We review current ICH data and discuss future directions notably on prostaglandin receptors, which may lead to the development of unique therapeutic targets against hemorrhagic stroke and brain injuries alike.Each year, approximately 795,000 people experience a new or recurrent stroke. Of all strokes, 84% are ischemic, 13% are intracerebral hemorrhage (ICH) strokes, and 3% are subarachnoid hemorrhage strokes. Despite the decreased incidence of ischemic stroke, there has been no change in the incidence of hemorrhagic stroke in the last decade. ICH is a devastating disease 37–38% of patients between the ages of 45 and 64 die within 30 days. In an effort to prevent ischemic and hemorrhagic strokes we and others have been studying the role of prostaglandins and their receptors. Prostaglandins are bioactive lipids derived from the metabolism of arachidonic acid. They sustain homeostatic functions and mediate pathogenic mechanisms, including the inflammatory response. Most prostaglandins are produced from specific enzymes and act upon cells via distinct G-protein coupled receptors. The presence of multiple prostaglandin receptors cross-reactivity and coupling to different signal transduction pathways allow differentiated cells to respond to prostaglandins in a unique manner. Due to the number of prostaglandin receptors, prostaglandin-dependent signaling can function either to promote neuronal survival or injury following acute excitotoxicity, hypoxia, and stress induced by ICH. To better understand the mechanisms of neuronal survival and neurotoxicity mediated by prostaglandin receptors, it is essential to understand downstream signaling. Several groups including ours have discovered unique roles for prostaglandin receptors in rodent models of ischemic stroke, excitotoxicity, and Alzheimer disease, highlighting the emerging role of prostaglandin receptor signaling in hemorrhagic stroke with a focus on cyclic-adenosine monophosphate and calcium (Ca Stroke is a leading cause of long-term disability and accounts for one of every 18 deaths in the United States , which is synthesized in the vasculature of the brain (within neuron and glia cells), acts as a powerful endogenous pyrogenic mediator of the preoptic area by activating specific receptors  on the function of cells such as microglia and neurotrophils after ICH is still needed. More information on the general role of various cell types modulating inflammation after ICH can be found in a previous review from our group , and cytosolic calcium-dependent (sPLA2α) and calcium independent (iPLA2α) and metabolized to PGH2 by cyclooxygenase enzymes, COX-1 and COX-2 , prostaglandin I synthase (PGIS), prostaglandin D synthase , prostaglandin F synthase (PGFS), and thromboxane synthase (TxS). It should be noted that mPGES-1 was found to be significantly increased in the AD brains as compared to age matched controls  have been described  to increase intracellular levels of cyclic-adenosine monophosphate (cAMP), or Gαq  to increase intracellular levels of calcium or Gαs-proteins  to increase or decrease intracellular levels of cAMP and calcium. Table Prostaglandins exert cellular affects through their specific receptors: PGE2+ transients in response to β-adrenergic receptors, Epac2 activation induces exocytosis in human β cells, increasing the number of exocytic sites on the plasma membrane via its effects on Ca2+ signaling, and effects neuronal function following activation by potentiating the postsynaptic excitation currents . Epac1 and 2 function as guanine nucleotide exchange factors for the small G-protein Rap. Epac proteins are expressed throughout the body, with Epac1 specifically abundant in blood vessels, kidney, adipose tissue, CNS, ovary, and uterus, whereas Epac2 is mostly expressed in the CNS, adrenal gland, and pancreas  inhibited vascular endothelial growth factor-induced dye leakage from mouse dermal blood vessels  and permanent distal MCAO  in the CNS prevented oxygen-induced neuronal death and protected CA1 pyramidal neurons against ischemia in gerbils , the role of EP4 receptors in ICH may be more dynamic than in ischemic stroke  has been linked to exacerbating ICH injury  used as an antithrombotic agent, could also be used to prevent atherothrombosis and ischemic stroke , specific surface PGD2 receptor (Nagata et al., αi-proteins, leading to the inhibition of cAMP and increase in Ca2+ in a variety of cell types (Sawyer et al., One of two PGD2+ activation, we hypothesize that activation of this receptor could lead to aggravated brain damage and its inhibition could lead to better functional and anatomical outcomes after ICH. Moreover, blockade of this receptor could result in more availability of PGD2 to DP1 receptor, which could then lead to increased cAMP level and subsequent neuroprotection as we have previously reported (Saleem et al., −/− mice following ischemic stroke and acute NMDA-induced excitotoxicity due to similar binding affinity to DP1 receptor, DP2 receptors would have little therapeutic potential in ICH (Ahmad et al., Despite its expression in the spinal cord and its anti-inflammatory effect as an activator of peroxisome proliferator-activated receptors, little is known about the role of DP2 receptors after stroke (Genovese et al., Intracerebral hemorrhage accounts for 13% of all strokes in the United States each year. Following ICH, extravasated blood accumulates and compresses the surrounding brain tissue and treatment is primarily supportive with poor clinical outcome. To improve the outcome of ICH patients, a better understanding of the pathogenesis of ICH-induced brain injury is needed. We and others have identified prostaglandin receptors as potential therapeutic agents against ischemic stroke. Prostaglandins and their receptors regulate many physiological, inflammatory, and immunological processes. In the CNS, the role of prostaglandin receptors has been essentially first elucidated by gene-deletion studies and evidence suggests these receptors are a therapeutic target for the treatment of stroke. However, the therapeutic potential of prostaglandin receptors in ICH remains to be elucidated. Understanding the integration of a network of prostaglandin receptor signaling would improve our knowledge of these putative therapeutic targets for the effective treatment of neurological diseases. Evidence supports that prostaglandin receptors play a cooperative and/or sequential role in ICH-mediated inflammation and neurotoxicity. Intracellular signaling pathways activated by prostaglandin receptors in ICH are likely to be different than in ischemic stroke. However, we hypothesize that evolutionary regulated prostaglandin receptor signaling will be conserved and therefore this group of receptors, found to have a given role in ischemia may also have a unique role in hemorrhagic stroke. However it remains to be determined whether the effect of these receptors will be similar. Also, identification of a multitude of intracellular protein interactions with prostaglandin receptors may prove to regulate their neuronal expression, selectivity toward ligands, and crosstalk with cytokines, chemokines, and neurotransmitters. Studying these interactions and the cell-specific functions will help map a detailed network of signaling that would be necessary for an injury-induced cellular response. The discovery of a prostaglandin receptor specific intracellular response may enable us to navigate a complex pathway leading to the discovery of beneficial drugs for the treatment of hemorrhagic stroke.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.


Delivery and final fusion of the secretory vesicles with the relevant target membrane are hierarchically organized and reciprocally interconnected multi-step processes involving not only specific protein–protein interactions, but also specific protein–phospholipid interactions. The exocyst was discovered as a tethering complex mediating initial encounter of arriving exocytic vesicles with the plasma membrane. The exocyst complex is regulated by Rab and Rho small GTPases, resulting in docking of exocytic vesicles to the plasma membrane (PM) and finally their fusion mediated by specific SNARE complexes. In model Opisthokont cells, the exocyst was shown to directly interact with both microtubule and microfilament cytoskeleton and related motor proteins as well as with the PM via phosphatidylinositol 4, 5-bisphosphate specific binding, which directly affects cortical cytoskeleton and PM dynamics. Here we summarize the current knowledge on exocyst-cytoskeleton-PM interactions in order to open a perspective for future research in this area in plant cells. Figure 1). A growing number of papers document functions of the plant exocyst in similar processes with high demand for exocytosis, including root hair growth, hypocotyl cell elongation, cytokinesis, seed coat formation and papilla formation after a pathogen attack in plants (Polarized surface growth in eukaryotic cells involves interactions between the cytoskeleton and membrane transport pathways. The last steps of the secretory pathway taking place in the vicinity of the plasma membrane (PM) are regulated by an array of small GTPases, the exocyst tethering complex, and SNARE proteins. The exocyst is a protein complex comprising eight subunits  engaged in docking and tethering of secretory vesicles, providing a spatial and temporal regulation of exocytosis  and inten plants .Deep insight into exocyst functions and their mechanisms came from genetic studies on budding yeast, where the exocyst was originally discovered as a protein complex . In buddSEC10 and SEC15 exocyst components and CDC42 and RHO3 GTPases regulating the exocyst polar targeting  are more efficiently transported to the cell apex by the Myo52 myosin V along microfilaments . Either Unexpectedly, the fission yeast Sec3 not only acts in exocytosis but also marks sites for actin recruitment and controls overall actin organization via direct binding of For3 . Mutantsin vitro and in vivo with the yeast and rat Arpc1/Arc40 subunit of the Arp2/3 complex, a key regulator of actin polymerization. Inhibition of the Exo70 function in rat kidney cells blocks formation of actin-based membrane protrusions and affects cell migration .In mammalian cells, actin organization, as well as membrane trafficking, cell growth and differentiation, is regulated by RalA and RalB, ubiquitous small GTPases from the Ras superfamily . ActivatCells of mouse oocytes can use secretory (Rab11-positive) vesicles associated with the exocyst components via the Rab11–Sec15 interaction  as adaptAlthough we can find no dynamic membrane protrusions analogous to filopodia in plant cells, fine F-actin meshwork is essential for polar growth of root hairs, pollen tubes, or stigmatic papillae and this type of growth demanding precise regulation of exocytosis is also strongly dependent on the exocyst function .Arabidopsis via a plant specific adaptor protein ICR1 which is implied in the regulation of auxin polar transport  are present and due to some plant specific features they are called Rop (Rho of plant). Rop GTPases were clearly implied in the cortical cytoskeleton regulation mostly possibly via plant specific Rop-interacting adaptors RICs; . Very siin vitro, the exocyst complex reconstituted from recombinant subunits inhibits tubulin polymerization. However, deletions of any of Sec5, Sec6, Sec15, or Exo70 exocyst subunits diminished the inhibition activity. Surprisingly, Exo70 itself could inhibit tubulin polymerization, albeit the exocyst complex lacking the Exo70 subunit did not lose its activity completely. On the other hand, when Exo70 was overexpressed, the microtubule network became disrupted and filopodia-like PM protrusions were formed . However, both cytoskeletal systems in plant cells strongly interact  so that in the real biological context it will be challenging to separate their functions.In contrast to budding yeast, typical vertebral cells use microtubules for long-range cargo transport and microfilaments for short-range transport in cell cortex during later steps of vesicles traffic . SeveralMammalian cell migration involves cooperative reorganization of the actin and microtubule cytoskeletons under the control of Rho GTPases . Proper Interestingly, despite the different mechanisms of cytokinesis between plants and animals/fission yeast (contraction versus building of a cell plate), the exocyst is involved in both types of cytokinesis .IQGAP1 is another important regulator of both actin and microtubular cytoskeleton associated with the exocyst. The active RhoA and Cdc42 trigger association of Sec3 and Sec8 exocyst subunits with IQGAP1. This interaction is essential for MT1-MMP protease localization at invadopodia and thus for proper invadopodia functioning . IQGAP1 2) and indirectly by association with Rho GTPases . Sec15p binds to the membrane of secretory vesicles via the Sec4p Rab GTPase .In fission yeast, Sec6 and Sec8 exocyst subunits localize to cell tips largely independent of the actin cytoskeleton, but in a Cdc42 and PIPrganisms . Bendezúin vitro independent of the exocyst function. This represents a mechanism creating protrusions even in the absence of actin, albeit it is not clear to what extent stimulated actin polymerization, membrane delivery, and membrane deformation contribute to cell shape changes in vivo including formation of membrane protrusions. Thus, Exo70 as a membrane-bending protein may couple the actin dynamics and PM remodeling in morphogenesis.Very recently The exocyst is also essential for large-particle phagocytosis , SalmoneRegulation of the cytoskeleton structure and dynamics in plant cells is very much affected by the cell wall, implying close proximity between secretory pathway, cell wall biogenesis and cortical cytoskeleton. These cellular systems are regulated by small GTPases, especially from the ARF, RAB, and ROP families, major regulators of the cell polarity and morphogenesis closely related to their fungal or animal counterparts . Work inArabidopsis in endowed with 23 EXO70 paralogs) possibly provides a potential for fine targeting into specific cortical areas .On the contrary, dense cortical microfilament meshwork might block exocytosis in both animal and plant cells . For insl models  and currArabidopsis epidermal cells, was unaffected by actin or microtubule cytoskeleton disruption after short (10 min) treatment with inhibitors, however, prolonged actin cytoskeleton disruption (1 h) resulted in exocyst redistribution and aggregation at the PM and impaired dynamics (The dynamics of several exocyst subunits at the PM, as monitored by TIRF microscopy in dynamics . This isdynamics .Interestingly, exocyst complexes show almost no lateral movement within the PM in both plant and animal cells, as analyzed by the TIRF microscopy, and very similar time of persistence at the PM of about 10 s was recorded . SimilarDirect as well as a circumstantial evidence accumulated over the years concerning interactions and cooperation between the exocyst and cytoskeleton indicates that the exocyst, cytoskeleton, and membrane traffic meet at the active cellular cortex. The exocyst serves an important role in co-ordination of the vesicle trafficking with the cytoskeleton in eukaryotes, in addition to its canonical role in exocytosis. In plant cells, however, we have currently only limited and indirect evidence for this regulatory interplay, urging further research in this direction.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.


Heparin Induced Thrombocytopenia (HIT) is caused by antibodies that recognize platelet factor 4 (PF4) associated with polyanionic glycosaminoglycan drugs or displayed on vascular cell membranes. These antibodies are elicited by multimolecular complexes that can occur when heparin is administered in clinical settings associated with abundant PF4. Heparin binding alters native PF4 and elicits immune recognition and response. While the presence of heparin is integral to immunogenesis, the HIT antibody binding site is within PF4. Thus HIT antibodies develop and function to cause thrombocytopenia and/or thrombosis only in the presence of PF4. Future emphasis on understanding the biology, turnover and regulation of PF4 may lead to insights into the prevention and treatment of HIT. With the introduction of heparin into clinical practice in the 1940s, surgeons were able to perform complex operations using this anticoagulant to prevent and/or treat obstructive clots. The availability, over-all safety and performance of heparin opened the door for procedures such as hemodialysis, open-heart surgery and organ transplants . An estiIn the decades following its introduction however, a paradoxical, adverse effect of heparin was recognized . In a smEarly investigators determined that the agent responsible for HIT was a platelet-activating antibody that caused platelet consumption and a hypercoagulable state . InitialPlatelet factor 4 (PF4), also known as chemokine CXCL4, is a cationic, 7.8 kDa protein which forms tetramers at physiological pH and ionic strength -26. PF4 In addition to the presence of heparin during anticoagulant therapy the formation of immunogenic complexes that provoke antibody depends on the availability of PF4 . The plaThe availability of PF4 is influenced both by acute and chronic platelet activation, and logically plays a role in the risk for generation of PF4/heparin antibodies in the context of anticoagulant therapy. This suggests an explanation for the common observation that specific patient populations are known to be at an increased risk of developing HIT antibodies ,62-64. TIt is well documented in the literature that the presence of HIT antibody does not cause thrombocytopenia or thrombosis in the majority of seropositive patients ,67-69. IIn contrast to immunogenesis (formation of antibodies), which is dependent on the presence of heparin, HIT pathogenesis (antibody function) can occur after cessation of anticoagulant therapy, referred to as “delayed HIT” -76. StudThe development and functionality of HIT antibodies are dependent not only on availability of PF4, but more importantly on the level of PF4 in relation to heparin (or other GAG) ,91. The de novo antibody generation [Experimental and clinical studies find a distinction between antigenicity and immunogenicity, that is, between antibody binding or cross-reactivity, and antibody formation or seroconversion. Experiments employing a mouse model to investigate HIT immunogenesis have demonstrated that mouse PF4 (mPF4)/heparin complexes, but not mPF4 alone cause development of mPF4/heparin-reactive antibodies. The higher the concentration of mPF4/heparin complexes the greater was the antibody formation . A somewneration .It is also interesting to consider that anticoagulant ratios of PF4 and heparin differ from those of antigenic or immunogenic complexes. Only heparin in excess of PF4 has anticoagulant effect . Heparinde novo antibody formation, suggesting that the impetus for the HIT immune response may be more complex than the presence of a conformational change in a self-protein.With the discovery that a specific PF4/heparin complex was the HIT antigen ,115 it sThe HIT immune response has several unique aspects, and is as yet, not completely understood . AdaptivIn addition to their role in hemostasis, platelets are increasingly recognized as immune effector cells ,123. PF4This innate immune role of PF4 may help explain the unusual immune response to PF4 in the presence of heparin. In its antimicrobial role, PF4 binds to anionic components of bacterial surfaces. It has been discovered that PF4 bound to bacteria can be used to affinity enrich HIT antibodies from patient sera, thus demonstrating that antibodies generated in response to heparin therapy cross react with PF4 epitopes exposed on bacterial cells . There iA direct test of the concept that endogenous PF4-bound target antigens resemble the antigen generated by heparin during anticoagulant therapy used a mouse model of polymicrobial bacteria sepsis, and demonstrated that bacterial exposure resulted in development of PF4/heparin-reactive antibodies with a time course of a typical primary immune response . These sNaturally occurring soluble proteins are poorly immunogenic in the absence of an adjuvant such as alum or various oil emulsions, which have been used empirically as immunostimulatory agents . AdjuvanCells of the immune system express a variety of pattern recognition receptors (PRRs), including toll-like receptors (TLRs). These receptors respond to pathogen associated molecular patterns (PAMPs) that are characteristic of pathogen groups, but distinct from “self”, allowing a limited number of receptors to recognize a great variety of pathogens . PatternTLR activation is central to both innate and adaptive immune responses. Specific TLRs respond to particular pathogen classes, generating a context-specific, unique profile of cytokine signals which modulate the magnitude and fine structure of the B cell antibody response . In thisTo date, strategies to prevent or treat HIT have focused on minimizing the use of unfractionated heparin in favor of LMWHs or direct thrombin inhibitors . These aAs discussed above, HIT antibodies are necessary but not sufficient to cause the intense platelet activation that leads to thrombocytopenia and/or thrombosis. Formation of platelet-activating immune complexes depends on availability of the PF4 target antigen, and the risk of HIT is therefore highest in settings characterized by intense PF4 release. It is logical that minimizing the availability of PF4 or otherwise preventing formation of PF4/heparin complexes would be strategies to abrogate the risk of immunogenesis and pathogenesis of HIT antibodies .One such strategy was suggested by observation of patients with familial hypercholesterolemia. These patients do not achieve adequate low-density lipoprotein (LDL) cholesterol reduction through diet or statin therapy and may undergo frequent LDL apheresis treatments. Despite the repeated exposure to heparin and predisposition to vascular disease, the incidence of HIT is low in this population . Based oPresentation of the PF4 target antigen results from the physiochemical properties of complexes of heparin and PF4 tetramers formed and sustained at specific molar ratios . In thesIn general, antigenic epitopes are exposed when PF4 binds to any heparin-derived anticoagulant drug. In addition to its anticoagulant activity heparin has potent anti-inflammatory properties; however, the risk of bleeding prevents its use for non-thrombotic indications. Heparin which is desulfated at the 2-O and 3-O positions (ODSH) retains anti-inflammatory properties but has reduced anticoagulant activity . ODSH reA determining factor in the risk that HIT antibodies will be elicited as a result of heparin anticoagulant therapy is the presence of PF4. The presence of PF4 also determines whether HIT antibodies will lead to thrombocytopenia and/or thrombosis because only immune complexes of antibody plus target antigen, not antibodies alone, mediate the pathogenic platelet activation. This review presents the hypothesis that heparin serves as an adjuvant, which facilitates antibody formation by displaying PF4 in a motif recognized as a pathogen associated molecular pattern, an agonist for pattern recognition receptors on immune cells. Techniques aimed toward sequestering PF4 or minimizing its conformational alteration are promising areas of research toward developing effective clinical interventions to prevent or treat HIT.PF4: Platelet factor 4; PF4/H: Platelet factor 4/heparin; HIT: Heparin-induced thrombocytopenia; GAG: Glycosaminoglycan; ULC: Ultralarge complex; PAMP: Pathogen associated molecular pattern; PRR: Pattern recognition receptor; TLR: Toll like receptor; BCR: B-cell receptor: AT, antithrombin; PHR: PF4 to heparin ratio.The authors have no financial or non-financial competing interests to declare.MMP and JMW contributed to the conception, drafting and critical review of the manuscript, and both authors have given their final approval.


Over the last 15 years, considerable work has accumulated to support the role of the CNS in regulating postprandial glucose levels. As discussed in the first section of this review, the CNS receives and integrates information from afferent neurons, circulating hormones, and postprandially generated nutrients to subsequently direct changes in glucose output by the liver and glucose uptake by peripheral tissues. The second major component of this review focuses on the effects of external pressures, including high fat diet and changes to the light:dark cycle on CNS-regulating glucose homeostasis. We also discuss the interaction between these different pressures and how they contribute to the multifaceted mechanisms that we hypothesize contribute to the dysregulation of glucose in type 2 diabetes mellitus (T2DM). We argue that while current peripheral therapies serve to delay the progression of T2DM, generating combined obesity and T2DM therapies targeted at the CNS, the primary site of dysfunction for both diseases, would lead to a more profound impact on the progression of both diseases. Homeostasis refers to the process of maintaining basic physiological functions despite changes in the internal or external environment. This review will focus specifically on glucose homeostasis, which is the ability to maintain steady blood glucose levels (within 90–120 mg/dl) in spite of nutrient ingestion (feeding vs. fasting), stress , or other environmental challenges. Historically, the role of the CNS in regulating glucose has largely focused on the role of the autonomic nervous system (ANS) in defending against falling glucose levels (glucose counter regulation). However, a large body of work has accumulated over the last 15 years supporting the role of the CNS in regulating postprandial glucose levels. To do this, the CNS must sense and integrate information stemming from the activity of afferent neurons, and postprandial increases in circulating hormones and nutrients. The CNS then responds by increasing autonomic efferent activity to direct changes in glucose output by the liver and glucose uptake by peripheral tissues , the ventromedial hypothalamus (VMH), the lateral hypothalamus, and specific neurons within the hindbrain have all been shown to regulate both energy and glucose homeostasis. However, the role of the arcuate nucleus (ARC) on regulating both energy and glucose homeostasis is the most widely studied. Neurons within the ARC that express the anabolic peptides, neuropeptide Y (NPY) and Agouti-related peptide (AgRP), and the catabolic peptide proopiomelanocortin . Genetic inactivation of SOCS3 specifically within steroidogenic factor-1 (SF-1) neurons that are primarily expressed within the VMH, reduces both food intake and energy expenditure leading to no differences in body weight compared to control animals, but also normalizes high fat diet-induced hyperinsulinemia  channels is found in pancreatic β-cells where glucose metabolism generates ATP leading to KATP channel depolarization and eventually insulin exocytosis. Interestingly, neuronal activity in certain neurons within the ARC, VMH, and NTS is also regulated by KATP channel depolarization  and neurons regulate glucose homeostasis.The energy currency for all cells when nutrients are metabolized is adenosine triphosphate (ATP). Some membrane potassium channel activity (opening vs. closing of the channel) is directly regulated by ATP levels  is activated by low ATP levels, and mammalian target of rapamycin (mTOR) is activated by a surplus in ATP. Increases in hypothalamic AMPK drives increases in food intake . One specific isoform is PPARγ, a nuclear transcription factor which functions to direct lipid storage to adipose tissue and away from the liver and skeletal muscle as fat accumulation in these organs would impair insulin sensitivity. Clinically, PPARγ agonists (thiazolidinediones) effectively treat hyperglycemia in T2DM but also cause weight gain. Interestingly, PPARγ is expressed in the CNS, and in rats, both CNS administration of a PPARγ agonist, or viral induction of hypothalamic PPARγ expression  project to the ARC and ventromedial hypothalamic nucleus (VMH). It is hypothesized that glucose levels in the CSF can be detected by the tanycyte and this information is used to maintain glucose levels . 5HT is synthesized in the dorsal raphe nucleus of the midbrain and is released into the hypothalamus, including the ARC  is a gut hormone secreted postprandially from the distal small intestine. Initially studied for its important role in insulin secretion and thus glucose homeostasis, GLP-1 is now known to have a wide array of physiological effects including lipid homeostasis and cardiovascular function Drucker, . GLP-1 iPeripherally secreted GLP-1 likely has limited endocrine action as it has a short circulating half-life due to rapid degradation  is reduced in the ventromedial and dorsomedial hypothalamus in pre-diabetic compared to non-diabetic zucker rats  and encompass a broad range of behavioral and physiological characteristics from the sleep-wake cycle to hormonal fluctuations. Arguably, synching metabolic rhythms to the light:dark cycle is greatly beneficial to an organism. This synchronization allows for optimal energy balance tailored to the organisms’ active state as well as an increase in activity and the recruitment of various meal-related hormones  prior to a meal.Many organisms have evolved to predict daily environmental changes and incorporate 24 h rhythms within their physiology. These daily rhythms are known as circadian , located in the anterior hypothalamus, orchestrates circadian rhythms. The SCN derives “time of day” information from environmental light cues and relays this timing information to other areas of the CNS and periphery as depicted in Figure The PVN is a common nuclei downstream of both the SCN and the ARC. Dissimilar to the ARC however, rhythmic release of GABA from the SCN, rather than α-MSH, onto parasympathetic PVN neurons regulates circadian fluctuations of glucose metabolism  and lead to a lengthening of endogenous circadian period within peripheral tissues (Lamia et al., Furthermore, the SCN may also control glucose metabolism through two-way communication with the ARC. Indeed, the SCN synapses onto both the POMC and NPY/AgRP expressing neurons in the ARC Froy, . The SCNClock mutant mouse, which has a mutation in a core circadian gene, Clock. This mouse displays elevated body weight, fat mass, glucose, leptin, and lipid profiles (Turek et al., Cry1 and Cry2 results in elevated glucose and glucose intolerance (Zhang et al., Cry improves glucose tolerance in otherwise insulin resist and glucose intolerant db/db mice (Zhang et al., An increasing body of literature highlights the link between circadian disruption and disease including cardiovascular disease, Alzheimer’s, cancer, reproductive dysfunction, and important for this review, T2DM (Tenkanen et al., Taken together, the circadian system has a prominent effect on glucose metabolism. Working primarily through the ANS to generate daily rhythms in glucose parameters, the SCN, and circadian rhythms are also integrated to metabolic pathways at the brain, tissue, cellular, and genetic level.It is important to consider that many humans who experience circadian disruption due to shift work or altered sleep patterns, also consume high fat diets. While each of these challenges alone are known to affect glucose metabolism, it is possible that the combined effect of the two challenges are larger and more detrimental to glucose and/or overall health than the sum of each single factor. For example, merely consuming a high fat diet is known to alter the endogenous circadian clock resulting in a lengthened circadian rhythm and a delay in the expression of clock and clock controlled genes (Kohsaka et al., ATP channels and results in an increase in insulin secretion, acts centrally to cause hepatic insulin resistance (Pocai et al., A major limitation to targeting the CNS for treatment of obesity and/or T2DM is that in some signaling systems, CNS action may have opposite downstream metabolic effects. Two T2DM therapeutics have this problem: insulin, and glibenclamide. Although CNS insulin action is catabolic, T2DM patients gain weight as a result of insulin treatment. This likely results from the balance of the anorexigenic action of the hormone within the CNS (Woods et al., Interestingly, bariatric surgery is one form of combined therapy for both obesity and T2DM. Bariatric surgery has long been linked with sustained weight loss and recent research highlights the ability of bariatric surgery to act as an independent treatment of metabolic disease including T2DM (Cummings, However, the costs and invasiveness of bariatric surgeries prevents wide implementation and thus, additional treatment options are necessary. In principle, as circadian disruption has now been extensively linked to metabolic dysfunction, future treatment advances could use combined therapies that target both circadian disruption and obesity. Chronotherapy, which traditionally refers to utilizing the time of administration to optimize treatment, can now refer to targeting the circadian system as a form of treatment. Melatonin, for example, plays a key role in regulating circadian rhythms and also positively effects metabolism by increasing triglyceride clearance (Garaulet and Madrid, Thus, the CNS is clearly important in regulating glucose homeostasis under various physiological challenges including stress, meals, and circadian variations. Further, environmental pressures such as high fat diets and circadian disruptions alter the ability of the CNS orchestrate the peripheral organs to maintain glucose levels. We hypothesize that this contributes to the onset of T2DM. An increased understanding of how glucose metabolism responds to environmental challenges can lead to new treatments. Indeed, strides are currently being made to develop combination treatments that target both the cause and the symptoms of impaired glucose metabolism.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.


A sensory role for ATP was proposed long before general acceptance of its extracellular role. ATP activates and sensitizes signal transmission at multiple sites along the sensory axis, across multiple synapses. P2X and P2Y receptors mediate ATP modulation of sensory pathways and participate in dysregulation, where ATP action directly on primary afferent neurons (PANs), linking receptive field to CNS, has received much attention. Many PANs, especially C-fibers, are activated by ATP, via P2X3-containing trimers. P2X3 knock-out mice and knock-down in rats led to reduced nocifensive activity and visceral reflexes, suggesting that antagonism may offer benefit in sensory disorders. Recently, drug-like P2X3 antagonists, active in a many inflammatory and visceral pain models, have emerged. Significantly, these compounds have no overt CNS action and are inactive versus acute nociception. Selectively targeting ATP sensitization of PANs may lead to therapies that block inappropriate chronic signals at their source, decreasing drivers of peripheral and central wind-up, yet leaving defensive nociceptive and brain functions unperturbed. This article reviews this evidence, focusing on how ATP sensitization of PANs in visceral “hollow” organs primes them to chronic discomfort, irritation and pain (symptoms) as well as exacerbated autonomic reflexes (signs), and how the use of isolated organ-nerve preparations has revealed this mechanism. Urinary and airways systems share many features: dependence on continuous afferent traffic to brainstem centers to coordinate efferent autonomic outflow; loss of descending inhibitory influence in functional and sensory disorders; dependence on ATP in mediating sensory responses to diverse mechanical and chemical stimuli; a mechanistically overlapping array of existing medicines for pathological conditions. These similarities may also play out in terms of future treatment of signs and symptoms, in the potential for benefit of P2X3 antagonists. What is clear is that primary afferents are the first conduit for all sensory information, and thus the primary site that may undergo modulation and plasticity in chronic disease and injury, leading to persistently altered sensation or dysaesthesia , which link sites in the peripheral receptive field to the first synapse of the sensory pathway in the spinal dorsal horn and dorsal brainstem, and in particular how they process signals from hollow organs. PANs have their cells bodies in the dorsal root and cranial ganglia, their peripheral fibers en route to the receptive fields can be short or extremely long, and they exist in several distinctive types, with differential morphological properties, speeds of conduction and molecular markers and receptors. The diversity of sensorineuron types confers a wide range of functions from low threshold (non-nociceptive) proprioceptive, mechanosensitive and thermosensitive detection (mostly the faster fibers), to high threshold (nociceptive) fibers sensitive to noxious mechanical and/or chemical stimuli: some which transmit signals rapidly  systems, our focus is placed on the lower urinary tract (LUT) and airways. These systems share many traits from a morphological, functional and therapeutic perspective (as discussed in more detail below), and are notable in that the most common pathologies in either system are associated with a range of primary symptoms and signs that include persistent and heightened, inappropriate irritative sensations and exaggerated autonomic reflexes, as depicted in Figure Five decades ago it was reported that fluid from lysed red blood cells applied to exposed blister bases on the human forearm evoked pain and discomfort  injection into the paw of rat evokes nocifensive responses  attenuates pressure thresholds , and this has been widely studied as a model of bladder irritability  and signaling to adjacent afferent nerve endings. In this latter respect, it is well established that copious release of ATP accompanies such environmental changes, and this ATP communicates with the afferents via P2X3 receptor activation.δ, convey much of the ongoing information to the CNS, where primary coordination is modulated at the pontine level. Efferent excitatory function to support voiding is largely carried by pelvic nerve parasympathetic fibers, with sympathetic innervation of the bladder neck and urethra providing the autonomic support during the long periods of continence, coupled with suspension of parasympathetic drive and maybe some sympathetic activity maintaining detrusor muscle compliance during filling. A large proportion of C-fibers and some Aδ-fibers from the LUT express P2X3 subunits and respond to ATP.On a simplified level, the wiring control of these organ systems also exhibits similarities. In the LUT, sensory afferents of the pelvic, pudendal and splanchnic (hypogastric) nerves, with the unmyelinated C-fibers in significant numerical predominance over AThe upper and lower airways are densely innervated by sensory and autonomic fibers. Although a small percentage of fibers originate from dorsal root ganglia (DRG), most afferent fibers are carried by the vagus nerve, with cell bodies contained in the nodose  and jugular  cranial ganglia , in turn under descending cortical control  in the bladder wall and conveys this information to the pontine centers and beyond, to inform conscious and graded perception  and prepare consciously integrated autonomic reflex coordination. The second system, perhaps a more primitive one, engages unmyelinated C-fibers and detects filling at a broader range of volume thresholds, as well as signals of local distress , and can elicit local  initiation of efferent autonomic responses that lead to increased detrusor activity. de Groat (δ) pathway to ascertain bladder status and desire to void, with C-fiber signals failing to escape tonic suppression except when local pathological conditions arise (such as in infection). The “reflex bladder”  to pontine control centers : urgency, frequency, nocturia, incontinence, discomfort and even pain are mediated by the unsilencing of these C-fiber pathways and emergence of the reflex bladder. Thus, an optimal approach would be to target selectively these afferents for suppression, to provide relief for countless numbers of LUTS sufferers with conditions such as OAB, benign prostatic hyperplasia (BPH), BPS and chronic pelvic pain syndrome (CPPS): all representing forms of pelvic hypersensitivity, typified by inappropriate urgent sensations at modest filling that trigger unwelcome efferent responses (hyperreflexia). Ideally, an understanding of what substances  cause the unsilencing of the reflex bladder would be additionally helpful.As was described for the LUT, the airways depend upon the coordination of afferent inputs to central (in this case medullary) nuclei to regulate the critical and extensively rhythmic efferent autonomic influences upon upper and lower respiratory tissues. Given the functional complexity of airways and the second by second need to modulate and tune functions, exquisite surveillance of the tissues (and the blood gases) and timely modulation of efferent responses are all the more critical to homeostatic wellbeing, and thus the complexity of afferent inputs is amplified.What is clear is that the airways need to respond appropriately to subjectively perceived irritations  as well as the perception of “air hunger”. Likewise, they must respond to afferent signals with well-regulated autonomic reflexes that may variously govern airways caliber and conductance (bronchoconstriction and dilation), fluid and mucus secretion and ciliary clearance. As described for the LUT, very common airways pathologies arise that are associated with poorly coordinated afferent-efferent communication, inappropriate sensations (hypertussive perceptions and dyspnea) and markedly reduced sensory thresholds that lead to maladaptive reflexes (airways hyperreactivity and hypersecretion). Variously, these signs and symptoms are common in chronic cough, asthma, COPD and interstitial disease, with hypersensitivity and hyperreflexia being key manifestations of widespread diseases. Although these diseases may have etiological bases in immune dysregulation and chemical exposure, it remains clear that current therapeutics offer merely patchy resolution of some but not all signs and symptoms. Though the underlying causes may largely drive changes through inflammatory pathways and factors, it remains clear that afferent targets in the respiratory system could offer important opportunities for therapeutic intervention.An approach that has been employed to impressive effect in the study of both LUT and airways to elucidate the origination and composition of afferent nerve responses to mechanical and chemical stimulation signals is the intact “tissue-nerve” preparation from rodents. The bladder-nerve preparations , Namasivayam et al.  were theIn the airways, focus on ATP as a sensitizing autacoid or transmitter has been limited, indeed more focus has been placed on its potential roles in inflammation, ciliary motility and mucus clearance  and continence failure.β agonists . The measure of clinical effectiveness of such agents within these systems is difficult to compare; however, the accessibility of the airways for inhalation therapy allows local delivery of drug concentrations imparting more meaningful clinical response than seems to have been achieved so far in LUT, where intravesical delivery is much less convenient or well-tolerated. Currently, systemic adverse events (AEs) greatly impair dosing to therapeutic effectiveness and frequently discourage LUTS patients from persisting with treatment. Whether this latter situation is improved with the new β3 adrenoceptor agonist will be followed closely.In the face of this significant sensory plight, the pulmonary and urological patients both have access to two mechanistic classes in common that aim to reduce the parasympathetic excitation of smooth muscles and improve compliance: antimuscarinics  and Beyond these classes, airways patients also derive significant benefit from the local delivery of corticosteroids which go some way to reduce the inflammation and severity of some of the common symptoms (and indeed may even blunt afferent discharge). Such an option has been examined in the LUT, but here local approaches are not so feasible or convenient, and safety concerns with chronic systemic exposure would preclude routine steroid usage even if some benefit might be afforded . In both systems, direct pharmacological targeting of sensory targets has been of limited value so far, except in some exceptional situations such as using inhalation delivery of local anesthetics  for intractable dyspnea in cardiac patients or their intravesical instillation for pain in BPS.β-adrenoceptors receptors that are present (though in neither case likely well innervated). Overall, these approaches offer benefit but are potentially undermined by on-target AEs . Clearly, there is a great need for something directed at quelling the sensory pandemonium.Thus, the current therapeutic options for patients whose lives are seriously impacted by abundant sensory symptoms from both organ systems do not directly target the C-fibers and their triggered reflexes; rather they focus on the efferent limb on the reflex: block the parasympathetic drive to smooth muscle excitability, or increase detrusor compliance by activating 50) of AF-219 has been reported as ∼30 nM versus recombinant hP2X3 homotrimers and 100–250 nM at hP2X2/3 heterotrimeric receptors, potencies very similar to those reported for recombinant rat receptors, and it displays no inhibitory impact on any non-P2X3 subunit containing receptors . Reports from other related chemical members of this P2X3 selective pyrimidinediamine class have shown that the mechanism of inhibition is non-competitive  and have been mixed regarding species-independency of P2X3 receptor potency estimates: AF-353  whereas the more potent analog AF-792  was shown to be less potent at human versus rat P2X3 receptors in one report  as the IC50 is a parameter that will change with agonist concentration used and depends on agonist potency at the different trimers.So far, despite the efforts of a several pharmaceutical organizations , and comparably reduced patients subjective cough related symptoms.To date, AF-219 has completed four Phase 1  and one Phase 2 (patient) studies, with 3 additional Phase 2 studies in progress.This was a relatively small pilot study, with a single, high daily dose level compared with placebo in patients with considerable cough burden. Nevertheless, the unprecedented magnitude of efficacy observed and its objective nature, coupled with the apparent absence of benefit on placebo, would appear to offer strong initial validation of the P2X3 target in a patient group with a significant “hollow-organ” sensory disorder, in keeping with the potential identified from preclinical in vitro and organ-nerve preparations. These findings also augur well for other signs and symptoms of airways disease that are impacted by afferent hyperexcitability, as well as important and bothersome conditions emanating from other tube and sac systems, including LUT. We await their outcomes with anticipation and excitement.Anthony Ford is an employee of Afferent Pharmaceuticals; Brad Undem is a member of the SAB of Afferent Pharmaceuticals.


A-mediated transmission and disturbances in the glutamatergic, noradrenergic and cholinergic systems, among others, has also been demonstrated. DS cognitive dysfunction caused by neurodevelopmental alterations is worsened in later life stages by neurodegenerative processes. A number of pharmacological therapies have been shown to partially restore morphological anomalies concomitantly with cognition in these mice. In conclusion, the use of mouse models is enormously effective in the study of the neurobiological substrates of mental disabilities in DS and in the testing of therapies that rescue these alterations. These studies provide the basis for developing clinical trials in DS individuals and sustain the hope that some of these drugs will be useful in rescuing mental disabilities in DS individuals.Down syndrome (DS) is the most common genetic cause of mental disability. Based on the homology of Hsa21 and the murine chromosomes Mmu16, Mmu17 and Mmu10, several mouse models of DS have been developed. The most commonly used model, the Ts65Dn mouse, has been widely used to investigate the neural mechanisms underlying the mental disabilities seen in DS individuals. A wide array of neuromorphological alterations appears to compromise cognitive performance in trisomic mice. Enhanced inhibition due to alterations in GABA Trisomy 21, or Down syndrome (DS), is the most common genetic cause of intellectual disability. It affects 1 in 850–1000 infants , linguisIn recent years, the question of how trisomy of Hsa21 leads to this set of phenotypes has been a matter of debate. Two hypotheses have been proposed to account for this phenomenon: the “amplified developmental instability” hypothesis  and the The analysis of DS cases resulting from partial trisomies of Hsa21 and the development of a number of mouse models of this condition have provided insight on the causative role of dosage-sensitive genes on DS phenotypes. These studies have yielded evidence that support both theories; while the role of single dosage-sensitive genes on different phenotypes has been confirmed, research has also demonstrated that many of these DS features are due to the complex effects of multiple Hsa21 genes (see ) and theconstruct, face, and predictive validity [Construct validity relates to the similarity between the etiology of the human and the animal disorder . Face validity refers to how well the model mimics the molecular, cellular, physiological, and behavioral phenotypes observed in humans. Predictive validity requires that the new knowledge obtained in the animal model makes accurate predictions of what will be found in the human condition. This validity is particularly important for unraveling the neurobiological causes of the cognitive deficits found in DS that cannot be assessed in humans for ethical or practical reasons and for developing and testing new therapies.To identify the biological mechanisms underlying different pathologies and to evaluate the efficacy of novel therapies, thousands of animal models of human disorders have been developed. For an animal model of a human disorder to be considered valid, it has to satisfy three criteria: validity , 15. ConIn the following sections, we will summarize (1) the similarities between the genetic overdose of various mouse models of DS and human trisomy 21; (2) the concordance between the behavioral, neuromorphological, and neurochemical phenotypes of DS mouse models and the human condition; (3) the knowledge obtained in these animals about the neurobiology of DS that have yielded the development and analysis of several therapeutic strategies that could potentially be used to attenuate cognitive impairments in DS individuals.The long arm of Hsa21 contains approximately 552 genes, 166 of which are orthologous to genes localized in syntenic regions of three mouse chromosomes: Mmu16 (110 orthologous genes), Mmu17 (19 orthologous genes), and Mmu10 (37 orthologous genes) [The next approach adopted was the generation of mouse models with partial trisomies of sets of Mmu16 genes orthologous to those found in Hsa21. In 1993, Davisson et al.  created The Ts2Cje model carries the same segment of Mmu16 triplicated in the Ts65Dn mouse but is translocated to chromosome 12 . AlthougSeveral other segmental trisomic models of different segments of Mmu16, 17, and 10 have been created. In the late 90s, Sago and coworkers generated two mouse models with the triplication of two different regions of Mmu16: the Ts1Cje mouse, which presents a trisomy of 81 genes localized in the region of Mmu16 that extend from Sod1 to Znf295 ; the Ms1To model the trisomy of Hsa21 orthologous genes located in Mmu17, two mouse models have been created: the Ts1Yah mouse, trisomic for 12 genes in the Mmu17 region, syntenic to the subtelomeric region of Hsa21  and the The last segmental trisomic mouse generated is a mouse that models the trisomy of Hsa21 orthologous genes located in Mmu10. The Dp(10)1Yey mouse is trisomic for the region of Mmu10 syntenic to the distal part of Hsa21 containing 37 orthologous genes , 27.After the tree partial trisomic models for all the Hsa21 syntenic regions on Mmu10 (Dp(10)1Yey/+), Mmu16 (Dp(16)1Yey/+) and Mmu17 (Dep(17)1Yey/+) were established, Yu et al.  cross-brThe Tc1 model is a mouse in which the entire human Hsa21 has been triplicated . This moFinally, to study the role of particular genes in the DS phenotypes, a number of mouse models with the triplication of single genes and trisomic mice in which the expression of only one of the genes triplicated in DS have been normalized have been created (see ).This section describes the similarities between the cognitive and behavioral disturbances found in various mouse models of DS compared to the human condition .Motor dysfunction is a hallmark of DS. Hypotonia, hyporeflexia, reduced muscular strength, disturbances in striate muscle control, and delays in the acquisition of fine and gross motor skills are found in DS individuals from early childhood –35.Ts65Dn mice are not impaired in sensorimotor abilities such as forelimb strength, postural skills, equilibrium, and climbing , 37. HowSimilar to DS , Ts65Dn Ts1Cje and Ms1Ts65 mice display poor performance in hippocampal-dependent tasks such as the T-maze  and the Regarding the two mouse models trisomic for segments of Mmu17, Ts1Yah mice are impaired in the novel object recognition and Y-maze test, but their performance in the Morris water maze is enhanced ; howeverThe Dp(10)1Yey/+Dp(16)1Yey/+; Dep(17)1Yey/+ mouse is impaired in the Morris water maze and in the contextual fear conditioning test . FinallyIn summary, most of the above-mentioned mouse models show characteristic DS cognitive and behavioral phenotypes, although they differ in the degree of impairment.Several mechanisms have been proposed to be the neurobiological correlates of intellectual disability in DS, including altered neurogenesis, hypocellularity, altered synaptic development, increased inhibition, and neurodegeneration. In DS individuals, the volume of the brain is reduced, beginning at early developmental stages –62; in aThe brain volume of Ts65Dn, Ts1Cje, and Ts2Cje mice is also reduced during the embryonic period, but not after birth or during adulthood , 66–68. A number of studies demonstrated that brain areas are dissimilarly affected. Reduced volumes of the hippocampus, entorhinal, frontal, prefrontal, and temporal cortices, amygdala, cerebellum, brain stem nuclei, and mammillary bodies of the hypothalamus have been reported in children and adults with DS , 70–78.Consistently, size and anatomical alterations have been found in some brain regions of trisomic mice. The hippocampus and the cerebellum appear to be the most affected structures. Spatial learning is known to depend on the functional integrity of the hippocampus, a structure that plays a key role in information encoding and retrieving in the CNS , 80. In In DS individuals, cell density is not compromised in early gestation , 62, 84,Ts65Dn mice show reduced cell density during prenatal (E 18.5) and early postnatal stages in the neocortex (P8) . At 1 moThe cerebellum has been traditionally implicated in motor coordination, but there is increasing evidence for the role of this structure in higher cognitive processes, including attention, cognitive flexibility, and memory . ConsistAs expected from the reduced cerebellar volume of DS and trisomic mice, the cerebellum of Ts65Dn mice has a lower number of granule and Purkinje cells from early postnatal stages to adulthood , 93, 94.NGFR, a neurotrophin receptor localized in basal forebrain cholinergic neurons, immunoreactivity. Furthermore, there is a decrease in the size of these cholinergic neurons cell bodies [One of the anatomical substrates of learning and attention is the septohippocampal cholinergic system , 96. In l bodies , 99–102.In conclusion, persistent hypocellularity is one cause of intellectual disability in the trisomic condition. The following sections will describe the evidence showing that these neuroanatomical alterations could be caused by impaired cell proliferation, increased apoptosis and/or neurodegeneration.Neurogenesis is severely compromised in DS from early developmental stages. Impaired neuronal precursor proliferation, slowing of the cell cycle, and altered differentiation are thought to account for altered neurogenesis.In DS fetuses, a reduced number of dividing cells is found in the dentate gyrus (DG) and lateral ventricle , 89. RedReductions in neural progenitor cells and neuroblasts, leading to altered neurogenesis, in the embryonic neocortex and subventricular zone (SVZ) of Ts1Cje and Ts2Cje mice have also been reported , 105.It has been proposed that the extra copy of Hsa21 in DS delays the mitotic cell cycle of neuronal precursors, thereby affecting cell proliferation. Accordingly, a slowing of the cell cycle in different trisomic conditions has been demonstrated. In Ts65Dn mice, the cell cycle is extended during embryonic stages in CA3  and in eImpaired differentiation also appears to contribute to the smaller number of neurons in DS brains , 107. TsAdult hippocampal neurogenesis has been demonstrated in many species, including rodents –111. DurIn Ts65Dn mice, cell proliferation in the SVZ is reduced from birth to adulthood , 115. InCerebellar neurogenesis is also affected by trisomy. DS fetuses show reduced neurogenesis in the external granular layer (EGL) of the cerebellum and in the VZ . NewbornIt can be concluded that neurogenesis impairment, due to reductions in neural precursors, cell cycle timing and differentiation, is a hallmark of trisomic conditions from prenatal to adult stages. This altered proliferation is likely to be one of the mechanisms responsible for the widespread hypocellularity leading to altered synaptogenesis, connectivity, synaptic plasticity, and cognitive disabilities.A number of trisomic genes in DS have been proposed to play a role in the proliferation impairment found in this condition. One of the genes overexpressed in the DS brain is DYRK1A -phosphorylation regulated kinase 1A), an orthologous gene to the Drosophila gene minibrain . DYRK1A The Olig1 and Olig2 genes are also overexpressed in DS individuals. These genes encode transcription factors that have been implicated in neurogenesis and oligodendrogenesis –133. ChaThe APP gene is triplicated in DS and in most DS mouse models, and it is thought to play a role in different DS phenotypes, such as the development of AD pathology. It has been proposed that the APP gene may also be involved in the altered neurogenesis characteristic of the trisomic condition. Trazzi et al.  have recThe implication of adult neurogenesis defects in DS-related cognitive impairments suggests that therapies targeted to rescue neurogenesis may be of value in treating intellectual disability in DS individuals.The selective serotonin reuptake inhibitor fluoxetine is an antidepressant that has been shown to increase neurogenesis in the mouse DG and SVZ , 136. ChAnother drug that markedly increases neurogenesis in the DG of adult normal mice is lithium, a drug prescribed for the treatment of bipolar depression . LithiumAs mentioned above, the Shh pathway plays a key role in granule precursor cell (GPC) proliferation. Drugs targeting this pathway rescue neurogenesis alterations. Treatment of Ts65Dn mice with an activator of the Shh pathway, SAG 1.1, increased mitosis, restored cerebellar granule cell precursor populations , and resActive care programs are one of the most successful therapeutic interventions used in DS individuals. In rodents, environmental enrichment has been associated with morphological, physiological, and cognitive improvements. These changes include increases in cortical weight and thickness, hippocampal neurogenesis, dendritic branching, length, number of dendritic spines and size of dendritic spines –140, facExposure of Ts65Dn mice to environmental enrichment for 7 weeks modulated spatial memory in a sex-dependent manner . EnvironChakrabarti et al.  have recVoluntary exercise is beneficial for cognition in both normal rodents and mouse models of altered cognition , 147–149Apoptosis or programmed cell death is physiologically involved in nervous system development and aging. It has been proposed that the hypocellularity found in DS brains could also be due to increased cell death. However, thus far, studies on the apoptotic processes in the trisomic condition have led to contradictory results. Some groups have reported increases in the number of apoptotic cells in DS brains , 151 andAltered synaptic plasticity is an additional mechanism that may underlie intellectual disability in DS individuals. Synaptic plasticity includes alterations in the number and the functional characteristics of synapses, which are mostly localized to dendrites and dendritic spines , 160.Numerous studies have demonstrated impaired dendritic morphology in trisomic conditions. Although normal or even increased dendritic branching has been reported in DS fetuses and neonates –163, in Mouse models also resemble the dendritic pathology of DS. In adult Ts65Dn mice, neocortical pyramidal neurons show a reduction in the length and arborization of dendrites and in the density of spines . Spine dThese anomalies in dendritic tree arborization, spine density, and morphology lead to reduced density of synapses and compromised synaptic function in DS individuals. Several groups have tested the value of various therapeutic strategies for rescuing dendritic pathologies.Based on the observation that fluoxetine favors dendritic development in normal animals , BianchiThe enhancement of dendritic arborization and spine density has been firmly established as one of the positive effects of environmental enrichment , 176. BeAs predicted by the reduced length and number of branches of dendrites and density of spines, the trisomic condition is characterized by a reduced number of synaptic contacts and alterations in synaptic plasticity. Ts65Dn mice show a reduction in synaptic density in the neocortex and CA1 at P9  and in tNot only the number and characteristics of synapses but also the relative distribution of different types of synapses is altered in trisomic mice, leaving the balance between excitatory and inhibitory synapses shifted toward increased inhibition in the trisomic brain. Ts65Dn mice have less excitatory (asymmetric) synapses in the temporal cortex, DG, CA1, and CA3 , 177, anOverall, these morphological and functional disturbances compromise the physiological properties of synapses, possibly leading to cognitive impairments in DS and trisomic mice.DS individuals present small electroencephalographic (EEG) abnormalities. EEG alpha activity is relatively preserved in young individuals with DS, but older patients with dementia present abnormal activity . EEG cohIn mouse models of DS, altered synaptic plasticity in the hippocampus has been consistently reported. Hippocampal long-term potentiation (LTP) is considered to be the electrophysiological substrate of learning. Ts65Dn mice display reduced LTP in the hippocampal CA1 and DG regions , 182–186It has been proposed that altered synaptic plasticity in the hippocampus of DS mouse models results from increased inhibition due to unbalanced excitatory and inhibitory neurotransmission , 187. ReA receptors [A antagonist picrotoxin rescued the reduction in LTP induced by theta-burst stimulation (TBS) in these mice [A and GABAB receptor-mediated components of evoked inhibitory postsynaptic currents (IPSCs) were significantly higher in Ts65Dn mice, suggesting an increase in presynaptic release of GABA. Thus, both GABAA and GABAB receptors are implicated in the reduced synaptic efficiency found in the DG of Ts65Dn mice.In addition, overinhibition in the hippocampus of Ts65Dn mice has also been shown to be dependent on GABAeceptors  because ese mice . Furtherese mice  have shoB receptor-dependent slow inhibitory postsynaptic potential in hippocampal neurons [The G-protein-activated inwardly rectifying potassium channel 2 (Girk2) gene is overexpressed in DS individuals. Girk channels generate a GABA neurons . It has  neurons .Because overinhibition in the trisomic brain appears to underlie LTP impairments and, therefore, alter learning and memory processes, a number of studies have tested drugs that reduce GABA-mediated inhibition in an attempt to rescue the electrophysiological substrates of cognition.A receptor system plays an important role in cognition. Nonselective positive modulators of the GABAA receptor disrupt learning and memory processes [A antagonists picrotoxin (PTX), bilobalide (BB), or pentylenetetrazole (PTZ) restored LTP and cognition in the object recognition test in these mice [It is well established that the GABArocesses –194, whirocesses –197. Redese mice . Rueda eese mice  confirmeA negative modulators cannot be safely used to improve cognition due to their anxiogenic and proconvulsant effects [A receptor subtypes, GABAA  α5 subunit-containing receptors are known to facilitate cognition in hippocampal-dependent tasks [A  α5 negative allosteric modulators, also called inverse agonists, have cognition-enhancing effects without anxiogenic or proconvulsant side effects [A  α5 inverse agonist, α5IA, has been shown to rescue learning and memory deficits in TS mice without inducing anxiogenic and convulsant side effects [However, non-selective GABA effects . Among tnt tasks , 201. Mo effects –204. A f effects .Further support for the efficacy of reducing GABA-mediated overinhibition to improve cognition in trisomic mice comes from a recent report that demonstrated that environmental enrichment reduced the release of GABA in the hippocampus and visual cortex of Ts65Dn mice while rescuing spatial learning and hippocampal LTP .Alterations in several neurotransmitters and changes in the expression and function of their receptors, in both DS individuals and mouse models of this condition have been demonstrated. These impairments may be responsible for other phenotypes found in trisomic conditions, such as defects in neurogenesis, synaptic transmission, and cognition. Dopamine, taurine, and histamine levels have been shown to be altered in the brains of DS fetuses and adults , 207–209GABA is reduced in DS fetuses . Howeverα2 and downregulation of the α5 and β3 subunits of the GABAA receptor have been reported [β2 and β3 subunits of the GABAA receptor were found [A  α1 receptor expression [B receptor have also been reported in Ts65Dn mice [A or GABAB receptor subunits by western blot analysis.A number of alterations have been reported in the expression of various GABA receptor subunits. In neurospheres from fetuses with DS, upregulation of the reported . In the re found . Brain spression . Changes5Dn mice . However5Dn mice  did not A activity is known to regulate neuronal proliferation, migration, differentiation, and integration of newly generated neurons [A-mediated inhibition shown by Ts65Dn mice could, therefore, be implicated in the alterations in neuronal proliferation and survival found in these mice.GABA neurons –214. TheIncreased inhibition in the trisomic condition is also caused by alterations in excitatory transmission. Although similar levels of glutamate  are founAs detailed above, altered hippocampal LTP in trisomic mice suggests disturbances in NMDA receptor signaling. In Ts65Dn mice, a reduction of the GluR1 subunit of the AMPA receptor  and of tTs65Dn and Ts1Cje mice exhibit hypersensitivity to the locomotor stimulatory effect of MK-801, an NMDA receptor channel blocker .Alterations in the signaling mechanisms downstream of the NMDA receptor have also been reported; the hippocampi of Ts65Dn mice show disturbances in the calcium/calmodulin-dependent protein kinase II (CaMKII), phosphatidylinositol 3-kinase (PIP3K)/Akt, extracellular signal-regulated kinase (ERK), protein kinase A (PKA), and protein kinase C (PKC), all of which have been shown to be involved in synaptic plasticity .One of the targets of the NMDA receptor is the protein phosphatase calcineurin (CaN). The DSCR1 gene encodes a protein that inhibits CaN, and this gene is overexpressed in the Ts65Dn brain . The inhIn spite of the rescue induced by memantine of several DS-relevant phenotypes in the Ts65Dn mouse, a recent randomized double-blind clinical trial failed to find any benefit of memantine administration for 52 weeks on cognitive impairment and dementia in DS individuals over 40 years of age .Deficits in serotonin (5-HT) have been reported in the frontal cortex of DS fetuses  and in a1A receptor has also been implicated in the regulation of neurogenesis [1A receptor have been reported in the DS brain at birth [1A receptor expression may underlie the defective neurogenesis found in Ts65Dn mice [1A reuptake inhibitor fluoxetine rescued the expression levels of this receptor in Ts65Dn mice, suggesting that this effect may underlie the rescue of proliferation produced by this drug, as previously mentioned.The 5-HTogenesis , 227. Reat birth , in hippat birth . Thus, r5Dn mice . MoreoveOne of the anatomical substrates of learning and attention is the septohippocampal cholinergic system , 96. A nAlthough a normal number of cholinergic neurons is found in young Ts65Dn mice, basal forebrain cholinergic neurons (BFCNs) degenerate with age in these mice –102. HowThe levels of noradrenaline (NA) are normal in DS fetuses  but are β-adrenoceptors in the cortex and hippocampus; however, their function is altered. Basal production of cyclic AMP in the hippocampus of TS mice was impaired. In addition, the responses of adenylyl cyclase to the stimulation of β-adrenoceptors with isoprenaline and of the catalytic subunit with forskolin were both severely depressed [Ts65Dn mice show unchanged numbers of epressed , 234. Agepressed .A antagonists and NA-enhancing drugs improve learning in Ts65Dn mice.NA has been shown to play a role in neurogenesis, as neuron proliferation is enhanced or impaired following increases or reductions in NA transmission, respectively . TherefoThe role of neurotrophins (NT) in neuronal survival, differentiation, migration, and synaptic plasticity is well documented –240. ConThe reduced expression of BDNF has been observed in the hippocampus of DS fetuses , and theNT-3 is increased in the hippocampus of newborn and adult Ts65Dn mice , potentiNerve growth factor (NGF) is generated in the hippocampus and retrogradely transported to the soma of BFCNs . NGF levPeptide 6, an active region of ciliary neurotrophic factor (CNTF), modulates the CNTF pathway by inhibiting the antineurogenic activity of leukemia inhibitory factor, thereby increasing neurogenesis . AdminisConsidering the role of Dyrk1A in neuronal progenitor proliferation, neurogenesis, and neurodifferentiation, it has been suggested that molecules targeting this gene could provide therapeutic benefits to DS phenotypes. Epigallocatechin gallate (EGCG), an antioxidant extracted from green tea, is an inhibitor of the protein kinase DYRK1A .The chronic administration of EGCG from conception to adulthood rescued BDNF levels in the hippocampus of Dyrk1a transgenic mice . ConcomiFinally, Fukuda et al.  have recAlthough neurodevelopmental alterations occurring from early embryonic stages are likely to cause intellectual disability, there are a number of neurodegenerative mechanisms in DS that complicate this scenario. Atrophy of a number of structures, including the hippocampus, amygdala , 252, thDS and AD brains are characterized by activated microglia, and increased levels of proinflammatory cytokines that lead to neuroinflammation are likely involved in neurodegeneration , 254. ThMinocycline is a semisynthetic tetracycline that inhibits neuronal death and reduces inflammatory activity by blocking inflammatory mediators . The chrβ peptide [Vasoactive intestinal peptide (VIP) is neuroprotective, as it promotes the release of several survival factors from astrocytes and regulates neuropeptide release from glial cells, including activity-dependent neuroprotective protein (ADNP) and activity-dependent neurotrophic factor (ADNF) . The act peptide , 258.A receptor subunit β3 [In cultures of DS cortical neurons, treatment with SAL or NAP increases neuronal survival . In Ts65bunit β3 . Furtherbunit β3 .In DS individuals and in the Ts65Dn mouse, there is an overexpression of SOD1, the gene responsible for the formation of superoxide dismutase, an enzyme that modifies oxygen free radicals into hydrogen peroxide. The overproduction of hydrogen peroxide leads to the overproduction of highly reactive oxygen free radicals, which damage cell membranes, including the mitochondrial membrane, and deteriorate lipids, proteins, and mitochondrial DNA. This set of alterations is called oxidative stress. Evidence for increased mitochondrial superoxide production in DS individuals has been repeatedly demonstrated , 262. ThIn an attempt to reduce oxidative stress-induced neurodegeneration, several groups have tested the efficacy of various antioxidants to reduce the altered phenotypes in Ts65Dn mice. SGS-111, an analogue of the nootropic piracetam, has been shown to increase neuronal survival and prevent the accumulation of intracellular free radicals, peroxidative damage, and the development of neurodegenerative changes in both normal and DS cultured neurons . Howeverα-tocopherol, ascorbic acid, and the mitochondrial cofactor: α-lipoic acid) did not produce any improvement in cognitive functioning nor a stabilization of cognitive decline in adults with DS [A recent report  revealed with DS .Because estrogens maintain the function of basal forebrain cholinergic neurons, it has been proposed that the administration of estrogens may be useful in reducing the loss of these neurons in AD and DS individuals .The chronic administration of estrogens to aged female Ts65Dn mice enhanced cognition, increased the size and number of cholinergic neurons, increased the levels of NGF in the medial septum , restoreβ-amyloid, which is thought to be responsible for the amyloid plaque pathology and degeneration of BFCNs found in 100% of DS individuals over 40 years of age. Ts65Dn mice also show age-related elevations in the levels of the APP protein [β-amyloid peptide [One of the genes triplicated in the trisomic condition is APP. In DS individuals, the increased expression of this gene leads to the increased production of  protein  and the  peptide  in the c peptide . Thus, tβ-amyloid reductions on the Ts65Dn mice altered phenotypes, Netzer et al. [β-amyloid levels and rescued spatial learning in these mice. Because β-amyloid is a regulator of the glutamatergic system, the authors proposed that the cognitive enhancing effects of DAPT could be mediated by an enhancement and/or a regulation of excitatory synaptic transmission.To test the effect of r et al.  administGiven the role of the cholinergic system in cognition and the degeneration of this system in AD and DS individuals, it has been proposed that pharmacological enhancements of this system could help diminish cognitive deterioration in these conditions. Donepezil is an acetylcholinesterase inhibitor that is widely prescribed to enhance cholinergic transmission in the treatment of AD dementia. However, the chronic administration of donepezil did not improve learning and memory in Ts65Dn mice . SimilarPiracetam is a drug that shows cognitive-enhancing effects in patients with a number of cognitive disorders and dementia  and in sThe first partial trisomic models, the Ts65Dn and Ts1Cje models, demonstrated that DS phenotypes could be recapitulated in mice. More recently, knockout and transgenic mice for individual genes and new animals that are trisomic for different regions of orthologues of Hsa21 regions are helping to identify dosage-sensitive genes involved in DS phenotypes. Although some of these triplicated genes may play a role individually, it appears that DS phenotypes arise from the complex effects of groups of Hsa21 genes.In the last 20 years, the characterization of these animal models of DS, particularly the Ts65Dn mouse, has been enormously useful to understand of the neurobiological basis of intellectual disability. Several mechanisms have been proposed to underlie this altered cognition, including impaired neurogenesis leading to hypocellularity in the cortex, hippocampus, and cerebellum, altered dendritic morphology, altered synapses, increased inhibition and neurodegeneration. The new knowledge of the pathogenic mechanisms in DS individuals has been applied to the development of new pharmacotherapies. Several drugs have been shown to rescue neurogenesis, hypocellularity, electrophysiological deficits, and cognitive alterations in the Ts65Dn mouse. These studies provide the basis for developing clinical trials in DS individuals and sustain the hope that some of these drugs will be useful in rescuing intellectual disability in DS individuals.


Proteobacteria dominated in all shallow and deep waters, with Alphaproteobacteria and Gammaproteobacteria the two most abundant components, and SAR11 the most abundant group at family level in all regions. Cyanobacteria occurred mainly in the surface euphotic layer, and the majority of them in the tropical waters belonged to the GpIIa family including Prochlorococcus and Synechococcus, whilst those associated with Cryptophytes and diatoms were common in the subarctic waters. In general, species richness (Chao1) and diversity (Shannon index H′) were higher for the bacterial communities in the intermediate water layers than for those in surface and deep waters. Both NMDS plot and UPGMA clustering demonstrated that bacterial community composition in the deep waters (500 m ∼2000 m) of the three oceanic regions shared a high similarity and were distinct from those in the upper waters (5 m ∼100 m). Our study indicates that bacterial community composition in the DOC-poor deep water in both tropical and subarctic regions were rather stable, contrasting to those in the surface water layers, which could be strongly affected by the fluctuations of environmental factors.Community composition of Bacteria in the surface and deep water layers were examined at three oceanic sites in the Pacific Ocean separated by great distance, i.e., the South China Sea (SCS) in the western tropical Pacific, the Costa Rica Dome (CRD) in the eastern tropical Pacific and the western subarctic North Pacific (SNP), using high throughput DNA pyrosequencing of the 16S rRNA gene. Bioinformatic analysis rendered a total of 143600 high quality sequences with an average 11967 sequences per sample and mean read length of 449 bp. Phylogenetic analysis showed that  Bacteria are important components of marine ecosystems and play a critical role in the biogeochemical cycles Methodological constraints have limited the progress in the field of microbial diversity research, especially for microbial groups with high diversity but low abundance. Recently, the application of next generation sequencing technology, such as 454 pyrosequencing, has allowed us sequencing reads orders of magnitude greater than conventional sequencing approaches Pyrosequencing has been applied to investigate different microbial communities in various marine ecosystems, such as the North Pacific Ocean In our study, water samples were collected from three geographically separated oceanic sites located in both tropical and subarctic North Pacific Ocean, including the western tropical North Pacific characterized by permanent stratification and oligotrophic conditions, the eastern tropical North Pacific featured an shallow thermocline depth caused by upwelling and a permanent oxygen deficient zone between 400 and 700 m, and a subarctic site located in the Oyashio region where a spring phytoplankton bloom occurs annually. In each region, the microbial communities from four different depths covering from surface photic zone to deep water, along the steep gradients of light, temperature, pressure, macronutrient and trace metal concentrations, were examined by high throughput pyrosequencing based on 16S rRNA gene. The objectives of this study are to investigate the vertical profiles of microbial communities across the broad depth scale and to compare the microbial community structures in the tropical and subarctic North Pacific Ocean.Dongfanghong#2 in August 2007, 2) the subarctic northwest Pacific Ocean (SNP)  during an R/V Taisei Maru (JAMSTEC) cruise in May 2011 when a spring phytoplankton bloom occurred, and 3) the Costa Rica Dome (CRD) in eastern tropical Pacific  during the FLUZiE cruise in July 2010. No specific permissions were required for sampling in these locations, and our study did not involve endangered or protected species. Water from four different depths , was collected using a CTD carousel water sampler with Go-Flo or X-Niskin bottles . Between 1 and 2.4 liters of seawater were filtered onto 0.22 µm pore-size polycarbonate membranes  on board. The membranes were stored at −80°C until DNA extraction on land.Seawater samples were collected from 3 separate sites in tropical and subarctic North Pacific Ocean: 1) the Southeast Asia Time-Series Study station (SEATS) , which is located at the deep central basin of the South China Sea (SCS), during the GOE-2 cruise on board the research vessel Total genomic DNAs were recovered from biomass collected with the 0.22 µm filters following the instruction of PureLink™ Genomic DNA Mini Kit (Invitrogen). Extracted DNAs were precipitated with isopropanol and then stored at −80°C.The environmental DNAs were amplified with universal bacterial primers 16S-341F (5′-CCTAYGGGRBGCASCAG-3′) and 16S-806R (5′-GGACTACNNGGGTACTAAT-3′) flanking the V3 and V4 regions of 16S gene All forward-oriented reads generated from this study were separated according to their specific multiplex identifiers (MIDs), and quality control was performed using the RDP pyrosequencing pipeline. For example, reads were flagged as low quality when they had <300 bp in length, the start of the sequence did not exactly match a primer sequence, or one ambiguous base (N) was presented in the sequence H′), coverage (Chao) and operational taxonomic unit (OTU) numbers were calculated at the cutoff level of 3% using Mothur's summary.single routine. Rarefaction curves were calculated using rarefaction.single with 10000 iterations. Nonmetric Multidimensional Scaling (NMDS) analysis was applied to compare the bacterial community composition among all the samples using Primer 5 The richness estimator (Chao1), diversity  were remained after filtering out low-quality reads according to the applied criteria described in methodology . On averH′) were shown in all the three oceanic regions: higher in the intermediate waters (100 m and 500 m) than in the surface and deep waters. This pattern was also reflected by the rarefaction curves with both cutoff values of 3% on the species level  and diversity . Cyanobacteria was more abundant in the surface waters (11∼20 %) of the three oceanic regions.In total, seven bacterial phyla were identified from all samples and munities . Phyla oProteobacteria classes were identified from all samples  and was slightly more abundant in the tropical stations. Gammaproteobacteria was the second major group, but their abundance was much lower (9∼28 %) than Alphaproteobacteria. One general distribution pattern was the decrease of relative abundance of Alphaproteobacteria and increase of relative abundance of Gammaproteobacteria with water depth; and the abundance of these two groups became comparable in deep waters. In addition, Betaproteobacteria, Deltaproteobacteria and Epsilonproteobacteria were also found in different water bodies but they accounted for only small portions.Five  samples . AlphaprProteobacteria were consistently the most or second most abundant OTU throughout the water column of the three regions. It is noticeable that the top three most abundant OTUs in aphotic layer (deeper than 200 m) of all three sites were the same, namely SAR11, unclassified Proteobacteria and Gammaproteobacteria2, except CRD-500 m which saw the OTU of an unclassified bacteria took the first place. On the contrary, the composition of the most abundant OTUs in the euphotic layer was more variable. Cyanobacteria, GpIIa family in the tropical waters, and chloroplasts associated with Bacillariophyta and Cryptomonadaceae in the subarctic waters, was among the most abundant OTUs in the surface layers. In addition, certain OTUs, such as Alcanivoracaceae, Erythrobacteraceae, Moraxellaceae and Pseudoalteromonadaceae, appeared only in the deepest water layer (2000 m).The top 10 most abundant OTUs at the family levels in all the samples demonstrated a clear shift among the geographic locations and depths . SAR11 aVenn diagrams were plotted to show the similarities among different microbial communities at different depths in the same oceanic region in term of the overlapping of OTUs  . In the Bacteria play a central role in energy and matter fluxes in the sea and their metabolic activities are recognized as major drivers of the biogeochemical processes in the oceans, yet the genetic diversity and variability of their community composition among various oceanic realms are still poorly understood. It has been a long-standing interest in the field of marine microbiology to study how microbial assemblages vary in space along environmental gradients and among geographic locations separated with vast distance, particularly the debate of ‘Everything is everywhere’ and the importance of ‘the environment selects’ The recent development of high throughput tag pyrosequencing methods has allowed recovering of much more detailed data about microbial diversity, abundance and community structure, thus offered an excellent opportunity to evaluate bacterial distribution profiles along various environmental gradients. Previous investigations of microbial community structure in the Pacific Ocean using conventional molecular techniques such as denaturing gradient gel electrophoresis (DGGE) and clone library likely underestimated both richness and diversity due to technical limitations. For example, based on 16S rRNA gene clone library, much lower number of OTUs (23∼59) and diversity (Shannon index of 2.51∼3.74) were detected from the eastern tropical North Pacific Ocean Proteobacteria, confirming their global distribution. Proteobacteria display a large phylogenetic diversity that may contribute to their successful colonization over a wide range of ecological niches. Our finding was consistent with previous phylogenetic studies on tropical Pacific Ocean using clone library In the marine bacterial communities investigated here, a large proportion of the ribosomal sequences belonged to Proteobacteria, Alphaproteobacteria predominated in all three regions, particularly the upper water column (surface and 100 m), comprising primarily free-living oligotrophic SAR11 representatives. It is well established that SAR11 dominates the surface waters globally Gammaproteobacteria was another dominant group of Proteobacteria in our study and their importance increased in deep waters. They were also reported as the most abundant group in the northern Baltic Sea Gammaproteobacteria detected in SNP is in agreement with Kataoka et al Gammaproteobacteria in SNP based on estimation by DGGE.On the class level of Cyanobacteria represent 11∼20 % of the reads in the euphotic zone of all three locations, and the species composition was apparently different between the tropical and subarctic waters. GpIIa family including Prochlorococcus and Synechococcus were the most important Cyanobacteria in oligotrophic tropical surface water; this is well documented across the global oceans Cyanobacteria in the subarctic waters came from the symbionts associated with Bacillariophyta and CryptomonadaceaeCryptophytes were relatively abundant in SNP during the spring.The existing of large gradient of physico-chemical conditions along the water depths, such as the decreasing in light, temperature and labile organic matter availability, have been identified as important impact factors affecting the vertical distribution of marine microbial communities Regarding to the bacterial vertical profile alone, contradictory reports have been documented. Using fingerprinting techniques, bacterial richness and evenness in the deep waters were found as high and variable as those in the surface and subsurface waters in the North Pacific and Atlantic Oceans In our study, higher bacterial species richness and diversity in the intermediate water columns than those from surface and deep waters were recovered from the geographically distinctive oceanic regions of SNP and CRD. Whether such vertical distribution pattern represents a general feature of the global ocean, and the mechanistic reasons that cause such pattern remain unexplained, but it is clear that the high numbers of unique OTUs, representing rare phylotypes, detected in the intermediate waters contribute largely to the in situ high diversity Our finding that the bacterial communities of the three regions shared high similarities (>80 %) in the deep waters, but were distinctive from upper layers, is consistent with the previous finding that deep-sea assemblages forming a separate cluster from surface assemblages in the North Pacific Ocean Planctomycetes in CRD-500 m in our study clearly supports the existence of anammox process in the OMZ of CRD.It is worthwhile to note that one sample in this study (CRD-500 m) is located in the oxygen minimum zone (OMZ) characterized by stably depleted oxygen concentrations (e.g. <20 µM). The co-occurrence of anaerobic and aerobic processes makes the OMZ a microbial hotspot, resulting in unique microbial assemblages and high microbial diversity Moraxellaceae and Pseudoalteromonadaceae were among the most abundant families in the 2000 m depth of SCS and CRD, respectively, and they contain mainly psychrotolerant bacteria that are able to live in extremely cold habitats such as deep sea Furthermore, many bacterial groups were found only in the deep layers, particularly the tropical waters. For example, Everything is everywhere, but, the environment selects’ (Baas Becking 1934 Similar vertical variation patterns of Bacteria diversity were found in the three regions with different latitudes in the Pacific Ocean. Generally, bacterial community structures in the deep waters were quite different from those in upper water column, implying that the bacterial populations were affected by the prevailing physical and hydrochemical conditions along the water column. On the other hand, the bacterial community composition in the deep water of the three oceanic regions, which are thousands kilometers apart, were rather similar, supporting the old microbiological tenet ‘