approximately right angular .♂ , labeled1♀ , with savide supra).As for the genus : Body length 11.5 mm . Head including labrum coarsely and closely punctate, upper part of head somewhat more coarsely so than lower part; area in front of ocelli, extending down medially as frontal carina, smooth and impunctate; area around base of mandible, including malar space, smooth and shining; scape and basal half of mandible more finely punctate except for anterior apical smooth shining swelling of scape and smooth mandibular area near articulation. Mesosoma mostly coarsely and closely punctate, punctation similar to that of clypeus except somewhat coarser (like upper frons and vertex) in much of mesoscutum; median part of mesoscutum, except anteriorly, with many punctures separated from one another by smooth ground often one-third as wide as nearby punctures; lateral margin of mesoscutum (about as wide as nearby tegula), lateral extremity of mesoscutellum, and axilla much more finely punctate, these punctures as dense as they can be, variable but most about one-third as wide as those on disc of mesoscutum; posterior propodeal areas with sculpturing grading toward granular. Metasomal terga and sterna with coarse, dense, shallow punctures about size of punctures of frons; posterior margins of T1 to T3 narrowly impunctate, T4 more broadly impunctate, T5 with impunctate zone much broader than exposed punctate zone; S1 to S5 with narrow apical smooth margins.Setae mostly sparse enough that details of surface sculpturing visible ; anterior to these bands setae inconspicuous, short (about one ocellar diameter in length), dusky; T5 largely hidden by T4, thus setal characters not clear but PageBreakband of pale setae absent; S2 to S6 with long whitish setae, up to five ocellar diameters in length, sparse on S2 and S3, denser and forming preapical bands on S4 and S5.Setae of head and mesosoma mostly four to five ocellar diameters in length, those of clypeus shorter than elsewhere; setae largely absent on posterior three-fourths of mesoscutum, mostly not branched, dull whitish or cinereous but dusky brownish on genal and hypostomal areas, grading to brownish cinereous on labrum and clypeus  and black on lateral margin of clypeus, on lower paraocular area below level of antennal base, and on posterior lateral angle of mesoscutum, on upper part of axilla, and perhaps on extreme basolateral margin of mesoscutellum. Setae of antennal scape and basal parts of legs including upper surfaces of femora similar to cinereous setae of mesosoma but shorter; posterior margins of pro- and mesofemora with fringes of long, pale setae, slightly darkened apically on mesofemur; setae of flagellum and pedicel extremely short; under surfaces of femora with areas of brownish dusky setae, grading to dusky on outer surfaces ofIntegumental coloration black throughout. Wings transparent, with brownish dusky stain in distal halves, darker distal to marginal cell, clear near and for short distance distal to 1rs-m and 2m-cu and along anterior margins of both submarginal cells; veins and pterostigma black.Female (paratype): As described for holotype male except as follows: Apex of metasoma intact. Forewing length 9.5 mm. Head width 3.3 mm; head length 2.8 mm; distance between outer margins of tegulae 4.25 mm.PageBreak slightly longer than maximum tibial diameters. Metasomal T6 with well-defined pygidial plate, pointed at apex; T6 apparently produced as slender apical process above comparable slender apical process of S6 which has strongly notched apex exceeding tergal apex.Malar space a depressed groove, base of mandible separated from compound eye by about one-third of distal diameter of F1. Labrum broader than long, apical margin concave. Inner orbits more distinctly converging below than in male; ocellocular distance about 1.5 times interocellar distance, nearly three ocellar diameters; distance between lateral and median ocellus nearly one ocellar diameter. Apical width of F1 approximately equal to apical width of scape; pedicel fully exposed, broader than long; F1 about 2.5 times as long as its apical width, F2 about as long as broad, subsequent flagellomeres similar but progressively very slightly longer so that F7 is slightly longer than broad, F10 only slightly longer than F9 with apex broadly rounded; flagellum not tapering so that F1 and F10 approximately equal in width. Distitarsi broken off and lost except for one front leg that is difficult to see, although pretarsal claws smaller than in male; apical tibial spines represented by outer apical anterior and posterior protuberances except posterior spine of protibial apex present and that of mesotibia a large, long, blunt process; thickened spinelike setae also present on meso- and metatibial apices and on outer surface of metatibia; tibial spursArea in front of ocelli not entirely smooth, with somewhat irregular punctures; scape without smooth, shining apical swelling. Metasomal terga and sterna with punctures slightly better spaced than in male, middorsally separated by shining ground one-third puncture width to full puncture width; apical impunctate margins of T1 to T3 slightly wider than in male, not contrasting with that of T4.PageBreakMost setae of legs from coxae to upper surfaces of tarsi whitish; area of dense setae on outer surface of mesotibia absent. Discal area of mesoscutum, upper inner extremity of axilla, and anterior margin of mesoscutellum with setae blackish . Metasomal T2 to T4 with discal setae largely palid and longer than in male so that subapical white setal bands contrast with their background less than in male; T5 and T6 without subapical bands of white setae; S2 to S5 with abundant long whitish setae, those of S2 and S3 not contrastingly sparse, as in male.Sinomelecta oreina is not unheard of among bees and this condition is found in various genera. For example, this same reduced antennomere count is well known and fixed across species in the augochlorine genus Chlerogas Vachal .Note that the mention of “Brachymelecta mucida is wholly unique in the North American fauna. Not only is it completely dissimilar from the other known melectine fauna for the continent, superficially more similar to some Asiatic forms, but also the type specimen is the only known individual to have ever been collected, despite considerable effort in Nevada and neighboring states. It is even possible that the species is today extinct, although not too much can be made from decades of absence data as putatively lost species can still turn up , is merely an autapomorphic species of that genus with its two submarginal cells and reduced flagellar count in males. This might seem particularly likely given that 1rs-m is lost easily in apoids, thereby resulting in the two submarginal cell condition. From Baker’s (1997) account and figures there was no reason to readily exclude a placement within Melecta, perhaps considering the taxon as a subgenus. Indeed, the genital capsule is somewhat reminiscent of Pseudomelecta Radoszkowski in its broad, squat form with a broad gonostylus that is densely setose throughout, and with a broad ventral setose lobe : videMelecta and the form of the mesoscutellum, pubescence, and presence of arolia readily exclude Sinomelecta oreina from Thyreus. Naturally, cladistic work is needed throughout the Melectini, particularly to determine whether Melecta as constituted is monophyletic, and it is hoped that this fuller account of two rare genera might encourage and bolster such work.In regard to obe vide. The hid


PTEN) on chromosome 10q23.3 is a negative regulator of the PIK3/Akt survival pathway and is the most frequently deleted tumor suppressor gene in prostate cancer. Monoallelic loss of PTEN is present in up to 60% of localized prostate cancers and complete loss of PTEN in prostate cancer is linked to metastasis and androgen-independent progression. Studies on the genomic status of PTEN in prostate cancer initially used a two-color fluorescence in situ hybridization (FISH) assay for PTEN copy number detection in formalin fixed paraffin embedded tissue preparations. More recently, a four-color FISH assay containing two additional control probes flanking the PTEN locus with a lower false-positive rate was reported. Combined with the detection of other critical genomic biomarkers for prostate cancer such as ERG, androgen receptor, and MYC, the evaluation of PTEN genomic status has proven to be invaluable for patient stratification and management. Although less frequent than allelic deletions, point mutations in the gene and epigenetic silencing are also known to contribute to loss of PTEN function, and ultimately to prostate cancer initiation. Overall, it is clear that PTEN is a powerful biomarker for prostate cancer. Used as a companion diagnostic for emerging therapeutic drugs, FISH analysis of PTEN is promisingly moving human prostate cancer closer to more effective cancer management and therapies.The phosphatase and tensin homolog gene ( Prostate cancer is one of the leading causes of cancer mortality in men in the Western world. In the United States, it is the most commonly diagnosed cancer in men and second only to lung cancer in the number of male cancer deaths . ProstatGiven the broad spectrum of clinical and molecular behaviors, the wide range of clinical outcomes and their associated treatments, it is clear that prostate cancer is a highly heterogeneous disease that presents great complexities in determining risk stratification and appropriate treatment strategies. The main challenge for physicians remains to distinguish indolent from clinically significant tumors. With the goal of improving clinical management of the disease, current efforts are focusing on identifying the genes and understanding the pathways involved in mediating disease progression and treatment resistance. A further short term goal of genetic testing of tumor samples is the identification of appropriate companion diagnostics, allowing stratification of patients for treatment and monitoring of treatment.One pathway with a prominent role in prostate cancer is the phosphatidylinositide 3-kinases (PI3K) signaling pathway. Current estimates suggest that this signaling pathway is up-regulated in 30–50% of prostate cancers –8. PI3K Once activated, these receptors phosphorylate PI3K at the cell membrane. Phosphorylated PI3K in turn phosphorylates phosphatidylinositol-4,5-diphosphate (PIP2), leading to the accumulation of phosphatidylinositol-3,4,5-triphosphate (PIP3). PIP3 recruits Akt  and phosphoinositide dependent protein kinase 1 (PDK1) to the cell membrane, where Akt is phosphorylated by PDK1. Phosphorylated Akt activity extends over a wide range of substrates, but most importantly activates the mammalian target of rapamycin (mTOR), which play a significant role in tumorigenesis . mTOR isPTEN). PTEN is a dual specificity protein and lipid phosphatase that not only targets acidic residues in protein substrates, but more importantly, the 3-phosphate from PIP3, converting it back to PIP2  analyses have provided a robust evaluation of the genomic status of PTEN in prostate cancer. This assay design has a centromere probe as a chromosome copy number control, and the PTEN locus probe labeled in a different fluorochrome. Using this FISH method, early studies by Yoshimoto et al. , while PTEN deletions were found in 23% of High-Grade Prostatic Intra-epithelial Neoplasia (HGPIN), a pre-malignant stage of prostate carcinoma, and 68% of overt prostate cancer. The authors concluded that acquisition of a PTEN deletion is an important step toward prostatic tumorigenesis  that depart from the primary tumor will die, whereas an estimated 0.01% of CTCs are likely to give rise to metastases . Despitecurrence . An addicer CTCs . Their dTEN loss . With emTEN loss .PTEN genomic status for prostate cancer prognosis is compelling but understanding its co-operation with other genetic aberrations in the context of this highly heterogeneous disease is crucial for accurately predicting clinical outcomes and developing targeted therapies. One of the most pivotal interactions in both human and murine prostate cancer is that between PTEN and the ETS-related gene, ERG. Genomic alterations of ERG resulting from a fusion with the androgen responsive trans-membrane protease, serine 2 (TMPRSS2) gene are highly pervasive in prostate cancer and can be detected in about 40–70% of clinically significant diseases. Several studies have confirmed that tumors with TMPRSS2:ERG gene rearrangements are enriched for PTEN genomic deletions in localized prostate cancer and CRPC , suggesting a selective advantage for tumor cells harboring both PTEN deletion and ERG fusion . Since AR expression levels are low in hormone naive tumors, addition of an inhibitor of PI3K/mTOR to the standard ADT of advanced prostate cancer may therefore be beneficial to patients with PTEN deleted tumor. A number of other studies have implicated differential expression of AR and PTEN deletions/Akt activation as significant predictors for disease progression and the development of CRPC  studies have showed that gain of 8q, including 8q24 involving the MYC gene, is one of the most frequent alterations in prostate cancer (MYC (8q24), ∼40% of primary tumors and >90% of metastases showed varying levels of MYC copy number gains  and MYC  were associated with significantly increased genetic instability and biochemical relapse compared to tumors with normal PTEN and MYC status. In light of these results, triaging patients by the use of PTEN and MYC copy number changes within pre-treatment biopsies may allow for better use of systemic therapies to target sub-clinical metastases or locally recurrent disease and improve clinical outcomes   –53. Howee cancer , 54. PTEe cancer . Recent e cancer , 57. Thet cancer . Even the cancer –61, to drequired , 62.Because PTEN is a tumor suppressor that negatively regulates the PI3K pathway, up-regulation of this pathway’s downstream targets is commonly observed in PTEN-deficient prostate cancers. Both Akt and mTOR are two important PI3K targets that are frequently activated in human primary prostate cancer specimens, as evidenced by increased phosphorylation of both Akt and S6RP, a downstream target of mTOR , 65. Thein vitro  . Everoliin vitro , 74. In PTEN loss, is associated with the activation of PI3K signaling; conversely, PI3K inhibitors can reactivate androgen signaling and sensitivity to antiandrogen therapy. In particular, recent studies have identified several mechanisms for the crosstalk between AR by mTOR  governing the initiation and progression of prostate cancer. Although significant progress has been made in our ability to forecast outcomes for prostate cancer after therapy using clinical and histological variables, the ability to accurately predict response to a specific treatment remains elusive. Molecular and cytogenetic assays such as FISH analyses of 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.


In vivo magnetic resonance imaging showed significant preservation of the intact spinal cord parenchyma. rhHGF-treatment did not give rise to an abnormal outgrowth of calcitonin gene related peptide positive fibers compared to the control group, indicating that this treatment did not induce or exacerbate allodynia. This is the first study to report the efficacy of rhHGF for treating SCI in non-human primates. In addition, this is the first presentation of a novel scale for assessing neurological motor performance in non-human primates after contusive cervical SCI.Many therapeutic interventions for spinal cord injury (SCI) using neurotrophic factors have focused on reducing the area damaged by secondary, post-injury degeneration, to promote functional recovery. Hepatocyte growth factor (HGF), which is a potent mitogen for mature hepatocytes and a mediator of the inflammatory responses to tissue injury, was recently highlighted as a potent neurotrophic factor in the central nervous system. We previously reported that introducing exogenous HGF into the injured rodent spinal cord using a herpes simplex virus-1 vector significantly reduces the area of damaged tissue and promotes functional recovery. However, that study did not examine the therapeutic effects of administering HGF after injury, which is the most critical issue for clinical application. To translate this strategy to human treatment, we induced a contusive cervical SCI in the common marmoset, a primate, and then administered recombinant human HGF (rhHGF) intrathecally. Motor function was assessed using an original open field scoring system focusing on manual function, including reach-and-grasp performance and hand placement in walking. The intrathecal rhHGF preserved the corticospinal fibers and myelinated areas, thereby promoting functional recovery.  Spinal cord injury (SCI) is followed by secondary degeneration, which is characterized by progressive tissue necrosis. Many experimental interventions have focused on this posttraumatic inflammatory process, using neurotrophic factors to reduce the damaged area and to promote axonal regeneration through the lesion epicenter. Neurotrophins such as nerve growth factor (NGF) Hepatocyte growth factor (HGF) was first identified as a potent mitogen for mature hepatocytes In this study, a total of 400 µg of recombinant human HGF (rhHGF) was infused intrathecally for four weeks immediately after C5-level contusive SCI in adult common marmosets, as a preclinical trial. Contusive injury is considered the most relevant to human SCI when evaluating therapies and predicting their translation to human treatment. However, to assess hand dexterity recovery, we needed an open field rating scale for a primate cervical SCI model that would be comparable to the BBB scale in rodents To precisely evaluate motor functions in the marmoset SCI model, we focused on the wrist and finger extension movements, because these motions are the most impaired in incomplete cervical SCI We first investigated the distribution of spinal motoneurons that innervate the hand muscles in marmoset forelimbs, by injecting cholera toxin B subunit (CTB) into the forearm flexor and extensor muscles. The wrist and finger extensor motoneurons were mainly located in lamina IX of the cervical (C)4-C7 segments, and wrist flexor motoneurons were in lamina IX of the C6-thoracic (Th)1 segments . Based oWe next investigated the pattern of the corticospinal tract (CST) pathway and its terminations in common marmosets. Axial sections of intact and injured spinal cords were immunostained with an anti-calmodulin-dependent kinase IIα (CaMK IIα) antibody Whereas CaMK II α-IR in the lateral column was obviously reduced in segments caudal to the lesion site in the PBS group compared with the intact spinal cord control, the CaMK II α-IR was well maintained, even caudal to the lesion site, in the rhHGF group . CaMK IITo examine rhHGF's tissue-sparing effects, we stained axial sections with Luxol fast blue (LFB) 12 weeks after SCI. There was a significant rim of spared myelinated white matter, even at the lesion epicenter, in the rhHGF group . StereolIn vivo MRI and diffusion tensor tractography (DTT) in vivo images that might reflect the pathological changes after SCI. One animal from each group was subjected to MRI and DTT analysis on the same day, and the recovery of motor function was assessed over time using our open field rating scale. Sagittal T2-weighted images (T2WIs) from each individual showed a diffusely expanded high-intensity area around the lesion epicenter 1 week after SCI, which was gradually compacted thereafter with the low-intensity area inside . At 12 weeks after SCI, DTT showed a larger number of spinal tract fibers around the lesion epicenter in the rhHGF group than in the PBS group, especially in the ventral white matter, which may have been a precise reflection of the spared rim of myelinated white matter detected by LFB staining and axial T2WIs -immunoreactive sensory axons in the Rexed lamina III 12 weeks after SCI to determine whether rhHGF treatment induced histological changes that could cause allodynia. Dense CGRP-positive innervation of lamina I and the outer part of lamina II was observed before and after SCI, and abnormal innervations of lamina III were detected after SCI in both groups . StereolAll animals showed severe quadriplegia 1 day after injury; they lay on the floor in a prone position, with little limb movement, and could not roll over or move forward by themselves. After C5-level SCI, irrespective of therapeutic intervention, the animals showed progressive recovery of motor function as assessed in three main categories: upper limbs, lower limbs, and trunk stability . This stThe early phase of recovery was characterized initially by the animals' movement and placing of weight on the lower limbs. As the animals recovered, they showed slight to extensive movements of the three lower limb joints, and began to lift their upper body from the floor by bearing weight on the upper limbs in stance only. They also began to move forward by placing weight on the lower limbs, dragging the anterior chest on the floor without other observable movements, and placing weight on the upper limbs with the hands below shoulder level. The next phase of recovery was characterized by lifting the upper body by placing weight on the upper limbs, even when moving forward. When the animals progressed to raising the upper body from the floor in walking, they began to move their upper limbs to the rhythm of the walk, with their hands above shoulder level. However, more typically, the forearms were dragged on the floor, with the wrist joints dropped and the forearms not pronated. They were eventually able to bear weight on the upper limbs while walking, dragging only the hands, and finally recovered to walking without dragging the hands, in the most-recovered cases in the rhHGF group. The hand position also gradually recovered from being between the shoulder and head level to above the head. The most remarkable category, hand placement in walking, was assessed when the hands were above shoulder level, and was characterized by wrist extension, forearm pronation, and finger extension. Initially, the wrist joints were completely dropped, and the forearm was not pronated; thus, the animals supported their weight on the dorsal side of the hands  or on thThe assessment of reach-and-grasp performance highlighted finger flexion and extension, wrist extension, forearm pronation, and shoulder flexion. For several days after SCI, the animals could not raise their upper limbs, and could not actively flex their fingers even when a pen was placed in their hands. The animals eventually began to grasp a pen passively placed in their hands, and to lift their upper limbs to reach and grasp a pen that was suddenly presented in front of their eyes. They typically recovered the ability to elevate their hands up to head height, and to reach and grasp a pen perpendicular to the body plane, with incomplete extension of the fingers and wrist joint. Many animals did not recover sufficiently to grasp a pen parallel to the body plane, even at head height , becauseWhereas intact animals never dropped their limbs through the gaps in the cage bars, animals after SCI often dropped the limbs through the floor gaps. We evaluated the distance the limb was dropped, as follows: lower limbs up to the thigh, knee, or foot only, and upper limbs up to the humerus, elbow, or hand only. Initially, the animals dropped their upper limbs up to the humerus and lower limbs up to the thigh, and were not able to raise the limbs at all . Over thThe bar grip  and spon2 = 0.785), suggesting that the scale closely reflected the involvement of the CST pathway.Of the three methods assessing motor function , our original open field rating scale yielded the smoothest recovery curve after SCI. Using this scale, the rating for the upper limbs showed a smoother recovery curve than that for lower limbs, reflecting the fact that the cervical SCI model used in this study is not a complete injury model, but rather a severe central cord syndrome model, which shows better and earlier functional recovery in the lower limbs than in the upper ones . To asseWe recently demonstrated that applying exogenous HGF by HSV-1 vector into the injured spinal cord of adult rats after SCI exerts significant neuroprotective and anti-apoptotic effects on neurons and oligodendrocytes to promote their survival, and enhances angiogenesis around the lesion epicenter. These multiple effects significantly reduce the area of damage and provide a better scaffold for axonal regeneration, thereby promoting functional recovery after thoracic SCI in rodents To begin to translate this HGF strategy to the treatment of human patients, here we performed intrathecal rhHGF infusion during the acute phase of cervical SCI in marmosets. Although previous clinical studies revealed that cervical SCI, which is more frequent in humans than SCI at other levels, is the main cause of manual dysfunction The CST is a major pathway for voluntary movement control. It is a phylogenetically new system that appeared first in mammals and developed predominantly in primates Direct CM connections predominantly developed in higher primates such as macaque monkeys, apes, and humans In this study, as shown in Intrathecal rhHGF infusion significantly reduced the damaged areas and promoted functional recovery after SCI, consistent with our previous report Compared with the direct CM pathway, the role of indirect pathways from the cerebral cortex to motoneurons, via subcortical or spinal interneuronal systems, has received much less attention, even though such pathways contribute the majority of motoneuron input In the present study, we induced a C5-level contusive injury as shown in Beyond the CST, there are other possible mechanisms for functional recovery after SCI. First, the C3–C4 PNs could receive convergent inputs from various descending tracts, such as the rubro-, reticulo-, raphe-, and tectospinal tracts, as in rodents and cats in vivo MRI techniques, including DTT in vivo images and motor function recovery was observed before 12 weeks. To improve the predictive and analytical value of these techniques, precise quantitative comparisons of in vivo MR images, pathological findings, and motor function over time after SCI will be necessary, especially during the acute phase of SCI .With regard to clinical trials, methods for evaluating the spared and regenerating axons after SCI intervention are critical. If the extent and severity of the spinal cord lesion can be estimated by We believe that intrathecal administration would also be the most effective administration route in the clinical application of rhHGF for SCI patients, because the blood-brain barrier significantly limits the translocation of rhHGF from blood to the spinal cord. We observed that the subcutaneous injection of rhHGF was ineffective in ALS rats , while the intrathecal administration of rhHGF showed significant therapeutic effects in these animals Overall, intrathecal rhHGF administration after SCI significantly reduced the extent of damage to the spinal cord parenchyma, preserved the CST pathway in the lateral column, and promoted hand dexterity, presumably by involving indirect CM pathways connected by interneurons. Furthermore, marmosets did not show abnormal behaviors or signs of discomfort or pain when the examiner touched their bodies or manipulated their limbs. These findings suggest that rhHGF treatment did not induce allodynia; this was also indicated by immunohistological analysis with an anti-CGRP antibody Osmotic mini pumps  were filled with PBS, or with rhHGF  diluted with PBS so that each pump contained 400 µg of rhHGF. Each pump was connected to an intrathecal catheter , and the apparatuses were incubated in sterile PBS at 37°C for 48 hours before use.Adult female common marmosets  were used . All surgeries were performed under general anesthesia induced by intramuscular injection of ketamine  and xylazine  and maintained by isoflurane . The animal's pulse, arterial oxygen saturation, and rectal temperature were monitored during the surgical procedures. After a laminectomy at the C5 level, the dura mater was exposed and a 20-g weight was dropped from a height of 50 mm onto the dura using a modified-NYU impactor as reported previously Guide for the Care and Use of Laboratory Animals , the Guidelines and Policies for Animal Surgery provided by the Animal Study Committee of the Central Institute for Experimental Animals and Keio University and the guidelines outlined by Weatherall Report,and were approved by the Animal Study Committee of Keio University .All interventions and animal care procedures were performed in accordance with the Laboratory Animal Welfare Act, the In vivo MRI and diffusion tensor studies were carried out at 1, 3, and 12 weeks after SCI in two animals from each group, four animals in total, under general anesthesia as described above. MRI was performed using a 7.0 tesla MRI, PharmaScan 70/16  with a coil dedicated for small animals; diffusion tensor analyses were performed as previously described Two intact animals were anesthetized with intramuscular injections of ketamine and xylazine as described above. A small skin incision was made in the left forearm of each, and 5 µl of CTB  was manually injected into 4 points of the forearm flexor or extensor muscles. The animals were sacrificed 5 days after the CTB injection. Spinal cords were collected and immunohistological analysis performed as described below.Spinal cords were perfusion-fixed with 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS), and post-fixed in the same fixative (24 hr) with 10% sucrose in 0.1 M PBS (24 hr) and 30% sucrose in 0.1 M PBS (24 hr). Spinal cord segments were embedded in an optimal cutting-temperature compound and cut into 20-µm-thick sections on a cryostat. For diaminobenzidine staining, the sections were incubated at 48°C with polyclonal anti- CaMK II α , polyclonal anti-CGRP , and polyclonal anti-CTB  antibodies, followed by biotinylated secondary antibodies . Biotinylated antibodies were visualized using the Vectastain Elite ABC kit (Vector Laboratories), followed by diaminobenzidine (Sigma). All images were obtained by microscopy .To quantify the CaMK II α-positive area, LFB-positive myelinated area, and CGRP-positive fiber length, axial section images were obtained and analyzed using grain counting with light intensity by a Micro Computer Imaging Device . Threshold values were maintained at constant levels for all analyses. After obtaining images of axial sections stained with an anti- CaMK II α antibody, the number of CaMK II α-positive neurons in the intermediate zone (IMZ) of the gray matter was counted.The bar grip test, which tests the animal's gripping reflex (the motion undertaken when attempting to grasp an object placed in front of their eyes) and evaluates the grip power, was carried out as previously reported Spontaneous motor activity tests were carried out as previously reported To develop an original open field rating scale, the natural course of motor function recovery after cervical SCI was precisely observed in 6 marmosets before starting experiments using rhHGF. Two blinded examiners participated in all the open field tests and were positioned across from each other to observe both sides of the marmoset. Marmosets were tested alone for 5 minutes on the floor and in the cage 3 times per week until 4 weeks after SCI, and once per week thereafter. During the open field test, marmosets were encouraged by slight tapping when they remained stationary for longer than 15 seconds. In cases of borderline locomotor performance or disagreement between examiners, scores indicating the greater deficit were assigned. All lower limb movements were counted except those that were obviously part of a reflex . The bilateral upper limbs were assessed separately, and the score was defined as the mean of the bilateral scores for motor function.All data are reported as the mean±SEM. In all the histological examinations, an unpaired two-tailed Student's t-test was used for single comparisons between the rhHGF and PBS groups. The results of the original open field rating scale, bar grip test, and spontaneous motor recovery test were analyzed using the Mann-Whitney U-test. To validate the original open field rating scale, correlations between the results of this scale and the spontaneous motor activity or histological findings  were analyzed by simple linear regression analyses.


Thyroid-stimulating hormone receptor (TSHR) loss-of-function (LOF) mutations lead to a wide spectrum of phenotypes, ranging from severe congenital hypothyroidism (CH) to mild euthyroid hyperthyrotropinemia. The degree of TSH resistance depends on the severity of the impairment of the receptor function caused by the mutation and on the number of mutated alleles In this review data about genotype-phenotype correlation and criteria for clinical work-up will be presented and discussed. Complete TSH resistance due to biallelic LOF TSHR mutations must be suspected in all patients with severe not syndromic CH and severe thyroid hypoplasia diagnosed at birth by neonatal screening. Partial forms of TSH resistance show a more heterogeneous hormonal and clinical pattern . In these cases TSH serum levels are above the upper limit of normal range for the age but with a very variable pattern, free thyroxine (T4) concentrations are within the normal range and thyroid size can be normal or hypoplastic at ultrasound scan. An early substitutive treatment with L-T4 must be mandatory in all patients with severe CH due to complete uncompensated TSH resistance diagnosed at birth by neonatal screening. The usefulness of substitutive treatment appears much more controversial in patients with subclinical hypothyroidism due to partial TSH resistance in whom the increased TSH concentration should be able to compensate the mild functional impairment of the mutant receptor. Together with standard criteria we recommend also an accurate clinical work-up to select patients who are candidates for a LOF TSHR mutation. Conflict of interest:None declared. Congenital hypothyroidism (CH) is the most common endocrine disorder in infancy. It shows a considerable variability in etiopathogenesis and clinical aspects. Among these various disorders, resistance to thyroid-stimulating hormone (TSH) has been defined as a genetic disease, characterized by TSH serum levels above the normal range, normal or reduced thyroid hormone concentrations, normal sized or hypoplastic thyroid gland, in absence of anti-thyroid antibodies. Recently, the OMIM database for genetic disease (at the website www.ncbi.nlm.nih.gov) classified TSH resistance among the six forms of nongoitrous CH (CHNG) and loss-of-function (LOF) mutations in the TSH receptor (TSHR) gene were identified as the most frequent cause of TSH resistance  1,2).,2.1,2).This review will focus on the TSH resistance due to LOF TSHR gene mutations. Data about the spectrum of the TSHR gene mutations, genotype-phenotype correlation, phenotypic variability, and criteria for clinical work-up will be presented and discussed with the help of recent studies on TSHR gene analysis in large populations.TSHR Physiology TSHR encoded by the TSHR gene (chromosome 14q31) . TSHR ECThe TSHR ECD contains a leucine-rich region (LRR) involved in hormone binding specificity and two cysteine-rich domains that would contribute to the 3D structure of the ECD and are involved in the maintenance of the two-subunit structure of the receptor. Between the LRR region and the TMD, there is a structural linker (hinge region) probably involved in structural conformationThere are several pieces of evidence indicating that TSHR is present as dimers or higher-order oligomers on the thyroid cell surface. TSHR oligomerization is an early event in receptor maturation and it may play an important role in receptor trafficking and targeting to the cell membrane .Spectrum of LOF TSHR Gene MutationsTo date, more than 60 mutations in the TSHR gene have been described in association with different degrees of TSH resistance , 4,5,6,,40,41,42Recently, Tao  suggesteThe mutations in the TSHR gene that cause an absence or strong reduction of the protein product, such as frame-shift, nonsense and splicing mutations, usually affect the amount of the receptor expressed on the plasma membrane with consequent severe impairment of the protein function. Fourteen of 25 patients reported in the literature , with thThe functional effects of the other TSHR gene missense mutations range from a slight alteration in biological function of the receptor to a condition of pseudo-dominance, in which a mutated receptor is able to alter the function of the normal receptor. Among the four missense mutations reported in the N-terminal region of ECD (exon 1), two (P27T and E34K) showed a slight activity reduction in functional studies, while the other two showed a severe loss of function ,45,46,47Eleven mutations map in the LRR domain , whose 3In total, 33 subjects are reported with mutations in the LRR domain, of whom 20 are monoallelic-only four mutations  showed a loss of function both in cAMP production and binding activity ,28,31, wThe last five ECD mutations map in the so-called hinge region that plays a role in structural conformation and undergoes post-transductional modifications. In this group, the mutations R310C  and C390Finally, most of the missense mutations lie in TMD, involved in signal transduction through Gs protein interaction and dimerization. As recently reported, it is assumed that the biochemical function of the TSHR is not guaranteed only by the cAMP generation via Gα proteins ,51,52. TGenotype-phenotype CorrelationsTSHR LOF mutations lead to a wide spectrum of phenotypes, ranging from severe CH to mild euthyroid hyperthyrotropinemia. The sensitivity of thyroid tissue to TSH stimulation can be fully lost with severe impairment of thyroid function and growth despite an enhanced TSH secretion or partially conserved with compensatory effect of the elevation of TSH levels  .The degree of TSH resistance depends on the severity of the impairment of the receptor function caused by the mutation and on the number of mutated alleles . When boHowever, phenotypic variability is a characteristic of TSHR gene mutations. The same mutation can be associated with different thyroid function in different subjects or in the members of the same family, and thyroid hypoplasia, which is preferably associated with null mutations in homozygous state, is found also in patients carrying monoallelic mutations. One emblematic mutation correlating with this phenotypic variability is the P162A mutation, reported in different families , both inTherefore, these findings suggest that other factors, such as genetic heterogeneity, such as digenic inheritance ,25, limiMost likely, the pediatric age of patients with NASH examined in our study  could exDiagnostic and Therapeutic Work-upWhen to InvestigateThe great phenotypic variability makes often difficult the diagnostic approach to TSH resistance due to LOF TSHR mutations. First of all, we have to point out that LOF TSHR mutations should be differentiated from other pathological mechanisms able to cause similar clinical features. The secretion of bioinactive TSH with conserved immuno-reactivity can be hypothesized, but no such cases have been reported . MutatioComplete TSH resistance due to biallelic LOF TSHR mutations must be suspected in all patients with severe non-syndromic CH and severe thyroid hypoplasia diagnosed at birth by neonatal screening. The prevalence of this form of CH varies among different screening programs, but it is considered a rare condition. Likely, its real  incidence can be underestimated due to its being strongly dependant on the experience of the operator for a correct US diagnosis. In fact, a mistaken diagnosis of athyreosis should be made at scintigraphy due to impaired radioiodine uptake by the very small amount of thyroid tissue . The detThe diagnostic approach becomes very puzzling in partial forms of TSH resistance due to LOF TSHR mutations, since these cases show a more heterogeneous hormonal and clinical pattern . In thesToday, due to the increasing sensitivity of laboratory assays for TSH, the occurrence of hormonal findings suggestive of partial TSH resistance in infancy or in childhood is very frequent. Only a minority of these subjects will be carriers of LOF TSHR mutations, so the criteria of selection of the patients who are candidates for genetic analysis become very important. The possibility of a mild form of hypothyroidism in heterozygous subjects for a TSHR gene mutation determined an increase of genetic studies in patients not selected by neonatal screening with the consequent increase of mutation detection previously identified in a single family. There are many Italian studies on this topic which reached different conclusions, most likely due to different selection criteria applied to the population examined. Alberti et al  found 4/In these subjects, besides the evaluation of the above-mentioned diseases, the diagnostic work-up must exclude AITD as the most frequent potential cause of subclinical hypothyroidism in late infancy and childhood. The differential diagnosis is based on the clinical history , the positivity of anti-thyroid antibodies and/or the finding of the typical non-homogeneous hypoechoic pattern at thyroid US.Therefore, partial TSH resistance due to LOF TSHR mutations must be suspected in all subjects with:1) TSH serum levels persistently above the upper limit of normal range or fluctuating around the upper limit of normal range with tendency to increase when increased thyroid activity is required  2) normal/near-normal fT4 concentrations3) absence of anti-thyroid antibodies4) normal or hypoplastic thyroid gland in situ at US with normoechoic pattern 5) positive family history for thyroid pathology 4) absence of other defects/malformations typical of syndromic CH or PHP1a5) negative perchlorate discharge test in selected casesWhen to TreatAn early substitution treatment with Levo-T4 (L-T4) should be mandatory in all patients with severe CH due to complete uncompensated TSH resistance diagnosed at birth by neonatal screening. Recently, Dimitropoulos et al  reportedThe usefulness of substitution treatment appears to be much more controversial in patients with subclinical hypothyroidism due to partial TSH resistance in whom the increased TSH concentration should be able to compensate the mild functional impairment of the mutant receptor. Certainly, there are many reports of both biallelic and monoallelic TSHR mutations diagnosed during childhood and adulthood by chance in patients who showed normal physical and neurological development ,12,28,29Since TSH is thought to be the most sensitive and early indicator of insufficient thyroid production, there is general agreement among pediatric endocrinologists to consider a TSH serum value higher than 10 mU/L (after 2 weeks of life) as a criterium for beginning substitution therapy. In our study, following these criteria, L-T4 therapy was started in 6 of 16 subjects with TSHR gene mutations and in 8 of 27 subjects with similar NASH phenotype but without TSHR mutations . None of the treated children showed clinical signs of hypothyroidism, but in some parents, carriers of the mutation, the treatment became necessary for the onset of clinical features of hypothyroidism concomitant with AITD ,53. TeneMizuno et al  reportedObviously, more prospective longitudinal studies are needed to clarify the genotype-phenotype correlation in patients with partial TSH resistance and to improve the therapeutic approach. Today, the choice of treat or not to treat must be individualized according to biochemical and clinical parameters. In any case, a careful follow-up is needed in all untreated cases because some patients with fully or partially compensated TSH resistance could develop uncompensated resistance over time, in particular with concomitant AITD ,53.In summary, a review of available data on patients with loss-of-function mutations of TSHR genes shows that: 1) A mutation in the TSHR gene is a rare event in terms of mutated alleles/health alleles but may be a frequent cause of NAHT when the patients are accurately selected; 2) Together with standard criteria  also an accurate clinical work-up is needed to select patients who are candidates for a LOF TSHR mutation testing; 3) The need for substitution therapy with L-T4 remains controversial in particular in pediatric patients with partial TSH resistance. A careful long-term follow-up is recommended in these cases.


Regulatory T (Treg) cells constitute a distinct T cell subset, which plays a key role in immune tolerance and homeostasis. The transcription factor Foxp3 controls a substantial part of Treg cell development and function. Yet its expression alone is insufficient for conferring developmental and functional characteristics of Treg cells. There is accumulating evidence that concurrent induction of Treg-specific epigenetic changes and Foxp3 expression is crucial for lineage specification and functional stability of Treg cells. This review discusses recent progress in our understanding of molecular features of Treg cells, in particular, the molecular basis of how a population of developing T cells is driven to the Treg cell lineage and how its function is stably maintained. While the majority of Treg cells develop in the thymus, some are induced from naïve CD4+ T cells in the periphery. In order for Treg cells to exert their regulatory functions, constitutive expression of the transcription factor Foxp3 is essential  cells, Foxp3 expression, combined with T cell receptor (TCR) stimulation, enables the acquisition of Treg properties including suppressive function, hyporesponsiveness to TCR stimulation, and up-regulation of Treg-associated molecules including CTLA-4, GITR, and CD25  cells represent a subset of CD4In addition to the expression of Foxp3, several comprehensive analyses have recently revealed possible involvement of other molecular mechanisms in the development of Treg cells. For example, genome-wide comparison of DNA methylation status in Tconv and Treg cells has demonstrated the presence of Treg-specific DNA hypomethylation in the genes associated with Treg function  cells have been revealed to differ from thymus-derived Treg (tTreg) or periphery-derived Treg (pTreg) cells in vivo. Firstly, they have only partial coverage of Treg-type gene expression profile   and it was shown to be required for stable expression of Foxp3 , Ikzf4 (Eos), and Tnfrsf18 (GITR) , molecular definition (CD4+Foxp3+ T cells), and epigenetic definition (cells with Treg-specific DNA methylation status) of Treg cells are negligible in most physiological settings. However, in the contexts of various immunological diseases, the accuracy of Treg definition matters. Some of the human naïve T cells and effector T cells are capable of expressing Foxp3 in response to TCR activation (Allan et al., + T cells are present at a normal or increased frequency. With the epigenetic features of Treg cells revealed, it may be used as a new tool for assessing Treg function and for better understanding of disease pathology.In this review, we have discussed how Treg cells can be molecularly defined as a cellular entity. Differences among functional definition (CD4ex vivo or induced in vitro is promising. The ultimate goal of this approach is to control inflammation with minimum adverse effects by using antigen-specific Treg cells. However, little progress has been made toward practical application of this idea due to the plasticity of some Foxp3+ T cells and the lack of reliable cell surface markers for differentiating human Treg cells from other activated T cells, which would increase the chances of non-Treg cells or unstable Treg cells being contaminated and thus raise the concerns regarding safety and efficacy of Treg cell therapy (Riley et al., in vitro from Tconv cells. Yet, current method of iTreg generation using TGF-β and IL-2 can induce Foxp3 protein expression, but these iTreg cells are significantly different from in vivo Treg cells in terms of gene expression, epigenetics, stability, and function (Ohkura et al., Treg cells have crucial roles in maintaining immunological self-tolerance and homeostasis and are suspected to be involved in a variety of immunological disorders Shevach, . Treg cein vitro; however, often it is not accompanied by epigenetic changes or Treg-type gene expression. Foxp3 requires its co-factors to potentiate its function in gene regulation and confer suppressive activity on Treg cells. Furthermore, for long-term lineage commitment of Treg cells, the stability of Foxp3 and other Treg signature molecules need to be ensured by epigenetic modification represented by DNA methylation status. Further investigation will elucidate the mechanisms of epigenetic changes as well as Foxp3 induction in the course of Treg cell development, enabling us to devise new approaches for clinical application of Treg cells.Treg cell development involves concurrent induction of Foxp3 expression and epigenetic conversion, which cooperatively generate Treg-type gene expression. It is possible to induce the expression of Foxp3 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.


It has been known for more than a century that most of the plant cells are connected to their neighbors through membranous pores perforating the cell wall, namely plasmodesmata (PDs). The recent discovery of tunneling nanotubes (TNTs), thin membrane bridges established between distant mammalian cells, suggests that intercellular communication mediated through cytoplasmic continuity could be a conserved feature of eukaryotic organisms. Although TNTs differ from PDs in their formation and architecture, both are characterized by a continuity of the plasma membrane between two cells, delimiting a nanotubular channel supported by actin-based cytoskeleton. Due to this unusual membrane organization, lipids are likely to play critical roles in the formation and stability of intercellular conduits like TNTs and PDs, but also in regulating the transfer through these structures. While it is crucial for a better understanding of those fascinating communication highways, the study of TNT lipid composition and dynamics turned out to be extremely challenging. The present review aims to give an overview of the recent findings in this context. We will also discuss some of the promising imaging approaches, which might be the key for future breakthroughs in the field and could also benefit the research on PDs. Figure 1A), suggests that this type of communication is not a hallmark of plant cells . These structures are dynamic and heterogenous and contrary to other types of membrane protrusions, such as filopodia, do not touch the substrate in cell culture .The existence of “cytoplasmic bridges” between plant cells was first reported by nt cells . Like PD culture . Althoug. Indeed, different studies using artificial membrane nanotubes and theoretical predictions highlighted the reciprocal influence of membrane curvature and lipid segregation  domain-containing proteins Figure . In contFigure 2D). Notably, the inner leaflet of membrane rafts is enriched with phosphoinositides, such as PIP2 and phosphatidylinositol 3,4,5-trisphosphate (PIP3), which emerged as major regulators of cytoskeleton structure and dynamics . They also contribute to anchor actin filament to the PM through protein linkers, such as ezrin-radixin-moesin (ERM) proteins, key proteins in the formation of PM protrusions  Iglič and coworkers recently highlighted the presence of cholesterol-sphingomyelin nanodomains within TNTs in a T24 urothelial cancer cell line . In cont3 in TNT formation. Wang and collaborators (2O2 treatment. Interestingly, they observed a drastic reduction of stress-induced TNT formation in astrocytes treated with PI3K and mTor inhibitors or expressing the Akt dominant negative mutant, whereas expression of the constitutive form of Akt increased the number of connections. They also reported an increase of Akt and PI3K phosphorylated forms upon H2O2 treatment. In this context, PIP3 formation thus appears as an important step for TNT genesis.To our knowledge, no other data regarding the lipid composition of TNTs has been reported in the literature thus far. However, indirect evidences support the role of PIPborators  recently3 for TNT generation (3, hindered the ability of Myo10 to induce TNT formation. Thus, in accordance with what has been reported for filopodia (3 binding seems to be necessary for Myo10 role in TNT induction. However, contrary to what has been shown for astrocytes, no correlation between Akt activation and TNT formation has been observed in CAD cells, suggesting a PI3K dependent but Akt independent pathway (In addition, recent data obtained in our laboratory using CAD cells, a mouse neuronal cell line of catecholaminergic origin, also suggested the importance of PIPneration . It was ilopodia , Myo10 r pathway .Although the cues for a critical role of lipids in TNT are considerable, research in the field has been hampered by technical limitations. While PD-enriched fractions can be obtained from plant cell walls, thus allowing biochemical approaches , isolati3-enriched compartments of the PM. Therefore a similar approach could yield important information on lipid/actin interactions within intercellular conduits.While classical studies consist of imaging the distribution of the fluorescence intensity from a given fluorophore (lipid-binding probes, lipid analogs, or fluorescently tagged proteins used as raft markers; Because lateral organization of the membrane affects the mobility of its constituents, sub-resolution membrane domains can also be analyzed thanks to fluorescence microscopy techniques based on molecular dynamics assessment . This inA major issue when studying lipid organization in cellular membranes is that the predicted size of lipid nanodomains is below the resolution limit of classical optical microscopes . The devAn alternative to fluorescence microscopy for high-resolution study of membrane organization within intercellular conduits might be the utilization of scanning or transmission electron microscopy EM; . HoweverFinally, because separating TNTs from the cell constitutes a technical challenge that may be difficult to overcome, an alternative non-targeted approach to resolve lipid distribution in intercellular conduits may come from the developments of imaging mass spectrometry techniques IMS; . Althougon TNT can be informative on PD membrane and vice versa. In these review we have underlined the importance of studying the lipid composition and dynamics in these structures, as they are likely to be key elements regulating their structure and function. In addition to the imaging techniques described above, biophysical approaches and computational models may also greatly contribute to extend our knowledge on this important subject. As it is often the case for emerging fields, we believe that the key for a better understanding of those fascinating intercellular communication highways lies in pushing the current technology to new applications and in the development of transkingdom and interdisciplinary studies.Despite differences in their formation and architecture, TNTs and PDs present striking functional and structural similarities, which need to be thoroughly explored. The questions raised by their unusual nanotubular membrane conformation are largely overlapping; therefore data obtainedThe 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.