Otx2-(midbrain)/Gbx2-(hindbrain) expressing border. The early interactions between these two pre-specified areas confer positional identities and induce the generation of specific diffusible morphogenes at this interface, in particular FGF8 and WNT1. These signaling pathways are responsible for the gradual histogenetic specifications and cellular identity acquisitions with in the MH domain. This review is focused on the cellular and molecular mechanisms involved in the specification of the midbrain/hindbrain territory and the formation of the isthmic organizer. Emphasis will be placed on the chick/quail chimeric experiments leading to the acquisition of the first fate mapping and experimental data to, in this way, better understand pioneering morphological studies and innovative gain/loss-of-function analysis.A crucial event during the development of the central nervous system (CNS) is the early subdivision of the neural tube along its anterior-to-posterior axis to form neuromeres, morphogenetic units separated by transversal constrictions and programed for particular genetic cascades. The narrower portions observed in the developing neural tube are responsible for relevant cellular and molecular processes, such as clonal restrictions, expression of specific regulatory genes, and differential fate specification, as well as inductive activities. In this developmental context, the gradual formation of the midbrain-hindbrain (MH) constriction has been an excellent model to study the specification of two major subdivisions of the CNS containing the mesencephalic and isthmo-cerebellar primordia. This MH boundary is coincident with the common  In the chick embryos, classical anatomical studies showed that the incipient neuromeric units grouped in three primordial vesicles at the 10–12 somites stage  of a vertebrate is a result of both ontogenic and evolutionary events. The first morphological evidence of the organization of the developing neural tube was reported by Orr  introduce Vaage, , represee Vaage, . It is we Vaage, .Otx2, Shh, Nk2.2, and Pax7, and the distribution of dopaminergic and serotonergic neurons, among other molecular and cellular characteristics, the above-mentioned neuromeres could be grouped into three broad areas or tagmatic regions: forebrain , hindbrain, and spinal cord  and the Gbx2-expressing  domains in which the double decreasing gradients of Engrailed and Pax2 expressions are centered, is associated with complex regulatory activities of a secondary organizer center, the isthmic organizer (IsO), mediated mainly by FGF8 and WNT1, two relevant morphogenes involved in the specification of the MH domain, including the meso-isthmo-cerebellar territory.To obtain this conserved arrangement along the longitudinal axis of the brain, the development of the vertebrate neural tube is conducted by multi-step mechanisms, involving inductive and morphogenetic events. Long- and short-range diffusible molecules, morphogens, govern cell survival and fate specification through the control of dynamic spatial-temporal expression patterns of key transcription factors, which provides positional identities. Innovative genoarchitectural studies and fate mapping studies established correlations between how this intricate genetic network modulates the specification of neuroepithelial territories in vertebrate CNS , the cerebellar primordium could be raised from the pro-rhombomere A1 (RhA1), the rostral most neuromere of the developing hind brain at this developmental stage, located therefore just caudal to the so-called mesencephalic/metencephalic (mes/met) constriction. This clearly defined vesicle of the developing neural tube would contain the presumptive territories of r1 and r2 Vaage, . AlvaradDrosophila orthodenticle (otd) gene and its vertebrate homolog (Otx) are clearly involved in the correct early specification of the anterior part of the developing head  a transient Otx2-negative domain was observed in the caudalmost portion of the “mesencephalic” vesicle between stages HH10 and HH17/18, which gradually reduces its size until it disappears completely from stages HH17/18  so t before ; (4) therebellum  as shownrebellum .Gbx) genes, vertebrate homologs of the unplugged (unp) gene of Drosophila, are also directly involved in the specification of the MH domain . Therefore, the Gbx2 mutant shows a relevant alteration of derivatives of r1–3, giving rise to a shortened area with abnormal histological characteristics. In the caudal midbrain, the III motor nucleus (midbrain) developed in the correct position. However, the midbrain displays abnormalities in its anterior/posterior patterning, suggesting that the caudal midbrain, inferior colliculi, could suffer a caudal extension to the r3/r4 border according to a caudal shift of the Otx2 domain  requirement could be necessary for the correct development of specific rhombomeres in the rostral hindbrain .In addition, gbx genes play a redundant role in morphogenesis and differentiation of the cerebellum  model, based on complementary degenerative mutations in a pair of duplicated genes, was used to elucidate how the mechanism diverged between tetrapod and teleost for vertebrate Gbx genes , homologs to the Drosophila engrailed gene  and rhombencephalic IV (trochlear) cranial nerves were lacking in homozygous mutant embryos. This phenotype may be caused by regional control of cell precursor proliferation instead of by cell fate determination; there is a loss or lack of proliferation of mid-hindbrain cells in En1 mutants  to rostral (weaker). The works of Nakamura’s group have shown that En2 determines the exact position of the rostral mesencephalic boundary by repressing diencephalic markers, such as Pax6, and activating mesencephalic genes Krd mutant mice exhibited a normal development of the MH domain  and a perturbation of the cerebellum foliation pattern  Krd(+/–)Pax5  mouse embryos show a significant absence of the caudal midbrain and alteration of the cerebellar vermis, whereas (−/−) Krd(+/–)Pax5 embryos display a complete loss of the midbrain and the rostral hindbrain  mutation  Krd (+/–)Pax5  mutant mice display an absence of En1/2 expressions  at the 10-somite stage , but not the dp3 and the secondary prosencephalon, was competent to express En2 expression pattern of the inverted portion was abnormal, displaying two mirror-duplicated areas of high En2 expression on both its rostral and caudal halves. As expected, the caudal diencephalon was induced to express the En2 gene. Cytoarchitecture analysis of long-survival resulting chimeras showed that the inverted graft developed an inverted isthmocerebellar structure in its rostral aspect plus an altered midbrain whose rostral and caudal halves show a caudal alar mesencepahilic grisea phenotype with symmetrical morphology and polarity, the rostral alar mesenphalic area  being absent  is required in embryonic development leading to growth and patterning events  mutant embryos show an absence of isthmus and cerebellum, whereas an enlarged tectum is formed  display an enlarged r1 but with some cerebellar cells, suggesting that neither the fgf8 gene nor other members of the Fgf family could be required for r1 specification and cerebellar development, as well as for cerebellar cell differentiation. However, fgf8 seems to be relevant to maintaining r1 devoid of Otx2 expression defining the MH boundary, in addition to being necessary for cell proliferation and cerebellar primordium morphogenesis  proteins, which trigger several intracellular signaling cascades, including Ras-ERK pathways as a subclass of mitogen-activated protein kinase pathway  and the anterior portion of the cerebellum (the vermis), the cerebellar hemispheres being present only with an altered foliation. Large deletions were also detected in the midbrain, mainly affecting the inferior colliculi. Similar effects were observed in mice homozygous for hypomorphic Fgfr1 alleles. In contrast, the basal plate of the MH domain  did not display any deletions or alterations in En1-Cre/+; flox/floxFgfr1 mice  gene in Drosophila  disruptions in mice  and IV motoneurons  were absent, which normally develop on both sides of the transverse Wnt1-positive ring. Therefore, the straight common border of swOtx2/Wnt1 expressions at the MH boundary failed to form correctly. In summary, Wnt1 could be needed firstly for the specification of the MH domain, probably by the maintenance of En expression territory  and isthmocerebellar  phenotypes. The latter effect could be mediated by correct adhesive properties of cell-cell interactions at the Otx2/Wnt1 MH boundary -mediated oligonucleotide knockdown showed that only a portion of the pax2a and en2a-expressing areas in the MH domain is dependent on the wnt1 and wnt10b activities. In these experimental embryos, the resulting phenotype was almost normal with only faint defects . Therefore, the wnt1 and wn10b genes seem not to be needed, surprisingly, for the correct development of the midbrain and cerebellum in zebrafish development  into the chick MH territory, always in direct contact with Gbx2-expressing RhA1  showed expression patterns similar to those described in normal development: Fgf8 and Wnt1 were expressed in two narrow bands, while Pax2 and En2 formed double decreasing gradients centered on the new intra-graft Otx2/Gbx2 boundary  and r1 (Gbx2-positive) were juxtaposed . The histological analysis of these +/Otx2lacZEn1 transgenic mice showed that the inferior colliculi were enlarged, prolonging caudally, whereas the cerebellum was smaller than in normal conditions with a loss of the anterior vermis and a deficiency of cerebellar midline fusion , it has been reported that Lmx1b seems to act as an effector of Fgf8 in the regulation of Wnt1 expression  protein, homolog of the Drosophila hairy-E(spl) family. In zebrafish, her5 expression is observed in the entire presumptive MH domain at gastrulation , her5 being necessary for the maintenance of the integrity of the MH domain but not for the establishment and early maintenance of the MH domain . Because her5 negatively regulates neural differentiation in the caudal midbrain, gbx2 could also promote neurogenesis in the anterior hindbrain by excluding her5 expression  and cranial nerve III and IV were also missing in the double mutant, whereas more caudal cranial nerves  were present  gene encodes a protein homologous to members of the HES family, very close to zebrafish Her5  zebrafish mutants, which carry loss-of-function mutations in the pou2 gene and display alterations in hindbrain segmentation  displays a complete absence of fgf8 expression in the isthmus at 24 hpf  and ace (fgf8) zebrafish mutants, suggesting that pou2 does not depend on fgf8 and pax2.1 expression  gene, ortholog of the zebrafish pou2 gene  displayed an altered expression pattern in the dorsal portion of the MH domain. As a consequence, the gross MH morphology was not altered  family of transcription factors are key regulatory elements in several developmental events  interface, as entities separated by a lineage restriction boundary was demonstrated using a TWnt1-CreER approach as long-term fate mapping, the TWnt1-CreER-stained cells being registered with Otx2 expression  cells generate from two separate cell populations during late gastrulation stages (Langenberg and Brand, Similar evidence of accurate neuroepithelial patterning and cell differentiation in the MH domain were also reported in the zebrafish MH domain. By staining live embryos with vital dyes and matching the positions of hundreds of stained nuclei within the In the present review, we provide tons of evidence in which different levels of transcription factors together with signaling molecules are orchestrated and are required to properly develop the specific regions within the embryonic midbrain and cerebellum. It has been also demonstrated that such differences in level and created gradients may underlie the anatomical variations between species. Future studies may reveal and relate the proper levels of these signaling molecules to build different brain regions in lower vertebrate brains. Moreover, we are far from understanding the precise spread mechanisms of these signaling molecules and the exact mechanism of cell-cell communication to acquire specific positional information and proper polarity in order to be a particular brain nucleus and how this communication between embryonic cells in a particular region is propagated.MH-S and DE conceived the research. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The current evidence suggests that OCT1 is expressed in the human intestine in small amounts (on gene and protein levels), while its cellular localization in the apical or basolateral membrane of the enterocytes remains to be finally defined, but functional data point to a secretory function of the transporter at the basolateral membrane. Thus, OCT1 should not be considered as a classical uptake transporter in the intestine but rather as an intestinal elimination pathway for cationic compounds from the systemic circulation.Intestinal transporter proteins are known to affect the pharmacokinetics and in turn the efficacy and safety of many orally administered drugs in a clinically relevant manner. This knowledge is especially well-established for intestinal ATP-binding cassette transporters such as P-gp and BCRP. In contrast to this, information about intestinal uptake carriers is much more limited although many hydrophilic or ionic drugs are not expected to undergo passive diffusion but probably require specific uptake transporters. A transporter which is controversially discussed with respect to its expression, localization and function in the human intestine is the organic cation transporter 1 (OCT1). This review article provides an up-to-date summary on the available data from expression analysis as well as functional studies  The intestinal epithelium is by far more than a simple passive diffusion barrier as assumed in earlier days. On the contrary, enterocytes are equipped with many physiologically highly relevant transporter proteins that mediate on the one hand a selective and specific absorption of important nutrients and endogenous compounds including peptides via the peptide transporter (PEPT)1 (SLC15A1), glucose via the sodium dependent glucose transporter 1 , fatty acids via the monocarboxylate transporter 1 , cholesterol and phytosterols via ABCG5/G8, bile acids via the apical sodium-dependent , and vitamins via the sodium-dependent multivitamin transporter   Drozdzi.On the other hand, intestinal transporters are recognized as significant determinants of intestinal absorption of many drugs and thus as important factors influencing their efficacy and safety . In thisin vitro, an important in vivo role in intestinal drug absorption was hypothesized  expressed at the sinusoidal membrane of human hepatocytes where it mediates the hepatic uptake of many drugs . In this studies . Associa studies . In this studies . Thus, i studies . HoweverAccording to former studies on human OCT1, the transporter was reported to be localized in the basolateral membrane of epithelial cells in kidney, intestine as well as the liver . Thus, iIn contrast to the well-established role of OCT1 in the hepatic disposition of drugs, its role in the intestine remains still unclear. This can be explained by the limited and in part controversial data on its expression there. Several studies unambiguously demonstrated mRNA expression of OCT1 in human intestinal tissue although the expression levels were much lower than that in the liver . More rein vitro and ex vivo studies or animal and clinical findings may provide more robust evidence on the expression and function of intestinal OCT1.With respect to the cellular localization of OCT1, immunohistochemistry analysis by Müller et al. demonstrated lateral localization . In contin vitro models for the identification of substrates and inhibitors of OCT1 and other SLC transporters  are well established sporters . Howeversporters .Caco-2 cell monolayers have been frequently used to study transepithelial transport of several OCT1 substrates . ConsideOn the contrary, Horie et al. observed a markedly higher cellular uptake of OCT1 substrates, i.e., tetraethylammonium (TEA), quinidine and metformin, after the apical side cells exposure in comparison with the basolateral side .® inserts) it becomes clear that the apical membrane is freely accessible to a drug, whereas the basolateral membrane is partly shielded by the artificial filter membrane. Thus, substantial differences in freely assessable membrane are neglected and it remains uncertain whether the substrates may stick to the filter membrane. In each case, polar OCT1 substrates are not expected to diffuse freely across the lipophilic membrane. This limitation would have been canceled out in comparative A-B and B-A transport studies. However, these control experiments have not been performed. The interpretation of those experiments is moreover complicated by the fact that the apical membrane forms brush border membrane-like microvilli resulting in a substantially higher surface area compared to the basolateral membrane which is so far not considered when calculating intestinal effective permeability (Peff) values. Due to these limitations, accumulation studies in Caco-2 cells are not expected to provide valid conclusions on the cellular localization of OCT1. Because Han et al. used the same methodical approach for transport studies of TEA and pentamidine, the derived conclusions on apical OCT1 remain questionable  and serum AUC of all investigated drugs were found to be markedly elevated in OCT1-knockout animals; oral bioavailability was not different or even increased. Assuming OCT1 as apically localized and therefore acting as an intestinal uptake transporter, one would expect a significantly decreased oral bioavailability in knockout mice. The study indicates that OCT1 is rather involved in transport from blood into deeper compartments than in uptake from intestinal lumen to blood. Consequently, OCT1 deficiency in knockout mice was associated with increased serum exposure (decreased serum clearance) and with a decreased volume of distribution of the respective substrates. This again suggests that OCT1 is most likely expressed in the basolateral membrane of the enterocytes.Similarly, the hepatic exposure and the duodenal content of sumatriptan, fenoterol, ondansetron, and tropisetron after their intravenous administration was lower in Oct1-knockout mice than that in their wild-type counterparts . FurtherIn contrast to this, again the already mentioned study by Ideally, confirmative transport studies should be performed as bidirectional transport studies using animal or human tissue mounted in the Ussing chamber, which represents so far the gold standard experiment to provide reliable and biorelevant information on the intestinal drug metabolism and transport . Althougin vivo; and fifth, the expression and function of intestinal and hepatic OCT1 results in opposite clinical effects in case of transporter inhibition. Hence, inhibition of intestinal OCT1  is expected to result in decreased oral drug absorption and systemic exposure, whereas inhibition of hepatic uptake will cause increased plasma levels of OCT1 substrates. Thus, a substantial overlap is expected which may mask the certain effect of intestinal OCT1. In the case of intestinal OCT1 on the basolateral membrane of the enterocyte, transporter inhibition may cause to some extent additionally increased serum levels caused by reduced direct intestinal excretion of the drug.Investigative approaches to estimate the function of intestinal OCT1 comprise pharmacogenetic studies and DDI studies with orally administered OCT1 substrates. The evidence from clinical studies showing that OCT1 might be a clinically relevant intestinal drugs uptake carrier is limited. This can be attributed to the following aspects which counteract reliable conclusions on the distinct role of OCT1 in the human intestine: first, substrates of OCT1 are partly subjected to extensive metabolism ; second, multiple other transporters can be involved in the pharmacokinetics of a certain OCT1 substrate ; third, OCT1 is not inducible by prototypical inducers of enzymes and transporters such as rifampin which disqualifies meaningful inductive studies; fourth, likewise, there is a lack of specific inhibitors that can be used In addition, renal OCT2 and MATE1/2 K may contribute also to clinical DDI studies because they accept many OCT1 substrates , i.e., iConsidering that there are no specific clinical inhibitors of OCT1 available, the use of pharmacogenetic studies in carriers of OCT1 null alleles (∼9% in Caucasians) is expected to provide additional evidence .N = 20) indicated that OCT1 significantly affected the serum exposure and efficacy of metformin. In detail, carriers of SCL22A1 loss-of-function alleles showed a reduced response to the drug ; second, no or only minor metabolism; third, no or only minor passive diffusion; and fourth, no other transporters influencing its pharmacokinetics in a significant manner.Applying these criteria to the substrates summarized in in vivo; e.g., in portal vein (up to 100-fold higher compared to the systemic blood concentration) relevant for OCT1-mediated uptake into the liver or in the intestinal lumen affecting interaction with intestinal uptake carriers   ). HoweveA well investigated drug in this regard is the antimigraine drug sumatriptan, which systemic exposure was over 2-fold increased after oral administration in carriers of SLC22A1 loss-of-function alleles . Althougmax and AUC of morphine and significantly increased opioid effects  and higher frequency of side effects have been observed in children after intravenous administration . FurtherAdditional arguments against OCT1 at the apical membrane in the human intestine provide interaction studies of atenolol with cimetidine, metoclopramide with ranitidine and metformin with trospium . In all Finally, the poorly absorbable bladder spasmolytic trospium  might be a good candidate to conclude on the function of intestinal OCT1 because this drug is given orally, is not metabolized and is not subjected to significant hepatic uptake but undergoes almost exclusively renal excretion . In thisHowever, as mentioned earlier, the interpretation of clinical interaction studies with OCT1 substrates and inhibitors as summarized in Assuming oversimplified that intestinal and hepatic uptake would contribute equally to the bioavailability of a certain OCT1 substrate, simultaneous inhibition of intestinal and hepatic OCT1 would result in only slightly changed systemic exposure of the victim drug . HoweverThere is no doubt that hepatic OCT1 can influence the pharmacokinetics and in turn the efficacy and safety of several drugs in a significant manner , 2020. IAs OCT1 was also shown to be expressed in the human intestine, it was assumed to be involved in the intestinal absorption of drugs. Despite its unequivocal intestinal abundance, the distinct localization in the enterocytes still remains uncertain as two independent studies identified OCT1 either in the apical or the basolateral membrane . HoweverAccordingly, most bidirectional transport studies of OCT1 substrates across Caco-2 cells demonstrated a markedly higher secretory transport compared to the opposite direction (B-A > A-B), which suggest a basolateral localization of OCT1 . As receFinally, also the available pharmacogenetic and DDI studies do not provide evidence for apically localized intestinal OCT1. However, the interpretation of clinical studies is complicated considering the complex contribution of intestinal, hepatic, and renal cation transporters. Moreover, confirmative induction studies as regularly performed for P-gp or cytochrome P450 enzymes are not possible for OCT1.in vitro and animal experiments as well as data from clinical studies suggest that OCT1 is localized in the basolateral membrane of the enterocytes and cannot be considered as an uptake transporter in the human intestine.In conclusion, available evidence from expression studies, Basolateral OCT1 in the enterocytes would imply its involvement in the intestinal excretion of drug from the systemic circulation. For this secretory net transport across the enterocytes, P-gp can be expected a relevant efflux transporter in the apical membrane because it accepts many OCT1 substrates. Indeed, this intestinal elimination pathway has been observed in several animal studies after intravenous administration of OCT1 substrates  but alsoOn the contrary, assuming OCT1 in the apical membrane of the enterocytes  would raGiven the assumption that OCT1 is not present in the apical membrane of the human enterocytes, which mechanisms may be involved in the intestinal uptake of cationic compounds? Beside mechanisms of paracellular transport as discussed elsewhere , the intFor an unequivocal proof for the localization of intestinal OCT1, targeted proteomic analysis of apical and basolateral membrane fractions of the human intestinal mucosa and bidirectional transport studies of established non-metabolized OCT1 substrates across human intestinal tissue from a sufficient number of volunteers  would be required.


The valley degree of freedom, like the spin degree of freedom in spintronics, is regarded as a new information carrier, promoting the emerging valley photonics. Although there exist topologically protected valley edge states which are immune to optical backscattering caused by defects and sharp edges at the inverse valley Hall phase interfaces composed of ordinary optical dielectric materials, the dispersion and the frequency range of the edge states cannot be tuned once the geometrical parameters of the materials are determined. In this paper, we propose a chirped valley graphene plasmonic metamaterial waveguide composed of the valley graphene plasmonic metamaterials (VGPMs) with regularly varying chemical potentials while keeping the geometrical parameters constant. Due to the excellent tunability of graphene, the proposed waveguide supports group velocity modulation and zero group velocity of the edge states, where the light field of different frequencies focuses at different specific locations. The proposed structures may find significant applications in the fields of slow light, micro–nano-optics, topological plasmonics, and on-chip light manipulation. Valley degrees of freedom (DOF), also known as valley pseudospins, mark discrete extreme energy states in the momentum space ,2,3,4,5.Graphene, which exhibits excellent properties, has promising applications in various fields ,34,35,36In this paper, we propose tunable valley GPMs (VGPMs) for group velocity modulation and light field focusing of surface plasmon polariton (SPPs) waves. It consists of graphene nanodisks arranged in a honeycomb lattice covered with a graphene monolayer on the top. Tunable VGPMs take the valley as the DOF and break the spatial inversion symmetry by changing the chemical potentials, thereby opening a complete photon bandgap in the entire BZ. The nontrivial topology phase transition is confirmed by verifying the valley Chern numbers. Furthermore, we designed a chirped VGPM waveguide composed of a supercell arrangement of VGPMs with gradually increasing chemical potential difference. By implementing the chirped VGPM waveguide, we demonstrated modulation of the edge state group velocity. Group velocity was slowed down to zero to realize the slow SPP wave and the light field of SPP wave focusing. The chirped VGPM waveguide has excellent potential in nanophotonic systems, alternative topological states, and the manipulation of spin–orbit interactions of light because of its excellent tunability, backscattering resistance, and low absorption.As depicted in 2:h1)1/2 . TherefoThe significance of the graphene chemical potential is wholly demonstrated in the dispersion relation of the transverse magnetic (TM) polarized SPP mode supported on the monolayer of graphene. This dispersion relation is derived by solving Maxwell’s equations with boundary conditions, which is described in  as(1)εAd regime , the vacHere,  formula :(3)σg=σiThe constants v = CK − CK’ was used to distinguish the topology, where CK/ CK’ is the valley-dependent index at the K/K’ valley. For VGPM1 with v = −1; for VGPM2 with v = 1. These characteristics theoretically reveal the valley Hall phase transition. Furthermore, this is also verified in the numerical simulation corresponding to the topological index of the left and right sides in 1 and K2 valleys  and anticlockwise (green arrow) power flows, i.e., pseudospins, around the field centers. This case was the opposite when 1 and K2 valleys were reversed with the increase of 1 and K2 were reversed and accompanied by a valley Hall phase transition.The valley topological edge state is a vital feature of a VGPM and an essential foundation for the designed chirped VGPM edge state waveguide. The valley topological interface used to realize the valley topological edge state is shown in i-column are denoted as The dispersion of the valley topological edge states is modulated by varying The group velocity dispersion curves and the light field distributions of edge SPP waves at different frequencies along the chirped VGPM waveguide interface are shown in In summary, we designed VGPMs, verified the valley pseudospin and valley hall phase transition, and numerically calculated the band structure. The valley topological interface was constructed by using the reverse valley Hall phases, and the dispersion curve of the valley topological edge mode was obtained. Then, the dispersion of the valley topological edge mode was tuned by changing the chemical potential difference of VGPMs, and the chirped VGPM waveguide was proposed accordingly. This waveguide offers multiple functionalities, including tuning of dispersion relations for valley topological edge states, modulating the group velocity of edge SPP waves, and realizing slow SPP waves. Finally, the intensity enhancement factor is introduced to verify the reliability of slow SPP waves and the light field focusing of the edge SPP waves. The proposed structure with excellent properties might find broad application in the fields of nanophotonic systems, alternative topological states, and manipulation of spin–orbit interactions of light.


The serine/threonine p21-activated kinases (PAKs), as main effectors of the Rho GTPases Cdc42 and Rac, represent a group of important molecular switches linking the complex cytoskeletal networks to broad neural activity. PAKs show wide expression in the brain, but they differ in specific cell types, brain regions, and developmental stages. PAKs play an essential and differential role in controlling neural cytoskeletal remodeling and are related to the development and fate of neurons as well as the structural and functional plasticity of dendritic spines. PAK-mediated actin signaling and interacting functional networks represent a common pathway frequently affected in multiple neurodevelopmental and neurodegenerative disorders. Considering specific small-molecule agonists and inhibitors for PAKs have been developed in cancer treatment, comprehensive knowledge about the role of PAKs in neural cytoskeletal remodeling will promote our understanding of the complex mechanisms underlying neurological diseases, which may also represent potential therapeutic targets of these diseases. Most neurons receive information from other neurons via synapses mainly formed on the surface of dendritic spines. Thus, the morphological plasticity and density of dendritic spines are crucial for the physiological functions of neurons. Dendritic spines consist of a network of actin filaments, whose polymerization and depolymerization can govern spine function and regulate synaptic plasticity. Dysfunction of synaptic cytoskeletal remodeling is closely related to diverse brain disorders  database  version] Platform in the HBT database from 6 brain regions of 59 donors. The expression value of each PAK gene was averaged using data from both the left and right brain hemispheres of same-age donors. We found that most PAK levels showed a decrease at the late prenatal stage, but each PAK presented a differential pattern in different brain regions, including the DFC , hippocampus, amygdala, mediodorsal nucleus of the thalamus, cerebellar cortex, and striatum, from 40 weeks before birth to 40 years after birth  and TCPY , but relatively low expression was found in the clusters of PBMC , MCPY , and FB (fibroblast)   Fig. A. To anaThe F-actin (filamentous actin) cytoskeleton is the main driving force behind dendritic spine remodeling and maintenance of synaptic plasticity. Cofilin is a major actin-depolymerizing factor that regulates the actin cytoskeleton dynamics and phosphorylation of serine residue at 3 (Ser 3) can inhibit its actin-depolymerizating activity   subunit in neurons -β can regulate the accumulation of PSD-associated PAKs by interacting with a large protein complex containing dPIX and Dock, which are crucial to the composition of neural circuits and the long-term plasticity of learning and memory at the neuromuscular junction , such as SAP102, can interact with EphB2 and Kalirin-7, a neuronal exchange factor for small GTPase, to activate their key downstream PAK signals  are the principal subtype of neuronal progenitors and can produce most cortical neurons in the neocortex  to distant targets, such as the cerebral cortex, is a critical step in GABAergic interneuron development  signaling, regulates the posterior-to-anterior migration of HSNs in developing embryos , a member of the Ig superfamily, to induce the activation of the PAK1-LIMK1-cofilin pathway and promote cytoskeletal remodeling processes and filopodium mobility , the receptor of anterior corner cells in the motor neurons, can recruit Dock and PAK1 to the plasma membrane and define the precise positioning of dendritogenesis in Pak3 knockout mice displayed delayed OPC differentiation and consequently myelination defects in the corpus callosum   to promote the activation of PAKs and phosphorylation of cofilin, eventually promoting NMDA excitotoxicity, a trigger of AD  and tau proteins, which trigger a complex cascade resulting in synaptic loss and neurotransmitter deficiencies  gene on the X chromosome  failed to activate and induce PAK phosphorylation in the spine, and newly formed actin filaments induced by theta stimulation remained unstable , the most common genetic form of MR, is caused by repeated CGG amplification of the PAK3 gene have been identified in different families of X-linked mental retardation with certain specific clinical features, such as microcephaly, mild to moderate mental retardation, oral motor dysfunction and aggressive behavior  has an autosomal dominant pattern of inheritance  arm of chromosome 3. Approximately 80%–99% of people with the 3q29 microdeletion have mild to moderate intellectual disability. They also have an increased risk of behavioral or psychiatric disorders, including autism, bipolar disorder, and schizophrenia  affects neuronal function and how it contributes to neurodevelopment and brain function requires further investigation.PAKs are widely present in neuronal and non-neuronal cells in the central and peripheral nerve system. In neuronal cells, each PAKs regulate synaptic plasticity by regulating both spine morphology and synaptic transmission/trafficking. PAKs can also regulate diverse neural functions and processes and are involved in broad neural activities in the brain. PAKs participate in various signaling pathways related to cytoskeletal remodeling and thus are involved in the molecular pathology of neurological disorders at different neurodevelopmental stages. PAKs thus have been found to play both direct  and indirect roles (NF1) in the pathology of neurological diseases. Although some of the molecular mechanisms remain unknown, PAKs, possessing specific small-molecule inhibitors in cancer treatment, also represent potential therapeutic targets of neurological diseases involving PAK dysfunction, which necessitates further investigation. Moreover, considering the differential roles of PAKs in neural function, small molecules specific for each PAK rather than a group of PAKs may provide more precise and accurate improvement for PAK-related neurological diseases.


Previous investigations described bioinformatic analyses based on the mRNA expression and somatic mutation as useful strategies for identifying cancer-associated molecules that were potential candidates of therapeutic targets. However, these data included secondary changes and non-functional alterations that do not influence tumor progression. Investigations, including our own studies, have shown that some RBPs shuttle cytoplasm and nuclei, and their affinity to RNAs is regulated by posttranslational modifications, such as phosphorylation. Therefore, the functional assessment of individual molecules is the most suitable strategy for identifying cancer-associated genes with or without expressional changes and mutations. This report showed for the first time that a functional assessment using an siRNA library was useful for identifying therapeutic targets from molecular groups, including RBPs, that had not been identified by expressional and mutational analyses.Previous investigations have indicated that RNA-binding proteins (RBPs) are key molecules for the development of organs, differentiation, cell growth and apoptosis in cancer cells as well as normal cells. A bioinformatics analysis based on the mRNA expression and a somatic mutational database revealed the association between aberrant expression/mutations of RBPs and cancer progression. However, this method failed to detect functional alterations in RBPs without changes in the expression, thus leading to false negatives. To identify major tumor-associated RBPs, we constructed an siRNA library based on the database of RBPs and assessed the influence on the growth of colorectal, pancreatic and esophageal cancer cells. A comprehensive analysis of siRNA functional screening findings using 1198 siRNAs targeting 416 RBPs identified 41 RBPs in which 50% inhibition of cell growth was observed in cancer cells. Among these RBPs, 12 showed no change in the mRNA expression and no growth suppression in non-cancerous cells when downregulated by specific siRNAs. We herein report for the first time cancer-promotive RBPs identified by a novel functional assessment using an siRNA library of RBPs combined with expressional and mutational analyses. Several RBPs are evolutionally conserved and ubiquitously expressed because of their essential roles in cellular events, such as cell growth, apoptosis and senescence  × 100. In this study, RBPs that showed a growth inhibition rate of >50% when siRNA was transfected were defined as tumor-promotive RBPs. The raw data of the cDNA array were analyzed using the Transcriptome Analysis Console (ThermoFisher Scientific). The overlaps of genes were detected using Microsoft Excel. The graphs of each experiment and Venn diagram were generated using the Microsoft Excel and PowerPoint software programs. The densitometry of Western blots was analyzed using the Image J software program. The interactome analysis of RBPs selected by the functional assessment and cDNA array analysis was performed using the MetaCore software program .t-test. p-values of <0.05 were considered statistically significant.The assay data were analyzed using Student’s To identify which RBPs are involved in tumor proliferation in gastrointestinal cancer cells, we constructed an siRNA library to perform a functional analysis of RBPs registered in the RBPDB. A total of 1198 siRNAs targeting human 416 RBPs were collected  siRNA t. A bioinformatics analysis, which highlights the expressional changes in cancer cells, reveals tumor-promotive or tumor-suppressive molecules. However, the expressional changes in cancer cells contain the secondary changes induced by cancer driver signaling, such as KRAS , HIF 1616 and p5To investigate the correlation between the growth-promoting effect and expressional changes in RBPs, we compared the gene expression between cancerous and non-cancerous cell lines by a cDNA array analysis in addition to the functional analysis using the siRNA library . A totalTo identify the tumor driver RBPs that were unable to be detected based on the expressional data in gastrointestinal cancer cells, we assessed a Venn’s diagram of the results of the functional assay and cDNA array analysis. siRNAs of 57, 66 and 68 RBPs strongly inhibited tumor cell growth without expressional changes in colorectal cancer cells (HCT116 or SW480), pancreatic cancer cells (SUIT2 or PANC-1) and esophageal cancer cells (OE33 or KYSE70), respectively . Among tTo confirm the growth suppression effect of these 12 RBPs, an MTT assay was performed. The growth of HCT116, SUIT2 and OE33 cells was significantly decreased in cells treated with siRNA of each RBP . To asseTo assess the interaction network of these 12 RBPs, an interactome analysis was performed using the MetaCore software program. These RBPs constructed a cell survival network mediated by classical pathways, including ERK, p53 and Myc signal transduction A. To evaTo identify organ-specific tumor-promotive RBPs, we compared the tumor-promotive function of RBPs among HCT116, SUIT-2 and OE33 cells. Our functional assay showed that 18 RBPs , 24 RBPs  and 35 RBPs  were specifically promoted in colorectal, pancreatic and esophageal cancer cells, respectively , suggestTo elucidate whether or not cancer cell proliferation was associated with genetic mutations of RBPs, information on genetic mutations of each cell line was obtained from COSMIC and compared with the findings of our functional growth inhibition analysis using an siRNA library. A total of 144 mutations of RBPs in HCT116, 6 in SW480, 27 in SUIT-2, 12 in PANC-1, 13 in OE33 and 42 in KYSE-70 were identified in the COSMIC database . FurtherWe showed that a global functional analysis was a useful and critical strategy for identifying therapeutic targets in cancer cells. In the present study, we focused on RBPs because of their tumor-associated functions and identified candidate driver RBPs in cancer cell growth. Interestingly, we identified 12 tumor growth-promotive RBPs with an expression that did not change in cancer cells. We found that the genetic mutations registered in the COSMIC database do not necessarily reflect the molecular function of RBPs. The molecular group of RBPs without expressional or mutational changes as well as those with expressional or mutational changes appear to be viable therapeutic targets for cancer treatment.In the present study, the list of RBPs that showed an altered expression in cancer cells was compared to the list of tumor-promotive RBPs identified by our functional analysis using the siRNA library. RBPs for which the growth-promotive function was associated with overexpression represented a relatively minor group in comparison to the RBPs without any significant expressional changes or non-tumor-promotive RBPs with significant expressional changes. These data indicated that the expressional changes in cancer cells include numerous secondary changes induced by alterations of tumor driver genes and that these changes are not suitable for therapeutic targets, even if they are useful as molecular markers. These results strongly suggested that an in silico database analysis to identify therapeutic targets based on significant expressional changes may miss many tumor growth-associated genes.Likewise, we identified common tumor-promotive RBPs by a functional screening assay that could not be detected based on the expression data in colorectal, esophageal and pancreatic cancer cells. We clarified that 12 RBPs  promoted tumor cell growth without expressional changes in all six cancer cell lines. Notably, strong growth inhibition was not detected in any non-cancerous cells  treated with the siRNA of nine RBPs , suggesting that these RBPs possess a tumor-specific growth effect and are therefore attractive therapeutic targets in digestive cancer. Interestingly, our functional analysis revealed that most RBPs were associated with cancer cell progression, irrespective of the expressional changes; thus, global RBP inhibition may be a multipotent therapeutic target in cancer.SNRPE is a component of spliceosomes that regulates pre-mRNA splicing through the formation of U snRNA assembly . Our funSF3A1 is considered to play an important role in spliceosome assembly and mRNA splicing . Our funSUPT6H plays a key role in the regulation of transcription elongation and mRNA processing . Our funDHX8, EIF3G, RBM22, U2AF1 UPF1 and YBX-1 are also candidates therapeutic targets for cancer therapy because the tumor cell progression was strongly inhibited by the downregulation of these RBPs in cancer cells, including HCT116, SUIT2 and OE33 cells, but not in non-cancerous cells. DHX8 regulates the splicing, including p21 , and is UPF1 is an RNA-dependent helicase, and ATPase is required for nonsense-mediated decay (NMD) of mRNAs containing premature stop codons . WesternYBX-1 can bind with DNA as well as RNA and is involved in various processes, including translational repression, RNA stabilization, mRNA splicing, DNA repair and transcription regulation ,31,32. IOur functional assay showed that growth suppression was induced in non-cancerous cells by the downregulation of three RBPs , suggesting that these molecules have pivotal functions in proliferation and apoptosis in non-cancerous cells as well as cancer cells. RPS3, which is a component of the 40S small ribosomal subunit and associated with translation, controls the inflammatory response through the transactivation and proteasomal degradation of NFκB . EIF2S1 Our functional assessment of RBP combined with a bioinformatics analysis showed that the classical cell survival pathways, including Akt and p53, were essential for the exertion of the tumor-promotive functions by the RBPs identified in this study. Likewise, this interactome analysis identified the comprehensive interactions between these RBPs and abnormally expressed mRNAs in cancer cells. As each RBP is associated with numerus mRNAs and each mRNA is controlled by numerous RBPs, interactions between RBPs and mRNA are quite complicated. Our functional assessment with an interactome analysis is a powerful tool for identifying pivotal mechanisms regarding the expression/stabilization of mRNAs by RBPs. Therefore, a functional assessment is a useful strategy for investigating unknown mechanisms of tumor progression, as well for identifying therapeutic targets for tumor therapy.Our microarray analysis using epithelial-like tumor cell lines showed significant differences in 12 RBPs between cancer and non-cancerous cells, whereas our in silico database analysis showed that two RBPs , eight RBPs  and two RBPs  were upregulated in colorectal, pancreatic and esophageal cancer tissue, respectively . VariousIn this study, we assessed the growth promotive function of RBPs in cancers with three different origins and different clinical processes and identified the tumor type-specific RBPs in colorectal, pancreatic and esophageal cancer cells. Our functional assay revealed that 18 RBPs in colorectal cancer cells, 24 in pancreatic cancer cells and 35 in esophageal cancer cells supported tumor cell growth in specific tissue. These data mean that the contribution of RNA regulation mediating RBPs to cancer progression differs according to the type of cancer and that these tissue-specific RBPs may be suitable as therapeutic targets for tumor therapy in each organ.Genetic mutations are well known to induce functional abnormalities of cancer-associated proteins, such as EGFR, KRAS and APC, resulting in cancer progression ,37,38,39As described above, our functional screening strategy can be used to identify additional novel cancer driver genes by targeting different molecular groups, such as transcriptional factors and growth factors. While this study used a procedure for screening cancer cell growth, similar strategies using migration or engraftment assays can identify key molecules for invasion and metastasis in cancers. Likewise, this strategy can be used to identify therapeutic targets of various disease, such as autoimmune diseases and neurodegenerative diseases.In conclusion, we identified 12 RBPs  with strong tumor proliferative effects without any marked changes in expression in colorectal, pancreatic and esophageal cancer using the functional assessment of RBPs with an siRNA library focused on cancer growth change. We also identified cancer driver genes from RBPs through the combination of a functional screening analysis and a cancer mutational database. These RBPs with or without changes in their expression or gene mutations are expected to be candidates for the development of novel molecular-targeting therapy.


Intracerebral hemorrhage (ICH) features extremely high rates of morbidity and mortality, with no specific and effective therapy. And local inflammation caused by the over-activated immune cells seriously damages the recovery of neurological function after ICH. Fortunately, immune intervention to microglia has provided new methods and ideas for ICH treatment. Microglia, as the resident immune cells in the brain, play vital roles in both tissue damage and repair processes after ICH. The perihematomal activated microglia not only arouse acute inflammatory responses, oxidative stress, excitotoxicity, and cytotoxicity to cause neuron death, but also show another phenotype that inhibit inflammation, clear hematoma and promote tissue regeneration. The proportion of microglia phenotypes determines the progression of brain tissue damage or repair after ICH. Therefore, microglia may be a promising and imperative therapeutic target for ICH. In this review, we discuss the dual functions of microglia in the brain after an ICH from immunological perspective, elaborate on the activation mechanism of perihematomal microglia, and summarize related therapeutic drugs researches. Intracerebral hemorrhage (ICH) has become one of the most common and lethal diseases in the last decades , and directivities in different distances from the hematoma , which promotes the adhesion and infiltration of peripheral leukocytes , Brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), neurotrophin 3 (NT-3), NT-4/5, which could promote neurogenesis and neural circuit reframing  and plasma proteins including thrombin and fibrinogen infiltrate into the brain parenchyma, and trigger the initiation of early cellular and molecular pathological processes. Hematoma not only contains the agents that directly activate microglia but also promote microglial M1-polarization indirectly through tissue damage. Because of energy exhaustion and cytotoxicity, RBCs in the hematoma begin to lyse within 1 day and continue for weeks after ICH  DNA binding activity  is a member of the serine/threonine kinase family, which includes P38, Extracellular Signal-Regulated Kinase1/2 (ERK1/2), c-Jun N-terminal kinase (JNK) pathways  is a kind of intracellular multi-molecular protein complex that is involved in inflammation  and Nuclear erythroid 2 related factor 2 (Nrf2) are important signals of M2-polarization  family exert their effect on both M1 and M2 polarization  achieved an early therapeutic effect in rodent experiments , and statins are relatively promising. Related clinical researches have been briefly summarized in Minocycline is an ordinary broad-spectrum antibiotic. It could pass through the blood-brain barrier freely and has a neuronal protection effect  are widely prescribed medications for the management of hypercholesterolemia. The potential of Statins for ICH treatment has been revealed recently (Chen Q. et al., Though how to regulate microglia to promote brain recovery remains worth pondering in some sense, there are latent misgivings that excessive inhibition of M1 microglia and promotion of M2 microglia may turn into adverse effects in ICH treatment, where we should keep watchful eyes.On the one hand, the immunoreactive materials secreted from M1 microglia appear to have delayed beneficial effects on brain repair. Solid evidence indicates that MMPs are necessary for angiogenesis, myelin remodeling, and axonal regeneration in ICH later stage (Lei et al., On the other hand, the early organizational disruption may build the basis of neogenesis. M1 microglia destroy dying and defunct neurons in pieces, which lends a convenience for M2 phagocytosis (Hu et al., As for M2 microglia, superfluous and prolonged existing growth factors will predictably cause abnormal tissue repair. Overexpression of Arg1 has been found to cause tissue scarring and brain dysfunction (Hesse et al., In summary, it is really improper to consider that M1 and M2 microglial phenotypes are thoroughly opposite. Instead, their interaction, cooperation, and even codependency are waiting to be explored in the future. A balance of M1 and M2 microglial, rather than extremely choosing M2 over M1, ought to be achieved for ICH individualized treatment, just like the balance of yin and yang.Although drugs with the pleiotropic ability of immune regulation may bring more benefits, not a few medical experiments failed just because of uncontrolled side effects. It is a neglected consensus that many microglial receptors and signaling molecules are meanwhile expressed or activated in other brain cells, such as astrocytes, oligodendrocytes, endothelial cells, and neurons. It is unwise to judge the holistic functions of concerned targets in the brain by taking only microglia into account. For example, CD163 helps microglia engulf and break down hemoglobin, whereas, inhibition of CD163 in the ICH acute phase unexpectedly reduces brain damage, possibly because inhibition of CD163 expressed on neurons decreases the Hb neurotoxicity induced neuronal death (Righy et al., Hence, we need a kind of drug that has a high targeting specificity to microglia. Preferably, it’s expected to have sufficient liposoluble ability to pass through BBB and concentrate on microglia. Furthermore, it’s recommended to conjunctive use advanced medical technology such as intranasal administration, nanomaterials, and genetic technologies to achieve better intervention results for ICH treatment.RB and ZF wrote and revised the manuscript. MY helped with the literature search and correction of the manuscript. BH and QH provided the conception and design of the review, and directed the writing of the manuscript. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The contribution of bone marrow stromal cells to the pathogenesis and therapy response of myeloid malignancies has gained significant attention over the last decade. Evidence suggests that the bone marrow stroma should not be neglected in the design of novel, targeted-therapies. In terms of gene-editing, the focus of gene therapies has mainly been on correcting mutations in hematopoietic cells. Here, we outline why alterations in the stroma should also be taken into consideration in the design of novel therapeutic strategies but also outline the challenges in specifically targeting mesenchymal stromal cells in myeloid malignancies caused by somatic and germline mutations. Under physiological conditions, hematopoietic stem cells (HSCs) are regulated by their bone marrow microenvironment (BMM) through cellular interactions and secreted factors to maintain a continuous pool of hematopoietic cells , myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) Medyouf, . Thus, tIn this article, we will outline the role of stromal cells  in myeloid malignancies in somatic disease, as well as germline conditions, and describe recent progress in dissecting the HSC-stroma crosstalk. Finally, we discuss possible application of the established murine disease models and future challenges in developing genetically targeted therapies for the BM stroma.SBDS gene with a cumulative probability of leukemic progression of >30% at the age of 30 years  introduced a new category of “myeloid malignancies with germline predisposition” to the 2016 Classification of hematopoietic tumors. Given that these “rare” mutations are only coming to light with increased use of parallel sequencing platforms in population and family studies Porter, , it can in vitro and in vivo  in non-hematopoietic, stromal cells were reported to give rise to a myeloproliferative phenotype + cells, as well-documented MSCs in the BM  indirect targeting of MSC function in vivo through genome editing of hematopoietic cells and cell-to-cell interactions, and (4) in vitro functional characterization of MSCs and potential therapeutic targets through CRISPR screens and 3D models.In this section we highlight some of the key challenges hampering the development of targeted genetic therapy of BM stromal populations: (1) identification of specific MSC population to edit, (2) targeting MSCs in their + Cre-driver previously studied as one MSC population, contains four subclusters of MSCs, with functional differences between them as current evidence suggests. As we gain knowledge of functionally distinct MSCs and their possibly common progenitors, it will become possible to target them. Importantly, the location of MSCs in relation to (mutant) HSPCs seems to predict biological functionality and these sinusoidal and CXCL12 niches will need to be further investigated , for the silencing of genes specifically in bone-marrow endothelial cells. Using nanoparticle enabled RNAi, the group targeted stromal-derived factor 1 (Sdf1) resulting in stem cell liberation into the blood, and monocyte chemotactic protein 1 (Mcp1) whose silencing retained monocytes in the BM. These modified nanoparticles lay the ground for editing non-hematopoietic cells in the bone marrow with a high efficacy and show that HSPCs biology can be altered through stroma alterations.Cas proteins need specially designed delivery vehicles for tissue-specific delivery as they cannot cross biological barriers themselves and have a high positive charge and molecular mass. Extracellular vesicles (EVs) are used as possible packing devices for sgRNA:Cas9 ribonucleoprotein complexes. It has been shown however that EVs are mainly taken up by the liver (~84%), whereas roughly 1.6% are found back in the bone marrow 4 h upon systemic administration, making delivery to the bone marrow quite challenging  ex vivo culturing environment greatly reprograms MSCs compared to direct sorting of primary cells for microarray analysis  and genome-editing including gene disruption and targeted integration of up to 3.2 kb of DNA with stable transgene expression, while retaining their in vivo on the basis of new possible germline/somatic mutations within the niche found with targeted sequencing in human disease to study disease pathogenesis, (2) targeting MSCs in vivo directly through MSC/EV-based approaches, (3) indirectly through modulation of hematopoietic cells, (4) modeling of the human hematopoietic niche using ossified scaffolds in xenotransplantations, and (5) in vitro Cas9-based screening methods. Targeted genome-editing will most likely become more feasible as we characterize the true MSCs as the target cell and improve engineering of carriers which will deliver the sgRNA:Cas9 cargo with high efficacy to the bone marrow.As we gain knowledge of the different functional subcomponents of the bone marrow niche, the disease model of myeloid malignancies will become more complex. It is evident that oncogenesis can arise from two non-mutually exclusive theories: niche-induced and niche-facilitated. In patients, we envision a future of personalized medicine in which the stroma can be pharmacologically targeted in combination with a hematopoietic cell-based therapy. We can use the accumulating knowledge with genome editing by (1) generation of murine disease models All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Visual information is conveyed from the eye to the brain through the axons of retinal ganglion cells (RGCs) that course through the optic nerve and synapse onto neurons in multiple subcortical visual relay areas. RGCs cannot regenerate their axons once they are damaged, similar to most mature neurons in the central nervous system (CNS), and soon undergo cell death. These phenomena of neurodegeneration and regenerative failure are widely viewed as being determined by cell-intrinsic mechanisms within RGCs or to be influenced by the extracellular environment, including glial or inflammatory cells. However, a new concept is emerging that the death or survival of RGCs and their ability to regenerate axons are also influenced by the complex circuitry of the retina and that the activation of a multicellular signaling cascade involving changes in inhibitory interneurons – the amacrine cells (AC) – contributes to the fate of RGCs. Here, we review our current understanding of the role that interneurons play in cell survival and axon regeneration after optic nerve injury. Ca2+ inGC death ; at the GC death . CertainGC death .While strategies to counteract the pathways leading to cell death can improve RGC survival, the effects are often transitory, or allow long-term survival in a compromised state, or even suppress regeneration . For exaThe low intrinsic capacity of mature neurons to regenerate axons within the CNS is caused in part by developmentally regulated expression of factors that prevent excessive cell growth and sprouting . ManipulAt the same time, many studies demonstrate the importance of the environment surrounding injured axons in suppressing or promoting regeneration. Modulation of extrinsic suppressors of growth such as myelin-associated inhibitors, components of extracellular matrix, microglia, or attenuation of pericyte-derived fibrosis, leads to modest improvements of axon regeneration . On the A factor that is now coming to light is the instructive role that amacrine cells (AC), the inhibitory interneurons of the retina, play in regulating RGC survival and axon regeneration. ACs either form direct, mostly (but not exclusively) inhibitory synapses (or gap junctions) onto RGC or modulate excitatory inputs from bipolar cells (BC) and inhibitory inputs from other ACs . GrowingIn vivo, the decline in RGCs’ growth state is associated with numerous changes in these cells’ program of gene expression, including an upregulation of the growth suppressive Kruppel-like transcription factors Klf-4 and Klf-9, and down-regulation of the growth-promoting transcription factors Klf-6 and Klf-7   , by expray mTOR) .in vivo; and conversely, activation of RGCs by bipolar cells or via silencing of ACs could be permissive for regeneration. More generally, circuit-level activity levels of the retina can alter the activity state of RGCs and thus influence axon regeneration in the optic nerve. RGC activity is reduced by the hyperpolarizing inhibitory drive from ACs, suggesting that such inhibition could suppress regeneration 2+ influx which elevates intracellular cAMP levels  can nevertheless increase axon regeneration, as we discussed above.Although RGCs can respond to some growth factors without elevating their physiological activity, such as SDF-1  and CCL5P levels  and mediP levels . Ca2+ inse genes . The actse genes . Convers2+)  of the retina, as demonstrated by selenite autometallography (AMG), is one of the earliest changes seen in mouse retina after optic nerve injury  . Elevatie injury  Figure +  when subjected to oxidative stress  sensor, Cu2FL2E , to prov2FL2E . One poshioneins . Alternal stores .2+ entering ACs upon activation of voltage-gated calcium channels or through NMDA or AMPA receptors  are abolished in mice lacking the zinc transporter protein ZnT3, the accumulation of Zn2+ in AC terminals following ONC is similarly absent in ZnT3 knock-out mice     . Blockadn (TeNT) . These d2+, it remains possible that other cations, e.g., Cu+ or Cu2+, could also be involved. Copper is stored in synaptic vesicles and released upon depolarization , enzymes that deacetylate histone proteins, thereby rendering chromatin more accessible for transcription. The deacetylating activity of HDACs depends on the binding of Zn2+ in the HDAC active site pocket . This NO-induced shift in ECl– is likely due to release of Cl– from intracellular stores  on RGC survival and regeneration (Silencing ACs or introducing chelators into the eye to suppress Znneration . These fAlthough the non-cell-autonomous regulation of neuronal survival and pathological functioning by other neurons is just starting to be recognized as being important after optic nerve injury, neuronal circuits have been implicated in various pathological processes and cell death in other neurodegenerative diseases . In amyoDespite considerable progress in the areas of RGC protection and optic nerve regeneration, there is still a long way to go before we achieve satisfactory levels of functional recovery. One factor that is now coming to be appreciated is the crosstalk between cell-intrinsic and cell-extrinsic factors, particularly the role of neural circuits and the activity of neurons that form synapses with the affected cells. A greater understanding of the role of circuit activity might substantially augment the outcome achieved by manipulating RGCs’ intrinsic growth potential and cell-extrinsic factors.All authors conceptualized the study and wrote the manuscript.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.


Adverse events and childhood trauma increase the susceptibility towards developing psychiatric disorders  in adulthood. Although there are treatment strategies that have utility in combating these psychiatric disorders, little attention is placed on how to therapeutically intervene in children exposed to early life stress (ELS) to prevent the development of later psychopathology. The lateral habenula (LHb) has been a topic of extensive investigation in mental health disorders due to its prominent role in emotion and mood regulation through modulation of brain reward and motivational neural circuits. Importantly, rodent models of ELS have been shown to promote LHb dysfunction. Moreover, one of the potential mechanisms contributing to LHb neuronal and synaptic dysfunction involves endocannabinoid (eCB) signaling, which has been observed to critically regulate emotion/mood and motivation. Many pre-clinical studies targeting eCB signaling suggest that this neuromodulatory system could be exploited as an intervention therapy to halt maladaptive processes that promote dysfunction in reward and motivational neural circuits involving the LHb. In this perspective article, we report what is currently known about the role of eCB signaling in LHb function and discuss our opinions on new research directions to determine whether the eCB system is a potentially attractive therapeutic intervention for the prevention and/or treatment of ELS-associated psychiatric illnesses. Early life stress (ELS) and childhood trauma have been recognized as critical risk factors that increase the probability of developing psychiatric disorders in both clinical observations and animal models  function in mental health disorders, identifying critical brain regions and neural circuits has remained of high importance. Stress-induced dysfunction in a number of brain regions following ELS has been linked to stress-related disorders including the amygdala  Institutional Animal Care and Use Committee (IACUC) and guidelines from the National Institutes of Health (NIH) for the care and use of laboratory animals.3, 2.5 KCl, 1.2 NaH2PO4, 2.4 CaCl2, 1.00 MgSO4, 11.1 glucose, 0.4 ascorbic acid, saturated with 95% O2–5% CO2. Sagittal brain sections were taken at 300 μm and the LHb was grossly dissected from each as done previously  was carried out as described previously  together with the stria medularis and pineal gland  and diacylglycerol lipase (DAGL), respectively, that are localized to postsynaptic neurons  and 2-arachidonoylglycerol (2-AG) which primarily act on cannabinoid receptors 1 and 2 (CB1R and CB2R) as retrograde signals, although they can also act on transient receptor potential vanilloid receptor type 1 (TRPV1) as non-retrograde signals  which once activated decrease the release of neurotransmitter presynaptically through a variety of mechanisms including decreased cyclic adenosine monophosphate (cAMP), membrane hyperpolarization through activation of potassium channels and decreased vesicular release of neurotransmitter via inhibition of voltage-gated calcium channels  and gene expression allow us to gain greater spatial and temporal control for cell type- and circuit-specific eCB neuromodulation and eCB-mediated behaviors. In addition to using preclinical animal models to study the role of eCB signaling, it is also important to understand whether pharmacological manipulation of eCB signaling in a clinical setting is a viable treatment method. Until recently, one problem with imaging the habenula was due to its small size which makes functional imaging a challenge . For example, CRIP1a is a CB1R-specific accessory protein (Niehaus et al., In summary, given the robust role of the LHb in psychiatric disorders as well as the role of CB1R-mediated regulation of LHb activity, targeting the eCB system for ELS-induced psychiatric disorders is a potential therapeutic option. Although a majority of the clinical data has been conducted in adults, greater observations to the role of eCB signaling in adolescents, as well as possible clinical administration of eCB-targeting compounds could provide data as to their safety and efficacy in younger individuals. Lastly, targeting receptor complexes (Rosenbaum et al., The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.The animal study was reviewed and approved by Uniformed Services University IACUC committee.FN and RS designed the experiments and wrote the manuscript. RS performed the experiments, analyzed the data, and prepared the figures. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In the human embryo, the genetic program that orchestrates germ cell specification involves the activation of epigenetic and transcriptional mechanisms that make the germline a unique cell population continuously poised between germness and pluripotency. Germ cell tumors, neoplasias originating from fetal or neonatal germ cells, maintain such dichotomy and can adopt either pluripotent features  or germness features (seminomas) with a wide range of phenotypes in between these histotypes. Here, we review the basic concepts of cell specification, migration and gonadal colonization of human primordial germ cells (hPGCs) highlighting the analogies of transcriptional/epigenetic programs between these two cell types. In mammals, the unique core regulatory circuitry of transcriptional factors, epigenetics and signaling inputs regulating germline specification is becoming revealed. In the present work, we critically review old and new evidence that the unique pluripotency status is imposed on hPGCs by epigenetics traits regulated by pluripotency, and germline transcription factors that make these cells prone to undergo tumorigenesis. Such notion is discussed under the view that PGCs might be part of a pluripotent stem/progenitor cell pool exhibiting common markers that contribute to various somatic cell lineages .Germ cell tumors represent neoplasms derived from germ cells that can contain both immature and mature elements differentiating into several tissue types. Gonads are the preferred location for the onset of these tumors; however, they can be also found in extragonadal sites (EGCTs) accounting for 1–5% of all germ cell tumors. The most widely accepted theory suggests that these tumors arise from PGCs, the embryonic precursors of adult gametes, misplaced during their migration to gonads. While extragonadal localizations likely represent primary sites before puberty, EGCTs are considered metastases from occult or “burned out” gonadal cancers when they occur in young adults. PGCs migrate across the embryo to reach developing gonads, where they differentiate into gametes. These cells require an intrinsic motility program and external guidance cues to survive and successfully migrate. Proper guidance involves both attractive and repulsive cues, expression of a variety of adhesion molecules and is mediated by protein and lipid signaling . In addiGunnar Teilum first proposed a theory concerning the malignant potential of extragonadal germ cells known as the “germ cell theory”, suggesting that EGCTs originate from stray PGCs, which have undergone a malignant transformation during embryonic development . SeveralEGCTs are usually seen in children or young adults and typically arise in midline locations. In adults, the most common sites of primary EGCTs are, in descending order, the mediastinum, retroperitoneum, and cranium , while iGenetic conditions such as Klinefelter syndrome (KS), Marfan syndrome or Down syndrome, as well as somatic mutations of genes involved in PGC proliferation have been shown to associate with EGCTs, which can appear either in childhood or in the post-puberal age ,14,15,16Histologically, EGCTs comprise seminomas (SEs), also known as germinoma, and nonseminomatous tumors (NSTs). The latter includes teratoma, embryonal carcinoma, endodermal sinus tumor (yolk sac tumor), choriocarcinoma, and tumors with mixed histology. SEs are composed of a homogeneous population of neoplastic gonocytes. NSTs are more aggressive with heterogeneous histological features, including partially differentiated populations . NonsemiThe genome of the germ cells, which is the only pathway to the next generation, must be maintained in an epigenetically reprogrammable status that allows at the same time the formation of unipotent gametes. In the mammalian embryo, the specification of the germline is inductive and requires a complex core regulatory circuitry of transcription factors locally activated by a specific combination of growth factors. During the last ten years, the genetically specified program of transcriptional activity governing the germline specification and determination in mammals has been intensely studied. Much of what is known today about mammalian germline development comes from work performed on mouse models. Recent studies revealed, however, critical species-specific differences between mouse and human PGC development, including differences in the transcription factor network ,21. Germ cell specification occurs between wpc (week post conception) 2 and 3 in humans, at 6.5 days post coitum/conception (dpc) in mice  and post-implantation (primed state) epiblasts [+ progenitors transiently expressing the mesodermal/endodermal transcription factors GATA3, EOMES, and T , have the potential for germ cell specification but also for differentiation into multiple somatic fates including amnion and cells from the gastrulating embryo. In this contest, prolonged exposure to BMP4 induces another transcription factor, TFAP2C that operates in the TFAP2A+ progenitors, upstream of PRDM1 (BLIMP1) to regulate the expression of the germ cell fate determinant SOX17, while simultaneously preventing germline cells to become somatic cells and re-wiring pluripotency in the newly specified PGCLCs to a naive-like state. A specific role of TFAP2C in humans involves the opening of naive-specific enhancers and the acquisition of naive-like pluripotency [These studies revealed that hPGCs likely originate from a lineage-primed progenitor expressing the transcription factor TFAP2A. At around day post conception (dpc) 11, in the implanted blastocyst, under the action of the growth factor BMP4, TFAP2Apiblasts . TFAP2A+ipotency . While iipotency ,24. Lin28a promotes mouse PGC development in vivo and in vitro via the let-7 precursor microRNA that targets PRDM1 [Lin28a knockdown compromises the size of the germ cell pool both in males and females [Seminomas typically express SOX17 in their nuclei and are negative for SOX2, known to be not expressed in human PGCs , while Ets PRDM1 . While L females , its ove females . After specification, PGCs migrate along the hindgut and through the dorsal mesentery to the gonadal anlage . Bax-dependent apoptotic pathway in the absence of KITL [In the human embryo, the dorsal part of the yolk sac containing the PGCs, becomes incorporated in the developing gut during the lateral folding 4 wpc, indicating that PGC translocation into the gut may be passive . Either  of KITL ,38,39,40 of KITL . During this transit, continuous epigenetic remodeling leads to a progressively global erasure of genomic imprints. Actually, gonadal hPGCs show the lowest levels of DNA methylation at CpG islands observed in the human genome to date . This deInterestingly, Mallol and colleagues  recentlyBoth mPGCs and hPGCs proliferate during migration and after reaching genital ridges (GRs), where they become surrounded by cords of somatic cells. The regulation of hPGC proliferation in both sexes in vivo is poorly understood. Data obtained from in vitro studies of human PGCs cultured on cell monolayers indicated that hPGCs are responsive to the same compounds  and growth factors  known to control survival and/or proliferation of mPGCs . Most imNext paragraph focuses on the latent pluripotency derived from specification and how the highly hypomethylated status of chromatin imposed on PGCs during migration represents a risk for tumorigenic transformation.As for other embryonic cells, growth factors are critical determinants for the program of transcriptional activity and of the epigenetic changes occurring in germline specification and during PGC migration. If PGCs fail to reach their destination, the combination of the same growth factors in different microenvironments could allow their survival and induce their transformation.It is likely that on way to the gonad anlage, PGC fate depends on whether they can preserve their identity by repressing differentiation cues towards somatic lineages. Multiple lines of evidence suggest that PGC fate remains undetermined until they colonize the gonads where they progressively stop expressing pluripotency markers, lose their somatic potential, and acquire the germ cell fate, undergoing meiosis to produce oocytes in females or mitotic arrest to give rise to prospermatogonia in males. DAZL, DDX4, MAEL, and TDRD12 have been reported to be transcription factors crucial to determine the germ cell fate within the gonads .Such latent pluripotency has been elegantly demonstrated by in vitro transformation of mouse or human PGCs in the presence of a cocktail of growth factors, such as Kitl, Lif, and bFgf ,49,50, tOne of the first mechanisms responsible for mouse germ cell transformation is the deregulation of cell cycle/survival signals linked to overactive Pi3k/Pten/Akt pathway ,52,53 foDnd1 gene in the 129Sv genetic background causes testicular teratoma at high rates [Dnd1 gene; moreover, enhancement of the cell cycle and suppression of apoptosis are likely key events for the conversion of PGCs into teratoma. For example, p21cip1 and p27kip1 mRNAs, which encode negative regulators of the cell cycle, were identified as targets of Dnd1, and a deficiency of Dnd1 resulted in downregulation of the p21cip1 and p27Kip proteins [Dnd1 resulted in downregulation of Ezh2, a core member of polycomb protein complex 2 (Prc2), responsible for catalyzing H3K27 tri-methylation in PGCs forming teratoma. As a consequence, Cyclin D1 was upregulated and the PGC cell cycle dysregulated. In fact, it is known that this cyclin forms a complex with CDK4/6 and enhances the G1-S transition of the cell cycle in response to mitotic signals. Similar changes in the expression of these molecules were found in PGCs transformed in vitro in EG cells [DND1 in humans are unlikely to contribute significantly to germ cell tumors [Another molecule regulating the conversion of mouse PGCs to pluripotent cells is the RNA-binding protein, Dnd1. Deficiency in the gh rates . Dnd1 ingh rates . Severalproteins . Interesproteins . MutatioEG cells . Althougl tumors , these rIn the epigenetic reprogramming of the human germline a central role is played by the transcription factors TFAP2C and PRDM1. Stella, Dazl, c-Kit, Cxcr4 and Nanos3, are severely impaired [+ PGC progenitors, upstream of PRDM1, to upregulate the expression of the germ cell fate determinant SOX17, while simultaneously preventing germline cells to become somatic cells and reprogramming pluripotency to a naive-like state. Although the factor/s that induce TFAP2C in the human embryo are not known, its induction in vitro is mainly driven by BMP4. In line with this role in hPGC specification, TFAP2C is upregulated during primed to naïve reversion of hESCs and becomes widely distributed along naïve-specific enhancers [The TFAP2C protein can act as either a homodimer or heterodimer with other family members and plays important roles in the regulation of both embryonic development and carcinogenesis including germinoma. In the mouse, loss of Tfap2c leads to derepression of somatic programs, and loss of germ cells. In addition, in such condition, the expression of genes involved in germ cell maintenance and differentiation such as impaired . Moreoveimpaired . E-cadheimpaired  and in timpaired . Of noteimpaired . In humanhancers . It is lnhancers . Relevant to the subject covered by the present paper, high levels of TFAP2C protein were observed in precursor lesions of CIS and classical seminomas ,71.PRDM14 is a member of the PR domain-containing family. There are 16 PRDM proteins, many of which are known as master epigenetic regulators of cellular differentiation, especially in hematopoietic, epithelial, and neural cells. PRDM proteins are among the most widely deregulated proteins involved in the early transformation of normal cells to cancer cells and their expression has been shown to be related to cancer properties such as proliferation, drug resistance, and differentiation.PRDM14 is characteristically expressed in pluripotent stem cells where it exhibits multiple functions, including a scaffold for chromatin remodeling, a transcription regulator required for maintaining pluripotency, and a required component for epigenetic reprogramming. In mouse ES cells, Prdm14 ensures naive pluripotency through a dual mechanism: antagonizing activation of the FgfR signaling by the core pluripotency transcriptional circuitry, and repressing expression of de novo DNA methyltransferases that modify the epigenome to a primed epiblast-like state. Prdm14 exerts these effects by recruiting polycomb repressive complex 2 (Prc2) specifically to key targets and repressing their expression. Prdm14 over-expression increases activation rates of the Pi3k/Akt/mTorc1 cell proliferation pathway, blockage of apoptotic pathways and down-regulation of the cyclin inhibitor p21. Prdm14 is essential for mouse PGC specification by assisting Blimp1-mediated repression of somatic transcripts, initiating global epigenetic reprogramming and upregulating germline-specific genes, in particular Nanos3. Accordingly, Prdm14 is sufficient to induce the PGC state in EpiLCs and its deletion causes proliferation and differentiation defects in PGCLCs . As reported above, Mallol and colleagues  recentlyAlthough it has been shown to play an important role in hPGCLC specification , PRDM14 On the basis of the evidence reported here, we postulate that up-or down-regulation of transcription factors, in particular of TFAP2C and PRDM1, in misplaced PGCs, resulting from or causing altered epigenetics germline program, might be among the major causes for GCT formation. It is surely not a coincidence that PGCs can be reprogrammed to pluripotency in culture without genetic manipulation. The variety of characteristics of these tumors support the notion that they could basically derive from germ cells that remain incompletely determined into the germline (seminoma) with a dysregulated cell cycle or from germ cells in which somatic differentiation programs are not maintained in the repressed state (nonseminomas) .Alternatively, we speculate that in microenvironment where PGCs do not receive any differentiation signals but remain viable, as their own fate determinants are gradually depleting, the cells cannot assume a specific fate. Under these conditions the PGCs possessing a latent pluripotent state are susceptible to transformation. Although they are mainly eliminated by cell death for the prevailing expression of cell death factors (probably BAX) over cell survival factors (perhaps BCL-X) , in someThe progression and eventual detection of the tumors occurs decades later in humans. Long-term maintenance of pluripotent stem cells in a naïve state may increase risk of chromosomal anomalies . One of Reflecting this “ambivalence” of the core regulatory circuitry, early PGCs could fluctuate from a naive and primed state regulated by transcriptional circuitry and signaling inputs. Associated to these remarks is the question of why such apparently risky strategy for PGC specification and determination has been selected during mammalian evolution. Many invertebrates and nonmammalian vertebrates express cytoplasmic germ plasm determinants at or soon after PGC specification instead of after gonad colonization. Is it possible that the extended duration of mammalian PGC migration benefits from an uncommitted pluripotent status, perhaps to counteract somatic development? Does the pluripotent state of PGCs facilitate cell migration? Or do mammalian PGCs postpone germ cell determination to contribute to non-gametogenic lineages? Of note, PGCs are suggested to have a pluripotent stem cell potential that may give rise to hematopoietic stem cells, that originate in the aorta-gonad-mesonephric region ,81 and pAll these results support the very old Simkins’ hypothesis , recentl


FMR1 gene and subsequent loss of its protein product FMRP. Despite this simple genetic origin, FXS is a phenotypically complex disorder with a range of physical and neurocognitive disruptions. While numerous molecular and cellular pathways are affected by FMRP loss, there is growing evidence that circuit hyperexcitability may be a common convergence point that can account for many of the wide-ranging phenotypes seen in FXS. The mechanisms for hyperexcitability in FXS include alterations to excitatory synaptic function and connectivity, reduced inhibitory neuron activity, as well as changes to ion channel expression and conductance. However, understanding the impact of FMR1 mutation on circuit function is complicated by the inherent plasticity in neural circuits, which display an array of homeostatic mechanisms to maintain activity near set levels. FMRP is also an important regulator of activity-dependent plasticity in the brain, meaning that dysregulated plasticity can be both a cause and consequence of hyperexcitable networks in FXS. This makes it difficult to separate the direct effects of FMR1 mutation from the myriad and pleiotropic compensatory changes associated with it, both of which are likely to contribute to FXS pathophysiology. Here we will: (1) review evidence for hyperexcitability and homeostatic plasticity phenotypes in FXS models, focusing on similarities/differences across brain regions, cell-types, and developmental time points; (2) examine how excitability and plasticity disruptions interact with each other to ultimately contribute to circuit dysfunction in FXS; and (3) discuss how these synaptic and circuit deficits contribute to disease-relevant behavioral phenotypes like epilepsy and sensory hypersensitivity. Through this discussion of where the current field stands, we aim to introduce perspectives moving forward in FXS research.Fragile X Syndrome (FXS) is a leading inherited cause of autism and intellectual disability, resulting from a mutation in the  FMR1 gene, leading to its transcriptional silencing and the subsequent loss of its protein product, Fragile x mental retardation protein  is the most common inherited form of intellectual disability (ID) and one of the leading known genetic causes of autism spectrum disorders -dependent long-term depression (LTD) at excitatory synapses  animals, which corresponded with a shift in the temporal window for LTP induction at these synapses , which promotes the degradation of the synaptic scaffolding protein PSD-95 , an auditory brainstem area important for sound localization, found evidence for increased sound-evoked activity and hyperexcitability at the population level in FMR1 KO mice  are reduced in the amygdala of adult  interneurons was deficient, resulting in reduced activity-dependent inhibition  inhibitory neurons , somatostatin positive (SST), and vasoactive intestinal peptide positive (VIP) interneurons , an enzyme involved in the degradation of perineuronal nets (PPNs) which preferentially stabilize synaptic connections with PV neurons  channels; and small- and big- conductance Ca2+- activated  K+ channels . Interestingly, increased INaP current in the entorhinal cortex was not due to direct modulation of ion channel expression or function by FMRP, but rather through exaggerated mGluR5 signaling  that plays a crucial role in setting resting membrane potential and dendritic excitability  are another class of ion channels important for dendritic excitability and the mRNA for several VGCCs have been shown to be targets of FMRP  cells  maintaining E/I balance in the network  and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs) in mediating homeostatic synaptic scaling at excitatory synapses  leads to a reduction of surface AMPARs and this has been observed to occur during synaptic downscaling  and retinoic acid receptor α (RARα) signaling in visual cortical circuits. RA regulates local homeostatic plasticity at the level of individual dendritic spines. In the case of activity blockade, a drop in CaFMR1 KO mice, as discussed above in “Ion Channel Dysregulation and Altered Intrinsic Excitability in FXS” section. Interestingly, in line with these findings on the intrinsic properties of FMR1 KO neurons, a recent study indicated that FMR1 KO neurons show a significant increase in input resistance along with distinct alterations in homeostatic intrinsic plasticity in different subsets of cortical neurons. Bülow et al.  being one of the most reliable and consistent approaches to assessing hyperexcitability in vivo. In AGS experiments, mice are presented with a 110–120 dB siren or alarm sound for a duration of 1–3 min. The mice are then scored for behavioral seizures with SE and death as a common final end point for the FMR1 KO mice , with FMR1 KO mice, however often in the opposite direction as seen in humans, with enhanced rather than reduced PPI magnitude , a modification of the paradigm where a startle-eliciting sound is preceded by a lower level auditory or tactile cue that reflexively reduces ASR magnitude  at suprathreshold intensities, suggestive of increased perceptual sensitivity. Indeed, RT-intensity functions have been shown to be a reliable psychoacoustic measure of loudness growth in both humans , no differences in overall whisker-evoked activity were seen in FMR1 KO mice in this study, as assessed by two-photon Ca2+ imaging of layer 2/3 neurons  FMRP is important for activity-dependent development and refinement of synaptic connectivity and loss of FMRP during early life critical periods can lead to abnormal excitatory and inhibitory synaptic connectivity, resulting in altered E/I balance that is likely to contribute to circuit hyperexcitability; (2) FMRP is required for ongoing activity-dependent plasticity in the mature brain and seems particularly important for regulating mGluR-dependent changes to excitatory synaptic function, inhibitory transmitter release, and cellular excitability; (3) FMRP regulates the expression and function of multiple ion channels through a variety of direct and indirect mechanisms. Changes to ion channel function with loss of FMRP not only directly affect intrinsic excitability in a manner to promote hyperactivity, but can lead to profound changes in pre-synaptic release properties and post-synaptic dendritic integration, which in turn will influence synaptic function and plasticity in a variety of ways; and (4) FMRP is an important regulator of homeostatic plasticity, which is essential for stabilizing activity levels in the brain, and impairments to this stabilization mechanism are likely to contribute to circuit hyperexcitability in FXS. The wide-ranging consequences of FMR1 deletion underscore the importance of examining multiple aspects of neuronal function  in FMR1 KO models under the same experimental conditions, ideally using approaches that span from single neurons to intact circuits to behavior. Future studies must also continue to make use of spatial and temporally restricted deletion of FMR1 to parse the contribution of different cell-types, brain regions, and developmental timepoints to FXS phenotypes. The development of FMRP-tat peptides to reintroduce different FMRP segments to FMR1 KO neurons is a powerful tool for dissociating FMRPs function via direct protein-protein interactions from its canonical role in translational regulation (Zhan et al., FMR1 KO rat (Till et al., Here we have highlighted the number of ways in which loss of FMRP can lead to neuronal hyperexcitability, and how these cellular and circuit changes contribute to the FXS phenotype. Because FMRP regulates multiple activity-dependent processes and is regulated in an activity-dependent manner itself, it is difficult to disentangle the direct effects of FMRP loss from secondary consequences. While some of the phenotypes described above are likely to be compensatory adaptions rather than direct pathologies related to All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.