Optic neuritis (ON) is an inflammatory attack of the optic nerve that leads to visual disability. It is the most common optic neuropathy affecting healthy young adults, most commonly women aged 20–45 years. It can be idiopathic and monophasic or as part of a neurologic disease such as multiple sclerosis with recurrence and cumulative damage. Currently, there is no therapy to repair the damage from optic neuritis. Animal models are an essential tool for the understanding of the pathogenesis of optic neuritis and for the development of potential treatment strategies. Experimental autoimmune encephalomyelitis (EAE) is the most commonly used experimental rodent model for human autoimmune inflammatory demyelinating diseases of the central nervous system (CNS). In this review, we discuss the latest rodent models regarding optic neuritis, focusing on EAE model, and on its recent achievements and developments. Optic neuritis is an inflammatory demyelinating disorder of the optic nerve. The typical form of optic neuritis is idiopathic or associated with multiple sclerosis (MS). Atypical forms of optic neuritis can occur in association with other inflammatory disorders or due to infections, immune-stimulating medications, and paraneoplastic disorders.The prevalence of optic neuritis is estimated to be as high as 115/100,000 depending upon geography and ethnicity . There iAccording to the involved site, optic neuritis can be classified as retrobulbar optic neuritis, affecting any part of the optic nerve behind its entry to the eyeball. This is the most common form of optic neuritis, and on examination, the disk often appears normal. Optic neuritis that includes inflammation of the optic disk is known as papillitis, which is visible on examination as hyperemia, swelling of the disk, blurring of disk margins, and distended veins . An affeThe common clinical presentation of a patient with optic neuritis is unilateral visual acuity loss, visual field loss, color vision deficits, decreased contrast, and brightness sense. There is also periocular pain precipitated by eye movements that may precede the visual loss by a few days. The extent of visual function damage may vary significantly according to the etiology of the optic neuritis. In typical optic neuritis and optic neuritis associated with MS, visual acuity loss is moderate, conversely, optic neuritis associated with neuromyelitis optica spectrum disorder (NMOSD) or myelin oligodendrocyte glycoprotein (MOG) often presents with severe vision loss , 6.Diagnostic investigations include MRI, visual evoked potentials (VEP), and cerebrospinal fluid (CSF) examination. MRI is performed to characterize the location and extent of inflammation and to rule out other etiologies such as multiple sclerosis. VEPs measure optic nerve function and may be useful when anatomic studies by MRI are equivocal. Degeneration of retinal ganglion cells (RGC) and retinal thinning which correlate with measures of persistent visual dysfunction after optic neuritis is demonstrated by optical coherence tomography (OCT) , 8.Treatment with high-dose corticosteroids shortens the period of acute visual dysfunction but does not affect the final visual outcome in typical optic neuritis . AtypicaAnimal models are essential for the understanding of etiology and pathogenesis of immune-mediated processes and to develop therapeutic strategies which eventually will lead to effective treatments for human diseases. Many studies have been conducted in order to understand the pathophysiological mechanisms of optic neuritis using different models.The EAE model is the most commonly used experimental rodent model for human autoimmune inflammatory demyelinating diseases of the CNS. EAE is a complex system of interaction between multiple immunological and neuropathological mechanisms leading to inflammation, demyelination, axonal loss, and gliosis. The regulatory mechanisms of resolution of inflammation and remyelination also occur in EAE.In EAE, the optic nerve lesions are, in most cases, a part of the whole CNS disease process which includes the brain and the spinal cord. There are subtypes that are found to be more associated with optic nerve involvement. For example, in the dark agouti rat model, acute optic nerve inflammation manifests earlier than spinal cord inflammation (PMID: 31267597) whereas in C57BL6 mice, optic nerve inflammation occurs chronically along with inflammation in the rest of the CNS (PMID: 29903027).Mycobacterium tuberculosis. CFA enhances peripheral immune response by promoting Th1 response and increases blood-brain barrier (BBB) permeability , or proteolipid protein (PLP), in the context of an inflammatory stimulus to induce encephalomyelitis. It is typically induced by either active immunization with myelin-derived proteins introduced into the CNS, emulsified in complete Freund's adjuvant (CFA), which contains heat-inactivated eability . PertussPassive immunization, by adoptive transfer of activated myelin-specific CD4+ T lymphocytes –16, alloThe inflammatory process in the CNS is first indicated by activated microglia, local macrophages, and peripheral T lymphocytes. Activated T cells undergo maturation and clonal expansion; later, they differentiate into effector cells and migrate through the blood circulation to breach the BBB. Adhesion molecules are expressed on endothelial cells in CNS microvasculature, allowing activated T cells to bind to these molecules and penetrate the endothelium. To further penetrate the subendothelial basement membrane which is mainly composed of type IV collagen, T cells utilize matrix-degrading enzymes . Once thMyelin loss leads to a disruption of axonal function, and axons may eventually die back depending on the severity and persistence of the inflammatory response. Axons are vulnerable to damage by inflammatory cytokines, enzymes, and nitric oxide which are expressed by activated immune cells during the inflammatory process and cause direct cytotoxic effects. Axons that survive demyelination when the inflammation resolves may become remyelinated; however, in most EAE phenotypes, inflammatory damage leads to neuronal cell death, axonal loss, and gliosis –26. IrreThe clinical evaluation of the disease progression can be done in several ways. Due to the fact that active EAE is an ascending progressive disease that progresses into paresis, it initially affects rodent tail tone, followed by limb motor deficits. Daily scoring of these parameters provides information regarding spinal cord inflammation and demyelination . The optEngineering a new MOG-specific TCR transgenic mouse, the 2D2 mouse made by Bettelli et al. in 2003 produces transgenic T cells with receptors capable of recognizing MOG presented by MHCII . ApproxiNeuromyelitis optica (NMO) is an inflammatory demyelinating disease involving the optic nerves and spinal cord. The damage is caused due, in part, to autoantibodies against aquaporin-4, a water channel on astrocytic foot processes at the blood-brain barrier. There are several animal models of NMO that have been developed . By crosSeveral studies showed that passive transfer alone of the aquaporin-4 antibody from patients with neuromyelitis optica is insufficient to reproduce the disease in rodents unless extremely high levels are infused. In the context of EAE with myelin-targeted T cells or even after an injection of Freund's adjuvant, the antibody can reach its target in the CNS and contribute to inflammatory demyelination –39. DireIn 1977, an experimental model for acute allergic optic neuritis was induced in guinea pigs. They exhibited two distinct clinical patterns: “retrobulbar optic neuritis” with normal fundus and “neuroretinitis” with hyperemia and swelling of the disk and retinal edema. Histopathology of the “retrobulbar neuritis” revealed that some of them had brain involvement without any involvement of the optic nerve, while others involved the retrobulbar portion of the optic nerve and chiasm with multiple foci of demyelination. In the neuroretinitic animals, the lesions were localized behind the lamina cribrosa and had an appearance characteristic of papilledema .Due to a unique anatomic structure, where the anterior part of the optic nerve within the lamina cribrosa is unmyelinated and supported by modified astrocytes, a barrier between the optic nerve and the retina restricts the inflammation to the optic nerve during ON, and prevents retinal inflammation as well , 45. In Theories regarding specific proteins playing a critical role in the mechanism of EAE optic neuritis have been proposed. Lipocalin-2, a protein that regulates diverse cellular processes, is expressed and secreted by microglia and astrocytes due to inflammatory stimuli in the central nervous system . LipocalAstrocytes in the optic nerve are known to play a pivotal role in neuroinflammatory processes in the CNS. In the human disease NMOSD, the astrocytic water channel aquaporin-4 is the naïve target of an aberrant immune response, especially in the optic nerve . The rolA study suggested that dietary supplementation with a balanced mixture of fatty acids (FAs) including omega 3 and omega 6, efficiently limit inflammation and prevent RGC degeneration, demonstrating a neuroprotective effect in EAE models . FurtherReactive oxygen species (ROS) are formed as byproducts in a variety of biochemical reactions. When generated in excess or not appropriately regulated, ROS may cause cellular damage and tissue injury. During an inflammatory process, activated immune cells release ROS, leading to oxidative stress and tissue damage and causing demyelination and axonal destruction . Reductiin vitro (Alpha lipoic acid (ALA) is a naturally occurring antioxidant which has recently been investigated for its neuroprotective capacities during inflammation . It has in vitro . When adin vitro . A studyin vitro . The amiin vitro .Unfortunately, studies suggesting antioxidative-based treatment for optic neuritis that showed benefit in the EAE model, did not progress to clinical trial in humans, and if so, did not show any benefit for the treatment in these cases.Optic neuritis may lead to permanent visual loss mediated by RGC damage. The goal of neuroprotection is to preserve axon structures and function; it is highly important given the poor regenerative capacity of neurons. The specific mechanism and timing of the RGC damage during the disease process are crucial for understanding the damage and how to prevent it. In a study aimed to determine whether axonal injury due to inflammation mediates apoptotic death of RGCs, EAE was induced followed by demyelination with significant axonal loss which was followed by loss of RGC. It was suggested that inflammatory cell infiltration mediates demyelination and leads to direct axonal injury. RGCs die by an apoptotic mechanism triggered by axonal injury . AnotherHigh-dose intravenous corticosteroids are the standard treatment for acute optic neuritis. The optic neuritis treatment trial (ONTT) demonstrated that intravenous methylprednisolone followed by oral prednisone helps recovery of visual acuity and results in slightly better vision at 6 months. Oral prednisone alone is not an effective treatment. It increases the risk of recurrent episodes of optic neuritis . HoweverBrimonidine (BMD) is a selective α2-adrenergic receptor agonist that is used clinically for the treatment of glaucoma. Topical administration of BMD at 0.2% was shown to result in vitreous concentration of 2 nM and above, which is known to activate alpha(2)-receptors . There hVarious mechanisms for neuroprotection by brimonidine have been suggested, including brain-derived neurotrophic factor (BDNF) activation in the retinal ganglion cells, glutamate inhibition, suppression of excitotoxicity in retinal ganglion cells, stimulating trophic factor release from Müller cells, and cell-survival signal upregulation, as well as apoptosis downregulation , 74.Voltage-gated sodium channels (Nav) are suggested to have a key role in the etiology of EAE by causing axonal degeneration . Nav1.6 Phenytoin is a voltage-gated sodium channel blocker used as an antiseizure medication. A study evaluating the effect of phenytoin on axonal degeneration in the optic nerve in EAE, reported that whereas ~50% of optic nerve axons are lost at 27–28 days in untreated EAE, only ~12% of the axons are lost if mice with EAE are treated with phenytoin .Myelin is a lipid-rich protective covering that surrounds axons. It provides an electrical isolation that allows fast nerve transmission and metabolic support to neurons. In demyelinating diseases, myelin loss leads to disruption in electrical conduction . FollowiA major target is lipid metabolism in oligodendrocytes. Lipids account for about 70% of the myelin membrane. A decrease in cholesterol synthesis in astrocytes might affect demyelination of autoimmune disease . CholestNormally, there is a balance between the rate of demyelination and remyelination processes. Myelin debris are phagocytosed by microglia; when this balance is disrupted, such as in inflammatory demyelinating diseases, it causes an accumulation of myelin debris that impairs remyelination .Another study hypothesized that the inability to repair the demyelination damage caused by the inflammation during EAE may result from decreased cholesterol synthesis by astrocytes. Upregulation of cholesterol-synthesis gene expression was observed in oligodendrocytes during remyelination .Another study demonstrated an accelerated remyelination after EAE induction by direct lineage analysis and hypothesized that newly formed myelin remains stable during the inflammation process due to the absence of MOG expression in immature myelin. Furthermore, withholding oligodendrocyte differentiation and myelination results in acceleration of remyelination, thus preventing axonal loss and improving functional recovery .Optic neuritis is an inflammatory demyelinating disorder of the optic nerve with up to 0.1% prevalence at certain regions and potentially devastating visual outcomes. Animal models using the EAE experimental rodent model are essential for the understanding of etiology, pathogenesis of immune-mediated processes and for the development of therapeutic strategies for optic neuritis.In this review, we discussed the latest rodent models regarding optic neuritis, focusing on the widely used EAE model, on its recent achievements and developments. So far, none of the therapeutic strategies have shown a substantial achievement regarding optic neuritis in the field of neuroprotection and remyelination. Further investigation is required.YR and ML contributed to the writing of the manuscript. Both authors contributed to the article and approved the submitted version.ML currently receives research support from the National Institutes of Health. He also received personal compensation for consultation with Alexion, Viela Bio, and Genentech and has served on the scientific advisory boards for Alexion, Genentech, and Viela Bio. The remaining author declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A novel bioassay for the Sonic Hh ligand in tissue specimens may assist HCC diagnosis with negative α-fetoprotein and predict early microvascular invasion. In-depth exploration of the Hh signaling deepens our understanding of its molecular modulation in HCC initiation, drug sensitivity and metastasis, and guides precise management of HCC on an individual basis.Hedgehog (Hh) signaling is a classic morphogen in controlling embryonic development and tissue repairing. Aberrant activation of Hh signaling has been well documented in liver cancer, including hepatoblastoma, hepatocellular carcinoma (HCC) and cholangiocarcinoma. The present review aims to update the current understanding on how abnormal Hh signaling molecules modulate initiation, progression, drug resistance and metastasis of HCC. The latest relevant literature was reviewed with our recent findings to provide an overview regarding the molecular interplay and clinical relevance of the Hh signaling in HCC management. Hh signaling molecules are involved in the transformation of pre-carcinogenic lesions to malignant features in chronic liver injury, such as nonalcoholic steatohepatitis. Activation of GLI target genes, such as ABCC1 and TAP1, is responsible for drug resistance in hepatoma cells, with a CD133 Hedgehog (Hh) signaling is a classic and conserved pathway, controlling embryonic development and tissue repairing under physiological conditions . DisruptAt the same time, molecular targeting agents and checkpoint inhibitors are optimal selections for refractory malignancies, such as pulmonary, hepatocellular and breast carcinoma, and primary and induced resistance to these therapeutics prompts basic and clinical investigations on the molecular interplay underlying drug resistance. Hh signaling has been found to be one of key signaling pathways in modulating drug resistance through ATP-binding cassette (ABC) transporters, such as ABCC1 and TAP1, in our previous studies ,17. Intrahepatic and extrahepatic invasion and distal metastasis are the main cause of death in HCC. Involvement of the Hh signaling in every step of progressive behaviors has been gradually elucidated at molecular levels and with clinical relevance. As one of the clinically important parameters, circulating tumor cells (CTCs) are the source of new metastatic lesions . Their cMost methodologies in detection of the Hh signaling molecules use molecular approaches, such as RT-PCR, for the mRNA levels, and Western blot or immunohistochemical staining for the protein expression levels and location in tissues. So far, quantitative determination of Hh signaling molecules has not been used to monitor drug sensitivity or prognosis prediction, although a sensitive ELISA kit is available for the Sonic Hh ligand (SHh) measurement . Since HThe present review aims to provide an overview covering the signaling mechanism, modulation and interaction with other signaling molecules, such as TGF-β. The major attention is focused on the molecular mechanism of Hh signaling in modulating drug resistance and metastasis of HCC. Additionally, a novel bioengineering assay for the detection of SHh in HCC specimens and its potential application are elaborated. The use of pharmacotherapeutic candidates for intervention of Hh signaling in cancer therapy has been covered recently by extensive reviews ,15,19,20Hedgehog (Hh) signaling is aberrantly activated from chronic inflammation to malignant transformation.Targeting Hh molecules, such as SMO or GLI, confers rational intervention for malignancies.Hh participates in drug resistance by maintaining stem-like properties, onset of EMT and induction of ABC transporters in HCC.Hh signaling contributes to HCC metastasis by mediating EMT, MMP production or involvement in multiple steps of metastasis.A sensitive aptasensor-based assay was developed to detect Sonic Hh ligand in HCC specimens with acceptable positivity and high specificity.Hh signaling is activated via canonical or non-canonical pathways under different conditions. The canonical Hh pathway includes secretory ligands , the 12-pass transmembrane protein patched1 (PTCH1), heptahelical transmembrane G-protein-coupled receptor smoothened (SMO) and transcription factor GLI proteins  . In the As mentioned above, Hh signaling in adult livers is normally silent. Hh ligands are expressed in injured hepatocytes, activated hepatic stellate cells (HSCs), Kupffer cells, endothelial cells, progenitor cells and natural killer cells; and activated Hh signaling is involved in tissue repair . During Detection of Hh molecules in HCC specimens indicates that the reactivation of Hh signaling contributes to hepatic carcinogenesis. SHh ligand was detected in 60% HCC tissues, especially in the tumor nests ,34. IncrFor canonical Hh signaling, primary cilia function as a signaling hub for SMO-dependent GLI activation. The non-canonical Hh signaling pathway does not involve the ligand and receptor (PCTH1) and is operated in a SMO-independent fashion. The non-canonical pathways of GLI activation were observed in hepatoma or cholangiocarcinoma cells with impaired cilia expression . The proHh signaling not only orchestrates embryogenesis but also regulates regeneration and inflammatory response to liver damage. Liver epithelial (hepatocyte or cholangiocyte) cells are induced to secrete Hh ligands under pathologic conditions, and in turn activate Hh signaling in neighboring Hh-responsive cells via a paracrine mechanism. Initial GLI2 activation upregulates the transcription of GLI1 and the entire signaling pathway. Persisting Hh signaling activation in injured hepatocytes and non-parenchymal cells contributes to hepatocellular carcinogenesis under pathologic conditions, such as nonalcoholic steatohepatitis (NASH) with significant fibrosis .Chronic infection with hepatitis B virus (HBV) and hepatitis C virus (HCV) increased hepatic expression of the Sonic and Indian ligands, which bound to the PTCH1 receptor on myofibroblast-like HSCs and hepatic sinusoidal endothelial cells (SECs) to initiate liver fibrogenesis and angiogenesis . Not onlfl/fl knockout (KO) mice displayed a decreased tumor burden. Hh-responsive HSCs and EpCAM+ ductal cells upregulated MYC and TGF-β2 expression and were transformed into fibrotic and pro-tumorigenic phenotypes [In nonalcoholic fatty liver disease (NAFLD), the Hh activity is positively correlated with the severity of liver damage. Lipotoxicity-injured hepatocytes with ballooning degeneration were the main source of Hh ligand production and accumulation of GLI2-positive Hh-responsive cells paralleled with the liver fibrosis stage in NAFLD patients ,50. The enotypes . With grenotypes ,58. FiguR172H and KRASG12D mutation, suggesting that Hh signaling activation facilitates malignant transformation in combination with other oncogenes, such as c-Myc and Wnt signaling [Hepatocellular adenoma (HCA) is a kind of benign tumor; however, it has an increased risk of malignant transformation when telomerase reverse transcriptase (TERT) was activated ,60. A neignaling . Clinical management of HCC needs to be balanced with tumor burden, liver reserve function and patients’ general health. According to the Barcelona Clinic Liver Cancer (BCLC) system, resection, liver transplantation and ablation are recommended to patients with a solitary nodule ≤ 2 cm or less than three nodules ≤ 3 cm (BCLC stage 0 and A); chemoembolization is recommended to patients with multinodular (BCLC stage B); and systemic therapies are recommended to patients with macrovascular invasion or extrahepatic spread (BCLC stage C) . Less th+ and CD133+ organoids or cells derived from HCC patients. HCC-derived CD44+ organoids displayed sorafenib-resistance, and a GLI inhibitor, GANT61, suppressed proliferation of both CD44+-HCC patient-derived organoids and CD44+ hepatoma cells, indicating that CD44+ cells with a sorafenib-resistant phenotype was maintained by the Hh signaling [−/CD133+ cells derived from primary HCC specimens, and was reported to suppress PTCH1 expression and consequently promote self-renewal of tumor-initiating cells through Hh signaling [In an adult liver, bilineage-potent hepatic progenitor cells (HPC) are located within the canals of Hering, and its appearance is in parallel with the extent of the ductular reaction driven by hepatocellular damage ,67. The ignaling . A long ignaling . Sestrinignaling ,72. Sestignaling . In summignaling ,58.Epithelial–mesenchymal transition (EMT) denotes a process by which epithelial cells lose their polarity and adhesion, acquire cell mobility and transform to the mesenchymal phenotype . This trThe ATP-binding cassette (ABC) transporter superfamily functions in transporting sugars, peptide, ions and drugs. ABCB1, ABCC1 and ABCG2 are three of the most characterized ABC transporters involved in multiple drug resistance through increasing the efflux of the chemotherapeutic agents ,83. Of nHCC often presents as early microvascular invasion (78.6% (22/28) in resected tissue)  and 10–4The particular feature of liver sinusoids allows easy local invasion of the HCC, and the malignant nodules may lack a connective capsule. Multiple studies have demonstrated that the positivity of Hh signaling molecules, such Sonic ligands, PTCH1 and GLIs, were about 50–70% in HCC specimens, which was much higher than the pericancerous liver tissue of a different base disease background, such as HBV or HCV infection or NAFLD ,36,106. −/EpCAM− Huh-7 cells, when passing through transwell selection, formed a unique subpopulation, Huh-7-trans, and had extremely high levels of MMP expression compared with their native Huh-7 hepatoma counterpart cells, and at the same time, the GLI1/2 expression levels were accordingly increased. Addition of an SMO inhibitor, LDE225, partially suppressed the expression of MMPs and GLI1/2 and attenuated their invasive behaviors [The metastasis involves a multiple-step process, including invasion, the disruption of the basement membrane by tumor cells, intravasation, transport and extravasation as well as colonization in other tissue . For eacehaviors , indicatehaviors . Additioehaviors ,17. Our ehaviors . −/EpCAM− hepatoma cells in the medium lobe of the liver in NOD-SCID-IL2-γR(-/-) NSG mice. These cells were transduced with a lentiviral vector encoding the firefly luciferase gene (LUX-PGK-EGFP) before injection [In order to study the metastatic properties of in situ xenografts in immunodeficient mice, we implanted 2000 HLF unsorted or HLF CD133njection . As shownjection ,11. As dnjection , and mayIt is common for cells to acquire mesenchymal features to be able to migrate from the original locus to distal sites through EMT. The EMT transcription factors   are ofteThe preferable sites for malignant metastasis depend on the CTCs, blood and lymphatic supply and microenvironmental niche of the tissue. Intrahepatic invasion, portal vein tumor thrombus and abdominal, pulmonary and intracranial metastasis are often seen with HCC. One unique phenomenon is the recurrence of HCC in implants of liver transplantation with the cell origin of the recipient, indicating that malignant cells spreading to other sites of the body re-circulate back to the preferable environment of the implant liver. This self-seeding happened in 5.7% of 60 cases 40 months after transplantation, and may be more often seen in living-donor recipients (31.8%) within 3 years ,116. TheOne phenotypic switch from CTCs to return the epithelial feature of HCC is mesenchymal–epithelial transition (MET), which is not yet fully understood regarding self-reseeding in the implants or establishment of new metastatic nodules in other sites. However, the HGF–cMET signaling axis may play a critical role in this process based on the evidence summarized in a recent review . The sigIn summary, metastasis is a complex process and involves coordinated action of multiple signaling pathways and is controlled by mutations, CSCs, the microenvironmental niche and other factors. Nevertheless, how Hh signaling participates in this multiple-step process is largely unknown, especially for HCC. It is relatively clear that Hh signaling modulates the synthesis and production of MMPs for assisting early extravascular process and governing EMT through modulating the production of transcription factors . More studies are warranted to elucidate whether and how Hh signaling controls the survival, formation of metastatic lesions in other tissue with other signaling molecules in a coordinating fashion in a reliable animal model, such as in situ xenograft tumor formation with live imaging of longitudinal tracking. Hh signaling molecules, such as ligands (SHh), receptors (PTCH1) or transcription factors (GLI1/2) in the tumor tissue or body fluid, may reflect tumor progression, metastasis, drug sensitivity or treatment response ,7,33. PrAs one of most refractory malignancies, the death rate of HCC ranks the third, whereas its prevalence lies in the sixth among malignancies. The high death rate is due to its primary or secondary resistance to drug therapy and early metastasis, such as local or portal vein invasion and distal metastasis. In other words, the refractory status and metastasis of HCC account for major causes of lower survival rate. As a classic morphogenic factor, the Hh signaling participates in the malignant transformation in chronic liver injury, oncogenic initiation, progression, drug resistance and metastatic process. The specific ligands, receptors, SMO transmembrane protein and transcription factors (GLI1/2) have become molecular targets for cancer therapy or biomarkers for prognosis prediction. The application of SMO inhibitors, vismodegib, sonidegib or saridegib, has been approved for the treatment of glioblastoma and base cell carcinoma. Clinical trials of these inhibitors in other malignancies, such as pancreatic, lung and colon cancers, are on-going. −/EpCAM− surface marker profile, and was associated with EMT, drug resistance and aggressive behaviors. Further studies have uncovered that increased TAP1 and ABCC1 through both GLI1/2 were largely responsible for the primary drug resistance in these CD133−/EpCAM− subpopulations. It is promising when GLI1/2, ABCC1 or TAP1 was suppressed, hepatoma cells became more sensitive to chemotherapeutics with a much lower half maximal inhibitory concentration (IC50), indicating the critical role of Hh signaling in modulating the drug sensitivity in these cells. Increasing evidence suggests that Hh signaling modulates the metastatic process through EMT, MMP production and may participate in the mobilization and survival of CTCs. For liver transplant recipients, self-seeding is a pivotal issue for HCC recurrence in transplanted liver, and affects the function of the implant organs and survival of the recipients. Although the literature is scarce regarding whether Hh signaling contributes to the establishment of new metastatic lesions, its oncogenic and promoting action on the microenvironmental niche may explain its role in facilitating the multiple steps of metastasis. Our previous studies have demonstrated that Hh signaling molecules, such as SHh ligand and GLI1/2, were increased at mRNA and/or protein levels in 50–70% of HCC arisen from HBV-positive patients, which were consistent with reports from other studies. Moreover, high signaling activity has been identified in hepatoma cells with a CD133With multidisciplinary efforts, a sensitive and reliable aptasensor-based bioassay has been established to directly measure SHh ligand in resected HCC specimens. This assay is able to detect SHh at the pmol levels. A pilot study has demonstrated that SHh levels in liver tissue and HCC were in a detectable range, and the measurement could distinguish malignant and pericancerous liver tissue with 100% specificity when the upper limit cut-off was set at a 99% confidence interval. Moreover, the measurement was compensatory for detection of AFP-negative HCC cases and could indicate early microvascular invasion, which is an early sign of portal vein thrombus and poor prognosis. In conclusion, upregulated Hh signaling molecules are frequently observed in HCC, and may participate in the initiation, progression, drug resistance and metastasis of the malignancy through EMT or other target genes, such as ABCC1, TAP1 or MMPs. Targeting Hh signaling molecules, SMO and GLI1/2 may confer therapeutic options for improvement of the treatment outcome. The quantitative determination of SHh may offer a sensitive and reliable assay for monitoring drug sensitivity, metastasis or prognosis, although additional clinical investigations in a larger cohort are needed for validation of its broad application.


BCR-ABL1 negative (aCML) is a rare myelodysplastic syndrome (MDS)/myeloproliferative neoplasm (MPN) with a high rate of transformation to acute myeloid leukemia, and poor survival. Until now, the diagnosis has been based on morphological grounds only, possibly making the real frequency of the disease underestimated. Only recently, new insights in the molecular biology of MDS/MPN syndromes have deepened our knowledge of aCML, enabling us to have a better molecular profile of the disease. The knowledge gleaned from next generation sequencing has complemented morphologic and laboratory WHO criteria for myeloid neoplasms and can provide greater specificity in distinguishing aCML from alternative MDS/MPN or MPNs. The most commonly mutated genes (>20%) in aCML are SETBP1, ASXL1, N/K-RAS, SRSF2, and TET2, and less frequently (< 10%) CBL, CSFR3, JAK2, EZH2, and ETNK1. Several of these mutations affect the JAK-STAT, MAPK, and ROCK signaling pathways, which are targetable by inhibitors that are already in clinical use and may lead to a personalized treatment of aCML patients unfit for allogeneic transplant, which is currently the only curative option for fit patients. In this review, we present two emblematic clinical cases and address the new molecular findings in aCML and the available treatment options.Atypical chronic myeloid leukemia,  BCR-ABL1 negative myelodysplastic syndrome (MDS)/myeloproliferative neoplasm (MPN) with a high rate of transformation to acute myeloid leukemia (AML), and is historically characterized by poor survival. The challenges of aCML management affect both diagnosis, due to the heterogeneity of the disease clinical features and the absence of unique biomarkers, and treatment choices, since no current standards of care exist.Atypical chronic myeloid leukemia (aCML) is a rare Here we describe two representative aCML clinical cases presenting in two different age groups and review the clinicopathologic features and management of the disease. The clinical characteristics and outcome of the two patients are summarized in 9/L with absolute neutrophil count (ANC) 34.2 × 109/L, hemoglobin (Hb) was normal and platelets (Plts) were 103 × 109/L. Manual differential revealed 46% hypogranulated and hypolobated neutrophils, 12% band forms, 12% metamyelocytes, 6% monocytes, 6% myelocytes, 2% promyelocytes, 2% eosinophils, and 14% lymphocytes. No hepatosplenomegaly was noted on physical examination. A bone marrow (BM) examination and trephine biopsy were performed and revealed hypercellularity with a significant myeloid expansion without excess of blasts and with mild dysplasia. Dyserythropoiesis and dysplastic megakaryocytes, including hypolobated forms, were noted  were 41.5 × 10re noted . KaryotyTET2  and one pathogenic mutation in EZH2 . After confirming the diagnosis of aCML, the ETNK1 gene was sequenced by Sanger method and resulted wild type.As a next step, the diagnostic sample was subjected to next generation sequencing (NGS) analysis using TruSight Myeloid Sequencing Panel (Illumina), covering 54 genes mutated frequently in myeloid malignancies see , which r9/L, ANC 14.7 × 109/L). He had a previous history of transitional cell carcinoma treated with radical cystectomy almost three years before referral and in complete remission ever since. In his medical history, he had a first hematology referral due to erythrocytosis at the time of the bladder neoplasia. At that time, a complete, first level molecular biology assessment for Philadelphia negative (Ph-) MPN was carried out and resulted negative, leading to the conclusion of a spurious erythrocytosis. An automated differential showed 90% neutrophils. The manual differential revealed 30% neutrophils with Pelger-Huet nuclear abnormality, 17% band forms, 6% metamyelocytes, 8% monocytes, 15% myelocytes, 14% promyelocytes, 1% blasts, and 9% lymphocytes. Fluorescence in situ hybridization (FISH) for t and molecular assessment for BCR/ABL1, JAK2V617F, CARL, and MPL were tested again at this time, and all resulted negative. The bone marrow (BM) aspirate was hypercellular without increased blasts but with subtle dyserythropoiesis and dysmegakaryopoiesis primarily consisting of hypolobated megakaryocytes with separate nuclear lobes , SRSF2 , and TET2 . A new mutation (p.H243P) in one of the hotspots described by Gambacorti-Passerini et al. [A 71 year old man was referred to our Hematology department due to leukocytosis , and the leukocyte alkaline phosphatase level may be low, normal, or increased, therefore lacking diagnostic utility [The clinico-pathologic characteristics of aCML include splenomegaly and a neutrophilic leukocytosis with left shift and prominent granulocytic dysplasia  . The WBCe marrow . Due to  utility ,5. A hyp utility ,7.9/L) at presentation, increased immature precursors in the PB, female gender, and age > 65 years [ASXL1, SETBP1, and TET2 genes have been associated with a more aggressive disease [The median overall survival (OS) of aCML patients is between 12 and 25 months ,10,11,1265 years ,10,13. I disease .The risk of progression to sAML seems to be higher in case of palpable hepato- or splenomegaly, monocytosis, BM blastosis > 5%, marked dyserythropoiesis, and transfusional requirement .Atypical CML is a challenging myeloid malignancy with features of both myeloproliferative and myelodysplastic syndromes. The MDS/MPN category was introduced in the 2011 WHO classification to include myeloid neoplasms with clinical, laboratory, and morphologic features that overlap MDS and MPN ,4. In adCurrently, the diagnosis of aCML is reached primarily on morphology-based criteria, as its clinical and molecular features overlap with those of other myeloid malignancies. No molecular abnormality detected to date is specific of aCML, and multiple mutations are often present in various combinations, due to the malignant multi-step clonal evolution of myeloid malignancies ,16.9/L with ≥ 10% of immature granulocytes, and ≤ 20% blasts in the blood and the BM [The main feature characterizing aCML is the presence of neutrophilic leukocytosis and marked dysgranulopoiesis. Moreover, to fulfil the diagnostic criteria, WBC should be ≥ 13 × 109/L), fewer immature granulocytes (< 10%), no granulocyte dysplasia, and no excess of blasts.These findings can distinguish aCML from CNL, that presents with higher WBC count , PDGFRB (5q31–33), FGFR1 (8p11), or JAK2 (9p24).The diagnostic work up should start with complete blood count with manual differential, morphological examination of PB smear, BM examination with assessment of dysplastic features, cytogenetic analysis and FISH to exclude Ph chromosome and rearrangements involving Once CML has been excluded, second level testing should include human leukocyte antigen (HLA) typing in transplant eligible patients, and assessment with a NGS myeloid panel, not only to confirm diagnosis but also to open the possibility of a target therapy.An algorithm summarizing the diagnostic work-up is reported in The molecular features of aCML include an increased frequency of gene fusions or an aneuploid karyotype. One or two chromosomal aberrations, e.g., trisomy 8 or 9, del 20q), −7/7q or isochromosomes 17q, are found in up to 50% of patients 0q, −7/7q but are Although in many myeloid malignancies there is prognostic significance associated with abnormalities of chromosome 7 and complex cytogenetics, no study to date has reported their prognostic role in aCML or in other MDS/MPN, with the exclusion of CMML.The heterogeneity of aCML, both clinical and molecular, is partly due to the difficulty in comparing reports of this rare disease in case series characterized over several decades, during which diagnostic criteria were redefined and novel DNA sequencing techniques emerged.The knowledge gleaned from NGS has complemented morphologic and laboratory WHO criteria for myeloid neoplasms and can often provide greater specificity in distinguishing aCML from alternative MDS/MPN or MPNs . HoweverASXL1 and TET2, a frequent finding in aCML, correlate with older age and monocytosis (TET2), are related to “clonal hematopoiesis of indeterminate potential” (CHIP), and are present in the founder clones of both MDS and MPN. [TET2 mutations [The mutational landscape of MDS/MPN represents an overlap between those of MDS and MPN. Similar to MDS, the majority of cases of MDS/MPN harbors at least two mutations involving epigenetic regulation and splicing factors in the dominant clone, with acquisition of additional mutations over time . Moreoveutations .SETBP1, ASXL1, N/K-RAS, SRSF2, and TET2, and less frequently (< 10%) CBL, CSFR3, JAK2, EZH2, and ETNK1 [JAK2, CALR, and MPL genes are wild type, although a few cases of CMML and aCML have been reported to harbor JAK2 mutations [The most commonly affected genes > 20%) in aCML are 0% in aCMnd ETNK1 ,22,23,24utations ,25.SETBP1 and ETNK1 appear to be the alterations most closely associated with aCML, even though they are not univocally disease-specific [SETBP1 and ETNK1 may be useful diagnostic tools for the disease [Mutations in specific ,21,22,26 disease ,21,22,26SETBP1 have been identified in up to one third of aCML patients as compared to 10% only of MDS/MPN-U and 6–15% of CMML, and occasionally in JMML or AML secondary to MPN or MDS [SETBP1 are associated with a more adverse clinical presentation , suggesting that this alteration is not only relevant to leukemic oncogenesis, but also provides important prognostic value [Recurrent mutations of N or MDS . Multiplic value .ETNK1 were identified at a prevalence of 8.8% in sixty-eight cases of aCML. All mutations were heterozygous, and in two cases occurred concomitantly with SETBP1 mutations. ETNK1 encodes an ethanolamine kinase, catalyzing the first step of the phosphatidylethanolamine biosynthesis pathway, which is critical for regulating the cell membrane architecture and the topology of transmembrane domains of membrane binding proteins [ETNK1 mutations is not completely understood. However, mutations appear to be relatively specific to aCML, as they are otherwise reported only in few cases of CMML and of systemic mastocytosis with associated eosinophilia [In 2015, recurrent mutations in nophilia .CSF3R gene, initially described in both CNL and aCML [CSF3R mutations are less frequent in aCML, being found in < 10% of patients, and are rare in MDS/MPN-U [CSF3R should prompt a careful revision of the diagnosis to rule out the possibility of CNL, as the molecular status would otherwise suggest.By contrast, mutations in the and aCML , appear DS/MPN-U ,8,27. ThSFSR2, especially in the context of monocytosis, may orient the diagnosis towards CMML, in which this gene is mutated in up to 50% of cases.Additionally, mutations in In conclusion, NGS studies could be useful for confirming the clonality of the disease, especially in cases with mild proliferative features and concomitant inflammatory comorbidities that might mimic a reactive leukocytosis process, and may help in the differential diagnosis among different MDS/MPN categories together with morphologic and clinic data.Moreover, the molecular findings may have crucial relevance in directing personalized therapies, since aCML-associated genetic alterations might be susceptible to specific therapeutic approaches, directly targeting the mutant proteins or their associated pathways .NRAS, JAK2, and CSF3R mutations directly impact on the MAPK and JAK/STAT pathways; SETBP1 encodes a protein named SET binding protein 1 (SEB) that impacts on AKT and MAPK pathways; mutations in the PTPN11 gene result in constitutively activated RAS signaling [Indeed in the last decade, the identification of oncogenic mutations in signal transduction proteins has clarified that specific pathways regulating the proliferation of myeloid lineage cells may be deregulated in leukemia . Severalignaling . InhibitASXL1, RUNX1, EZH2, and TP53 [In addition, in myeloid disorders, and particularly in MDS, mutations in several genes are adverse prognostic factors, namely alterations of and TP53 ,32,33. IFinally, the identification of molecular markers may be useful to monitor minimal residual disease (MRD), as validated in other myeloid diseases and shown in a few studies of aCML ,35.2 IV for 5 days on 28 day cycles) as an off-label prescription. After 3 cycles, the patient achieved a partial hematological response [Due to progressive leukocytosis and monocytosis, a BM assessment was repeated, documenting a stable disease without increase of blasts. However, considering the young age of the patient, the progressive leukocytosis despite cytoreduction and the progressive monocytosis that has been correlated with a higher risk of progression to AML ,23, we oresponse  with a nSETBP1 mutations and the generally poor risk related to SRSF2 alterations in the context of other myeloid neoplasms. Unfortunately, all the mutations detected in this case were not actionable, therefore pegylated interferon alpha (Peg–IFNα) 3 mcg/kg/week was chosen as first line therapy. After 3 months of treatment, a partial response was achieved and maintained over time with no toxicity.The patient was considered unfit for HSCT due to age and to the previous diagnosis of solid neoplasia. Although the patient’s WBC count was only mildly elevated and the Hb and Plts count were well preserved, his long term prognosis was poor due to the general high risk of AML progression related to the diagnosis of aCML itself and the unfavorable prognosis associated to his molecular profile, specifically with JAK2 inhibitors, are currently being investigated. However, up to date, the only curative treatment remains HSCT, an option available for the younger patients only. Participation on clinical trials should be considered in all cases. A possible treatment algorithm is reported in No standard of care is currently available for aCML management. This rare entity of MDS/MPN, as already detailed, is characterized by heterogeneous clinical and genetic features with a very poor prognosis. In addition, due to the low disease incidence and the absence of large randomized clinical trials, the physician’s choices remain challenging. Several treatment strategies validated in other myeloid diseases have been evaluated in aCML, starting from erythropoiesis stimulating agents (ESAs) to improve the anemia and up to cytoreductive drugs to control the proliferation and reduce the blast count , hydroxyurea (HU), or PEG–IFN–α. Given the recent clarification of the mutational landscape of MDS/MPN, different target therapies, including n = 4) or MUD (n = 4) or a syngeneic transplantation from a twin sibling (n = 1) [HSCT is considered as the sole potentially curative strategy for aCML. Data on this approach are scant and most evidence originates from studies including patients with heterogeneous MDS/MPNs. Lim et al. reported a series of 10 patients with MDS/MPN who received HSCT. The 5-year OS and disease-free survival (DFS) rates of the whole cohort were 42.2% and 46%, respectively. Only 2 out of 10 patients were aCML, and, at the median follow-up of 47.5 months, both were alive and in remission . Similar (n = 1) . All pat (n = 1) . A subse (n = 1) . These p (n = 1) . At a meSETBP1 mutation) prognostic factors and to offer HSCT at diagnosis in high risk cases and defer the procedures in the others [No evidence-based information on the best timing of transplantation is currently available, and indications rely on expert opinions. Gotlib et al. proposed HSCT for all candidate patients at the time of diagnosis , wherease others .The choice between these two approaches should also consider the availability of a suitable donor at the time of diagnosis and the presence of a targetable mutation that might be appropriate for an attempt with molecular therapies.In conclusion, allogeneic HSCT may be considered a promising approach for a subset of aCML patients and the new molecular findings may pave the way to a MRD driven approach to treat patients after HSCT; however, a large number of cases are elderly and may be not candidates for the HSCT.BCR/ABL1 positive and negative CML. Out of seven aCML patients, four obtained a clinically meaningful response but their median survival was only 13 months and the 2 year survival rate was 14% [Based on the use of the HMA azacytidine or decitabine in CMML, different studies have tried to use these drugs also in aCML . Kantarj was 14% . More re was 14% ,46,47,48HMA cannot be considered as a standard of care for aCML and their use is off-label. However, these drugs represent an alternative to standard chemotherapy for the pre-transplant cytoreduction in case of a low blast count, as in our clinical case 1, or in the presence of comorbidities. Moreover, given the evidence that the use of HMA as a bridge to HSCT in MDS does not worsen the transplant outcome and OS after transplant, it is tempting to extrapolate this hypothesis also to aCML ,50,51. HHMA may also be considered for patients who are ineligible to transplant that do not carry actionable somatic mutations and are intolerant to HU as an alternative to IFN.The role of AML-like intensive chemotherapy has not been explored extensively, but is usually offered to selected patients with high-risk proliferative clinical behavior as a bridge to HSCT .HU is commonly used to control splenomegaly and leukocytosis. Older studies reported that HU and IFN-alfa are able to induce a complete hematological response, however with short duration ,11. PEG–Several promising targeted therapies are currently being investigated, including the JAK inhibitor ruxolitinib, the SRC kinase inhibitor dasatinib, and the MEK inhibitor trametinib , with manageable or reversible toxicities [NRAS mutation who obtained a near complete hematologic response within several months of treatment with trametinib [CSF3R mutant aCML [Another option for ted mice . These pxicities . A singlametinib . Moreoveant aCML .Finally, there might be a future role for activators of PPA2, a major protein phosphatase which acts as a tumor suppressor, in the setting of aCML with SETBP1 overexpression. In fact, overexpression of SETBP1 protects SET from protease cleavage, increasing the formation of a SETBP1-SET-PP2A complexes that result in PP2A inhibition, promoting proliferation of the leukemic cells. Pharmacological activation of PP2A seems to offer a future therapeutic alternative, since in vitro PP2A restoration by PADs  reverseClinical trials including aCML patients are needed to come to clear treatment recommendation, even if the rarity of disease is an obstacle to the design of studies with high numerosity.At the moment of manuscript preparation, only 2 trials are actively recruiting aCML patients. Both are focused on the blastic phase of aCML. One is a phase I/II trial on a combination of azacitidine, venetoclax, and pevonedistat in patients with newly diagnosed AML secondary to other myeloid diseases including aCML (NCT03862157). The second study is a phase II trial testing the combination of topotecan hydrochloride and carboplatin with or without veliparib in AML secondary to myeloproliferative disorders or aCML in advanced phase (NCT03289910).The diagnosis of aCML is always a difficult task to accomplish. Until now, the diagnosis has been based on morphological grounds only, possibly underestimating the true frequency of the disease. Only recently, new insights in the molecular biology of MDS/MPN syndromes have deepened our knowledge in aCML. However, all the mutations described in aCML so far are neither specific nor diagnostic, being present in a wide range of other hematological malignancies and also in the normal elderly population, even though at a very low VAF. With a more comprehensive molecular profiling, the diagnostic criteria will be hopefully refined in the future to include also genetic features resulting in decreased reliance on absolute cutoffs in blood counts that may vary along with the disease history of the patients. Nonetheless, as discussed earlier, in some cases these alterations can be more than a simple diagnostic test and may allow personalized treatment. Large collaborative efforts are needed to design studies that will provide data so that the management of these diseases will be evidence-driven.


Brassinosteroids (BRs) play pivotal roles in the regulation of many dimensions of a plant’s life. Hence, through extensive efforts from many research groups, BR signaling has emerged as one of the best-characterized plant signaling pathways. The key molecular players of BR signaling from the cell surface to the nucleus important for the regulation of plant growth and development are well-established. Recent data show that BRs also modulate plant responses to environmental stresses such as drought and pathogen infection. In this mini review, we present the recent progress in BR signaling specifically in the post-translational SUMO modification of BR’s master regulators, BES1/BZR1. We also discuss recent findings on the crosstalk between BR, UV light, and jasmonic acid signaling pathways to balance growth during light stress and pathogen infections. Finally, we describe the current update on the molecular link between BR signaling and intracellular auxin transport that essential for plant development. Arabidopsis thaliana (Brassinosteroids (BRs) are plant steroid hormones that have a significant impact on plant growth and development, cell elongation, resistance to pathogens, and plant responses to environmental stresses. In the past decades, substantial progress in dissecting molecular mechanisms of the BR signaling pathway has been achieved using the model plant thaliana .Arabidopsis, BRs are perceived by a plasma membrane receptor, BRASSINOSTEROID INSENSITIVE1 (BRI1) leucine-rich repeat receptor-like kinase  like motif of DNA binding domain and are known as master regulators that modulate the expression of many target genes . These tRegulation of BES1 and BZR1 occurs in multiple ways. Phosphorylation of BES1/BZR proteins is well studied, and additional mechanisms that involve oxidation, ubiquitination, alternative splicing, and degradation are emerging 302 although this SUMOylation status is unaffected by brassinolide (BL) application. Compared to wild-type seedlings, the accumulation of BES1 and unphosphorylated BES1 protein is higher in a SIZ1 T-DNA insertion line (siz1-2). In the presence of cycloheximide, a protein synthesis inhibitor, BES1 protein in wild type is less stable than in siz1-2 seedlings with or without BR, suggesting a negative role of SIZ1 in BES1 stability. In agreement, a cell-free degradation assay shows that without a 26S proteasome inhibitor (MG132), MBP-BES1 protein degradation is slower when incubated with total protein extracts from siz1-2 seedlings than that of the wild type. Meanwhile, MG132 presence prevents MBP-BES1 degradation in both WT and siz1-2 extracts. Together, these data show that the SIZ1-mediated SUMOylation promotes BES1 instability and stimulates its proteasome-dependent degradation.Arabidopsis protoplast, they investigated the promoter activity of two BR repressed genes, DWAF4 and AT2G210, and one BR induced gene, SAUR-AC1. As expected, wild-type BES1 represses the activity of DWF4‐ and AT2G4210-LUC. Meanwhile, BES1K302R variant in which SIZ1 SUMOylation site is mutated, further represses the DWF4- and AT2G4210-LUC activity. Similarly, the activation level of SAUR-AC1-LUC activity is significantly higher in the presence of BES1K302R variant rather than wild-type BES1. To further confirm this finding, the binding ability of BES1 to the promoter of DWAF4, AT2G210, and SAUR-AC1 was investigated. Chromatin immunoprecipitation (ChIP) assay results showed that the BES1 binding ability increases in the siz1-2 compared to the wild type regardless of MG132. Based on these data, BES1 transcriptional activity is modulated by SIZ1-mediated SUMOylation . Using aulp1a mutant compared to wild-type since ulp1a mutant seedlings have longer root than wild-type seedlings in the presence of sodium chloride. Interestingly, ulp1a mutant is more tolerant to BR biosynthesis inhibitor brassinazole (BRZ), suggesting that the BR signaling in the ulp1a mutant is upregulated. To confirm the ulp1a BR response, the expression of BR biosynthesis genes and BR activated genes were investigated. BR availability activates BR induced genes under normal BR signaling, while BR biosynthesis genes are suppressed to maintain BR homeostasis. Indeed, in the ulp1a mutant, the expression of BR biosynthesis genes and BR activated genes decreases and increases, respectively, confirming that the ULP1a is negatively impacting BR signaling -qPCR, an electrophoretic mobility shift assay (EMSA), and a dual-luciferase (LUC) reporter assay confirmed that the BES1 directly binds the 3' downstream region of PDF1.2a and PDF1.2b and suppresses their expression.Recent data from rade-off ,C. Liao efensins . Second,efensins . In bes1bes1-D is more susceptible to Botrytis cinerea infection than wild-type, while stable overexpression lines of PDF1.2a show improved resistance to B. cinerea. In response to B. cinerea invasion, the double mutant, bes1-D and PDF1.2a overexpression lines, is more similar to PDF1.2a overexpression lines than bes1-D  and UDP-glucosyl transferase 74B1 (UGT74B1), as well as the expression of MYB34, MYB51, and MYB122 transcription factors that specifically regulate the expression indole-GS genes. Second, BES1 suppresses the transcriptional activity of MYC2 that involved in the activation of GS-biosynthetic genes. In the LUC reporter assay, the co-expression of both BES1 and MYC2 further represses the activity of CYP79B3-LUC and UGT74B1-LUC activity compared to MYC2 expression alone , and PIN-LIKES (PILS) family proteins  family, and BR signaling components, such as BIN2 alters the expression of auxin-responsive genes. Phosphorylation of ARF2 eliminates its DNA binding and repressor activity. Furthermore, around 40% of differentially expressed genes in e to BRZ . Recentlproteins ,D.shy2-31, and a wild-type background. The fluorescence signals of PIN1, PIN3, and PIN7 are higher in the shy2-31 than wild type, which argue that the SHY2 inhibits the expression of those PIN proteins. Moreover, in the shy2-31 background, BL further elevates fluorescence signals of PIN1, PIN3, and PIN7 while BRZ reduces the signals. Thus, PIN7 by BES1, the expression of some of PIN genes including PIN7 is partially regulated by BRs via BES1 and SHY2.BR also regulates PILS-mediated intracellular auxin transport. PILS proteins evolve independently from PIN proteins, although they are structurally similar . The rolOEPILS5) to identify secondary mutations with enhanced hypocotyl and root growth deficiency , carries a secondary mutation that surprisingly mapped to the BRI1 gene. The identified mutation creates a single amino acid change, glycine to serine, and the impact of this mutation resembles that of the previously identified weak loss-of-function bri1 mutant, bri1-6, or bri1-119  and the binding of BZR1 to PILS2 and PILS5 promoter was confirmed by chromatin immunoprecipitation (ChIP)-sequencing  than the wild type, suggesting a non-transcriptional regulation of PILS5 expression by BR signaling , a blue light receptor involved in the inhibition of hypocotyl elongation, interacts with BZR1 and suppresses BZR1 DNA binding ability. CRY1 also promotes the BZR1 phosphorylation to prevent its nuclear localization (In harsh environmental conditions, plants cannot move around; thus, fine-tuning their growth and development using sophisticated internal signaling networks enable plants to survive and to conclude their life cycle. BR signaling is essential in regulating plant growth in response to environmental stresses. For example, d growth . BES1 ald growth . Furtherd growth . New stulization . Meanwhilization . InvestiB. cinereal infection, BES1 directly suppresses the expression of defensins genes, PDF1.2a and PDF1.2b. On the other hand, upon exposure to S. exigua herbivory infection, BES1 negatively modulates MYC2 transcriptional activities. BES1 also represses the expression of indol-GS synthetic genes, CYP79B3 and UGT74B1 and GS-associated transcription factors. This negative regulation mediated by BES1 may be crucial to maintain homeostasis of defensins and GS compounds that are increasing upon pathogen attacks (Molecular mechanism behind the antagonistic interaction between BR and JA signaling in response to pathogen attacks gives some clues on how plants balance their growth in this condition. It appears that the BES1 is emerging as a node that connects BR and JA signaling and it negatively modulates different aspect of JA induced plant defense depending on the type of pathogens. In response to  attacks .Additional layers of hormone crosstalk have been depicted by how BR signaling modulates the intracellular auxin distribution. BR signaling is directly repressing the expression of PILS proteins that restrict nuclear auxin availability . BR signAK and YY conceived the idea and wrote the review. 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.


The medial prefrontal cortex (mPFC) integrates inputs from multiple subcortical regions including the mediodorsal nucleus of the thalamus (MD) and the ventral hippocampus (vHPC). How the mPFC differentially processes these inputs is not known. One possibility is that these two inputs target discreet populations of mPFC cells. Alternatively, individual prefrontal cells could receive convergent inputs but distinguish between both inputs based on synaptic differences, such as communication frequency. To address this, we utilized a dual wavelength optogenetic approach to stimulate MD and vHPC inputs onto single, genetically defined mPFC neuronal subtypes. Specifically, we compared the convergence and synaptic dynamics of both inputs onto mPFC pyramidal cells, and parvalbumin (PV)- and vasoactive intestinal peptide (VIP)-expressing interneurons. We found that all individual pyramidal neurons in layer 2/3 of the mPFC receive convergent input from both MD and vHPC. In contrast, PV neurons receive input biased from the MD, while VIP cells receive input biased from the vHPC. Independent of the target, MD inputs transferred information more reliably at higher frequencies (20 Hz) than vHPC inputs. Thus, MD and vHPC projections converge functionally onto mPFC pyramidal cells, but both inputs are distinguished by frequency-dependent synaptic dynamics and preferential engagement of discreet interneuron populations. The medial prefrontal cortex (mPFC) serves as an integrative hub for both cognitive and affective circuitry. The prelimbic region of the mPFC receives input from multiple subcortical regions including the mediodorsal nucleus of the thalamus (MD) and the ventral aspect of the hippocampus (vHPC) . IntriguOne possibility is that inputs coming from the vHPC versus MD activate discreet populations of mPFC cells, similar to the concept of “labeled lines” seen in the peripheral sensory system. Optogenetic and electrical stimulation studies suggest pyramidal neurons in layers 2, 3, and 5 may all receive functional inputs from both the vHPC and MD . HoweverAlternatively, prefrontal pyramidal cells could distinguish between both inputs based on qualitative differences, such as differences in their frequency-dependent dynamics. Neural oscillations, reflecting the rhythmic fluctuation of neural activity, have been shown to carry behaviorally relevant information. In this context, vHPC–PFC communication relevant to anxiety-related behaviors appears to preferentially occur at lower, theta-range (4–12 Hz) frequencies, while beta-range (20 Hz) frequencies have been shown to be relevant for MD–PFC communication during working memory maintenance . HoweverTo address directly the ways in which synaptic inputs from the MD and vHPC differentially target and communicate with prefrontal cells, we utilized optogenetic tools in combination with slice electrophysiology. We focused on neurons in superficial layers 2 and 3 in the prelimbic mPFC because prior work indicated that they are most densely contacted by the MD, while still receiving input from the vHPC . In addiAll animal procedures were approved by New York State Psychiatric Institute’s Animal Care and Use Committee. Parvalbumin Cre  or Vasoactive intestinal peptide Cre  mice were mated with Ai9 tdTomato reporter mice  to produce offspring for ChrimsonR and Chronos dual labeling studies. Adult (6–8 week-old) PV-Cre/Ai9 or VIP-Cre/Ai9 mice were used for viral injections  for in vitro optogenetic electrophysiology experiments. Animals were allowed to recover for 8 weeks prior to electrophysiology experiments. Animals were fed ad libitum and reared under normal lighting conditions (12/12 light/dark cycle). Male and female mice were used for all experiments. C57BL/6J  or 129SvEvTac  mice were used for single ChannelRhodopsin2 (ChR2) labeling studies. Adult (6–8 week old) C57BL/6J mice were injected with AAV5-hSyn-hChR2 (H134R)-EYFP in the MD and allowed to recover for 6 weeks prior to electrophysiological recordings. Adult (6–8 week old) 129SvEvTac (for 25 °C recordings) or C57BL/6J (for 32 °C recordings) mice were injected with AAV5-CaMKIIa-hChR2(H134R)-mCherry in the vHPC and allowed to recover for 8 weeks prior to electrophysiological recordings.12), AAV5-Syn-ChrimsonR-tdTomato (4.2 × 1012), AAV5-hSyn-hChR2(H134R)-EYFP (4.8 × 1012), AAV5-CaMKIIa-hChR2(H134R)-mCherry (4.5 × 1012).Sterile stereotactic survival surgery of C57BL/6J, PV-Cre/Ai9 and VIP-Cre/Ai9 mice was performed using the following viral injection volumes and unilateral coordinates: MD  and vHPC . Virus was injected at the rate of 0.1 μL/min and the pipette was allowed to remain in place between 2 and 5 min at the penetration sites. For the injection of the vHPC in 129SvEvTac mice five injections were done at −3.10 and at −3.30 AP levels for a total of 10 injections in one hemisphere. At each AP level, the five injection sites were −2.90, −4.0; −3.30, −3.60; −3.30, −1.7; −3.70, −3.2; −3.70, −2.5 . 129SvEvTac coordinates are in mm relative to Bregma  or brain surface at the most medial coordinate (DV) and virus was infused at a rate of 200 nL/min. Viruses were obtained from UNC Vector Core and viral titers are as follows: AAV5-Syn-Chronos-GFP  at a rate of 5 mL/min. A recording temperature of 25 °C was used for all experiments except for one subset of experiments in mice expressing ChR2(H134R) in the MD or vHPC, which was performed at 32 °C. Unless specifically indicated in the figure, recording temperatures were always 25 °C. Electrodes were pulled from 1.5 mm borosilicate-glass pipettes on a P-97 puller (Sutter Instruments). Electrode resistance was typically 3–5 MΩ when filled with internal solution consisting of (in mm): 130 CsOH monohydrate, 130 D-Gluconic acid (50%), 10 HEPES, 2 MgCl2, 0.2 EGTA, 2.5 MgATP and 0.3 NaGTP  or 130 K-gluconate, 5 NaCl, 10 HEPES, 0.5 EGTA, 2 MgATP and 0.3 NaGTP  single injection recordings).Whole-cell voltage clamp recordings were performed in visually identified pyramidal cells and genetically identified PV- and VIP-expressing interneurons in the prelimbic (PrL) region of layer 2/3 of the mPFC. Recordings were obtained with a Multiclamp 700B amplifier  and digitized using a Digidata 1440A acquisition system (Molecular Devices) with Clampex 10 (Molecular Devices) and analyzed with pClamp 10 (Molecular Devices). Following decapitation, 300 μm slices containing mPFC were incubated in artificial cerebral spinal fluid containing (in mM) 126 NaCl, 2.5 KCl, 2.0 MgClPyramidal cells were visually identified based on their shape and prominent apical dendrite at 40x magnification under infrared and diffusion interference contrast microscopy using an Olympus BX51W1 microscope coupled to a Hamamatsu C8484 camera (Olympus America). The recorded cell was placed in the center of the field of view, held at −70 mV in voltage clamp and the current response evoked by a four- or five-pulse train of 5 or 1-ms pulses of blue light (470 nm) or red light (635 nm) delivered at 8 or 20 Hz by a light-emitting diode  was recorded. For single ChR2(H134R) stimulation experiments, the LED intensity was varied between 5 and 20% of maximum intensity to produce detectable light-evoked postsynaptic currents (le-PSCs) of a comparable size, when possible. For dual ChrimsonR and Chronos stimulation experiments, the 470 nm LED intensity was kept at 1% and the 635 nm LED intensity was varied between 2 and 90% to produce detectable le-PSCs of a comparable size, when possible. Each experiment consisted of a four or five pulse train of 5 or 1-ms pulses of blue or red light delivered at 8 or 20 Hz; this stimulation paradigm was repeated 5 times for a given experiment and the current traces generated from all five stimulations were averaged and filtered at 2000 Hz with an eight-pole low-pass Bessel filter. Although minimal stimulation intensities were used, occasionally light stimulation provoked an action potential-like current instead of a le-PSC (identified based on response shape as well as amplitude). For the percent responding analyses, we included these large-amplitude responses in our averages. For the response ratio analyses, these large amplitude responses were excluded from the averages. In two instances, this resulted in the need to exclude a cell from the response ratio analysis that was included in the percent responding analysis, because all stimulus repetitions contained an action-potential like response. Le-PSCs from the averaged current traces were manually identified in MiniAnalysis (Synaptosoft). The relevant parameters for each response  were imported into Matlab where they could be sorted based on experimental condition.For a subset of experiments, 1 μM tetrodotoxin (TTX) in combination with 200 μM 4-aminopuridine (4-AP) was added to the bath is isolate monosynaptic transmission. We did not include TTX and 4-AP in all experiments because TTX/4-AP decreases the amplitude of the evoked postsynaptic response, and therefore biases towards not detecting a connection when one might in fact exist.Proper targeting of the MD and vHPC was verified for all mice used for electrophysiology. After mPFC slices were prepared, the remaining brain tissue was postfixed by immersion fixation in 4% paraformaldehyde at 4° for at least 48 h. The tissue was then washed for 24 h in phosphate buffered saline (PBS) before 100 μm slices through the MD and vHPC were prepared on a vibratome (Leica). Immunostaining to detect EYFP, GFP, or mCherry was performed using the following primary antibodies: EYFP or GFP , mCherry ; tdTomato was visible without additional amplification. Alexa Fluor-conjugated secondary antibodies  were used for secondary detection.For single injection experiments to compare cross-reactivity of ChrimsonR and Chronos to blue (470 nm) and red (635 nm) light, we used 3–6 cells per condition from 1 to 2 mice . For characterization of ChrimsonR and Chronos kinetics we used between 10 and 15 cells per condition from between 2 and 5 mice . To compare MD and vHPC inputs to pyramidal cells, we used 10–12 cells per condition from 6 mice . To compare MD and vHPC inputs to PV cells, we used 8–10 cells per condition from 5 mice . To compare MD and vHPC inputs to VIP cells, we used 6 cells per condition from 2 mice . For single injection experiments of ChR2(H134R) (including those at both 25° and 32 °C) in the MD or vHPC, we used 20 cells for the MD from 6 mice  and 16 cells for the vHPC from 6 mice .For analysis of effects of ChR2 variant stimulated and stimulation number, we used a two-way repeated measure ANOVA for continuous variables (le-PSC amplitude and ratios). For these analyses, we looked at both main effects of the two independent variables as well as if there was an interaction. Statistical significance was denoted in the figures based on Bonferroni-corrected posthoc analysis to assess the effect of ChR2 variant at each repeated stimulus. For analysis of effects of brain region stimulated, we used a Chi-square comparison for categorical variables (responding/non-responding). For analysis of effects of brain region stimulated and stimulation number, we used a two-way repeated measure ANOVA for continuous variables (le-PSC amplitude and ratios). For these analyses, we looked at both main effects of the two independent variables as well as if there was an interaction. Statistical significance was denoted in the figures based on Bonferroni-corrected posthoc analysis to assess the effect of brain region at each repeated stimulus. Statistical analysis was conducted using GraphPad Prism 8.To address directly whether MD and vHPC inputs converge onto the same populations of mPFC pyramidal cells, we implemented a dual optogenetic stimulation strategy using the ChrimsonR and Chronos system. This dual stimulation strategy relies on the separation of wavelength activation of the two opsins, with ChrimsonR being red-shifted (peak activation with 635 nm) while Chronos is blue-shifted (peak activation with 470 nm) . To valiIn order to utilize this dual opsin system to interrogate the frequency-dependent synaptic dynamics of MD and vHPC inputs, we needed to establish that both opsins produced similar frequency-dependent responses. To this end, we measured mPFC pyramidal neuron responses to five repeated stimulations at 8 and 20 Hz of MD terminals expressing either ChrimsonR or Chronos, respectively A. 8 Hz wn = 15 cells; ChrimsonR n = 10 cells; two-way rmANOVA, no main effect of ChR2 variant F = 0.2256, P = 0.6393) or 20 Hz  = 1.366, P = 0.2578). Similar to previously published characterizations of the kinetics of Chronos and ChrimsonR  = 0.05880, P = 0.8106; main effect of ratio number F = 11.14, P < 0.0001; ChR2 variant by ratio number interaction F = 3.706, P = 0.0156 but no significant Bonferroni-corrected posthoc differences between the ChR2 variants at any pulse). In response to stimulation at 20 Hz, ChrimsonR was slightly but significantly more desensitizing  = 4.481, P = 0.0485; main effect of ratio number F = 5.981, P = 0.0013; no ChR2 variant by ratio number interaction F = 2.592, P = 0.0621. However, there was a significant Bonferroni-corrected posthoc difference between ChR2 variants at the first pulse ratio tested . To offset this slight difference in the frequency-dependent synaptic dynamics, we varied whether Chronos or ChrimsonR was expressed in the MD or vHPC, respectively, as noted below.Using stimulation intensities of 1% for 470 nm and between 2 and 90% for 635 nm, the amplitudes of the le-PSCs following stimulation of either Chronos or ChrimsonR-expressing MD terminals did not differ by ChR2 variant at either 8 Hz  = 3.399, P = 0.0788; main effect of ratio number F = 26.07, P < 0.0001; no region by ratio number interaction, F = 0.5175, P = 0.6717 nor any significant Bonferroni-corrected posthoc differences between the brain regions at each ratio). At 20 Hz, differences in desensitization between brain regions were much more pronounced, as seen in a comparison of the ratio of le-PSC responses to subsequent pulses of light relative to the first pulse  = 6.309, P = 0.0224; main effect of ratio number F = 8.679, P < 0.0001; a region by ratio number interaction F = 3.051, P = 0.0367). Bonferroni-corrected posthoc comparisons of brain regions at each ratio revealed the vHPC responses were significantly more desensitized for ratios 2:1 and 3:1 . This desensitization of vHPC inputs to stimulation at 20 Hz was so pronounced that while MD inputs consistently provoked significant le-PSCs in response to repeated stimulations, vHPC inputs desensitized to the point that only 20% of the pyramidal cells recorded showed a significant le-PSC after the first stimulation . By comparison, all MD and the majority of vHPC inputs provoked significant le-PSCs in response to repeated stimulations at 8 Hz . To affirm that the opsin used to stimulate each of pathways did not influence these results, we compared the pulse ratios evoked by stimulation of the MD or vHPC at 8 or 20 Hz by opsin type and found they did not differ (data not shown).All recorded pyramidal cells received input from both the MD and vHPC. All 12 MD and vHPC cells stimulated at 8 Hz C and alln = 8/8 cells for MD, 9/9 cells for vHPC). Furthermore, we found that vHPC inputs displayed greater frequency-dependent synaptic depression at 20 Hz than MD inputs  = 13.58, P = 0.0022; main effect of ratio number F = 5.646, P = 0.0083 and a region by ratio number interaction F = 4.682, P = 0.0170). Bonferonni-corrected posthoc comparisons between the regions for each of the response ratios revealed significant differences between stimulation of the MD and vHPC terminals for all ratios examined . The desensitization of vHPC inputs to repeated 20 Hz stimulation was such that up to 50% of responses were not statistically distinguishable from the baseline noise, whereas stimulation of the MD inputs consistently evoked detectable postsynaptic le-PSCs . This difference in synaptic dynamics of the two inputs at 20 Hz was not an artifact of the stimulation conditions as similar results were obtained recording with a shorter pulse duration (1 ms) at 32 °C (H). Specifically, 12 of 12 pyramidal neurons responded to stimulation of MD inputs and 7 of 7 pyramidal cells responded to stimulation of vHPC inputs  = 7.439, P = 0.0143; main effect of ratio number F = 7.682, P = 0.0018 and no region by ratio number interaction F = 1.484, P = 0.2409). Bonferonni-corrected posthoc comparisons of the response ratios by region revealed significant differences between stimulation of the MD and vHPC terminals for ratios 2:1 and 3:1 . The desensitization of vHPC inputs to repeated 20 Hz stimulation was such that up to 85% of responses were not statistically distinguishable from the baseline noise, whereas stimulation of the MD inputs consistently evoked detectable postsynaptic le-PSCs . These data confirmed our findings from the dual stimulation experiments; while MD and vHPC inputs innervate all recorded layer 2/3 pyramidal cells in the ipsilateral prelimbic cortex, in response to stimulation at 20 Hz, the frequency-dependent synaptic dynamics of both inputs differ.As further confirmation of our dual stimulation results, when we expressed the commonly used H134R variant of ChR2 individually in the MD or vHPC and stimulated these individual inputs using stimulation conditions comparable to the dual stimulation ones (5-ms stimulation pulses recorded at 25 °C), we found that all layer 2/3 pyramidal cells tested displayed significant responses to stimulation of either input  = 21.06, P < 0.0001. Bonferroni posthoc PV vs. Pyr, multiplicity-adjusted ****P < 0.0001; VIP vs. Pyr, multiplicity-adjusted ***P = 0.0001). Importantly, to facilitate comparison of results from these different cell populations, the recordings from pyramidal neurons  was expressed in either the PV or the VIP population of interneurons by crossing Ai9 mice (tdTomato reporter mice) to either PV-Cre A,B or th neurons  were madF = 2.55, P = 0.12). In response to repeated stimulations at 8 Hz, both MD and vHPC inputs desensitized. When we compared the ratio of responses with repeated 8 Hz stimulations relative to the first to measure the degree of desensitization for MD versus vHPC inputs, we observed that vHPC inputs were more desensitized than MD inputs  = 5.358, P = 0.0352; main effect of ratio number F = 14.99, P < 0.0001; no interaction of region by ratio number F = 0.529, P = 0.6646 nor any Bonferroni-corrected posthoc differences between the regions for any of the ratios measured). Both inputs also desensitized following 20 Hz stimulation and this desensitization was significantly greater for vHPC inputs  = 7.263, P = 0.0184; main effect of ratio number F = 11.68, P < 0.0001; no interaction of region by ratio number F = 1.657, P = 0.1920). Bonferroni-corrected posthoc comparisons revealed significant differences between stimulation of the MD and vHPC terminals for ratios 2:1 and 3:1 . We then compared the percentage of PV neurons that showed a significant le-PSC in response to each stimulation of either MD or vHPC input. The fraction of neurons responding to repeated pulses was attenuated for vHPC stimulations relative to MD ones at 8 Hz . This attenuation was more significant at 20 Hz .Stimulation of MD inputs consistently evoked postsynaptic responses in PV interneurons C,D. Howen = 3 cells and vHPC n = 6 cells; 2-way rmANOVA, no main effect of region F = 2.198, P = 0.1817; main effect of ratio number F = 11.57, P = 0.0001; interaction of region by ratio number F = 3.411, P = 0.0363). However, there were no significant differences in the Bonferroni-corrected posthoc comparisons between regions at any ratio number. Both inputs also desensitized following 20 Hz stimulation, and the degree of desensitization was significantly greater for vHPC than MD inputs, similar to what was seen with pyramidal and PV cells  = 12.29, P = 0.0067; main effect of ratio number F = 3.039, P = 0.0461; no region by ratio number interaction F = 1.596, P = 0.2134). There was a significant difference in the Bonferroni-corrected posthoc comparison between regions at ratio 3:1 . There was no significant difference in the percent of VIP cells responding to repeated stimulation of the MD versus vHPC inputs at 8 Hz . Although VIP cells were initially more likely to receive input from the vHPC, at 20 Hz, the degree of desensitization of vHPC inputs was such that stimulation of this input was significantly less likely to provoke a response to repeated stimulations than MD inputs . As previously described, we successfully recorded responses in pyramidal cells following stimulation of MD inputs in the same slices from VIP interneurons that failed to respond to MD stimulation, and the amplitude of the interneuron and pyramidal cell responses was not significantly correlated  or vHPC input . Although the size of these groups is small, the mean pyramidal cell responses to stimulation of MD inputs were larger in VIP-Cre mice, where VIP interneurons appeared to receive less MD input.Next, we recorded VIP interneurons while stimulating MD or vHPC inputs with the same approach described above A,B. In crrelated , indicatFinally, to determine whether the excitatory le-PSC responses we were measuring contained a monosynaptic component and were mediated by direct inputs from either the MD or the vHPC to mPFC cells, we measured the onset latency of the responses. To this end, we first assessed the onset latency of le-PSC currents evoked under conditions that isolate monosynaptic transmission by applying tetrodotoxin (TTX) in combination with 4-aminopuridine (4-AP) to the bath. Tetrodotoxin blocks action potentials so that the only source of neurotransmitter release is via direct activation of ChR2 in the terminals. 4-AP was added to the bath to increase the amplitude of le-PSCs evoked under these conditions as le-PSC amplitude is greatly reduced in the presence of TTX. These conditions were not used for all experiments as they also decreased the amplitude of the evoked, postsynaptic responses, biasing towards not detecting a significant connection when one might in fact be there. Monosynaptic le-PSCs evoked under conditions of TTX and 4-AP had a mean onset latency of 10.10 ms with a standard deviation of 3.799 ms. We used the mean of the monosynaptic latencies plus two times the standard deviation of these latencies (17.7 ms) as the threshold below which we considered onset latencies under non-TTX experimental conditions as likely monosynaptic . Based oOne primary finding is that MD and vHPC inputs converge onto all layer 2/3 pyramidal cells in the prelimbic mPFC. While this result is consistent with previous work demonstrating widespread functional connectivity from the MD and vHPC onto layer 2/3 pyramidal cells of the prelimbic mPFC , our stuIt may be that the relative strength of these inputs onto individual pyramidal cells is an important determinant in their behavioral distinction. It is difficult to compare directly the amplitudes of light evoked currents from MD versus vHPC inputs to layer 2/3 neurons since this is determined not only by the synaptic strengths but also by the amount of virus injected and the intensity of light stimulation. A recent paper challenged the importance of vHPC input to layer 2/3 in prelimbic mPFC by suggesting the connections are weak onto layer 2/3 cells in prelimbic cortex relative to those onto pyramidal cells in layer 5 . In addiIn contrast to connectivity to pyramidal neurons, we found a bias in connections onto distinct populations of local interneurons. In particular, the populations expressing PV and VIP are of interest given that they are nonoverlapping, and form completely different types of connections within the local cortical microcircuitry . For exaIn addition to biases in functional connectivity from MD and vHPC inputs onto distinct populations of local interneurons, our results suggest that MD and vHPC inputs can also be distinguished based on frequency-dependent synaptic dynamics. Our dual injection experiments testing the response of MD and vHPC inputs to repeated stimulation at 20 Hz revealed that vHPC inputs showed much greater desensitization in response to repeated stimulations at 20 Hz than MD inputs. As a consequence, the probability of effective synaptic transmission onto pyramidal cells decreased dramatically by the second or third stimulation. Intriguingly, this difference in synaptic efficacy between the two inputs was frequency specific, as stimulation at a lower theta frequency (8 Hz) resulted in much more comparable synaptic dynamics between the two inputs. Importantly, because we are recording from the same cell in response to stimulation of both inputs, the differences in desensitization cannot be attributed to recording from two different postsynaptic neurons. Thus, MD and vHPC inputs onto layer 2/3 pyramidal cells can be distinguished by the most efficient frequencies with which they communicate with the PFC.These differences in synaptic dynamics could result from either pre- or post-synaptic mechanisms at MD–mPFC versus vHPC–mPFC synapses. Presynaptically, desensitization in response to repeated stimulation has been linked to high baseline release probability or decreases in the number of functional release sites, while postsynaptic changes in the accumulation of desensitized receptors may also contribute . The higIt is important to note that the responses observed under our recording conditions likely reflect the contribution of both monosynaptic and polysynaptic inputs. In particular, it is possible that feedforward inhibition initiated via activation of GABAergic interneurons may play a role in dampening down responses of pyramidal cells to repeated stimulations of vHPC inputs at 20 Hz. In the future, this would be an extremely interesting possibility to test by including blockers of GABAergic transmission in the bath.Interestingly, it appears that desensitization at high frequencies may not be a universal feature of vHPC synapses within the mPFC as vHPC inputs to layer 5 pyramidal cells were shown to sensitize at high frequencies such as 20 Hz . This diThe results of our in vitro experiments identifying preferential transmission from the vHPC to layer 2/3 of the mPFC following stimulation at 8 rather than 20 Hz are in line with in vivo experiments suggest that theta frequency oscillations (4–12 Hz) are one of the principal frequencies recorded in the vHPC . MoreoveOur study has focused on how inputs from the MD and vHPC can be distinguished by the mPFC but there may be cross-talk and modulation between the inputs. Some evidence that stimulation of one input might facilitate the other comes from our anecdotal observation that in one case, a functional connection between MD inputs and a VIP cell was revealed following stimulation of vHPC inputs at 8 Hz. A similar type of plasticity has been described in anesthetized mice where stimulation of thalamic inputs to the PFC could either enhance or inhibit subsequent PFC responsiveness to stimulation of hippocampal inputs depending on the frequency and duration of thalamic stimulation and the interstimulus interval . AlthougIn sum, we found that MD and vHPC inputs to layer 2/3 pyramidal cells are convergent, but are distinguished by their engagement of different elements of prefrontal interneuron circuitry as well as frequency-specific synaptic dynamics. Although there are many more elements of this connectivity that need to be established, these findings are an important first step in understanding how the prefrontal cortex is capable of both segregating and integrating multiple subcortical excitatory inputs.Supplementary_Figures_Publication_tgaa084Click here for additional data file.


The influence of uric acid (UA) on renal function and the significance of UA-lowering therapy are unclear. The purpose of the sub-analysis of the Assessment of Clinical Usefulness in chronic kidney disease patients with Atorvastatin (ASUCA) trial was to evaluate the influence of serum UA levels on renal function in Japanese chronic kidney disease patients with hyperlipidemia.Of 344 participants in the ASUCA trial, 279 participants whose UA levels at both baseline and 24 months were available were included. Based on UA level at baseline or mean UA level during the trial period, they were divided into four groups: < 5.0, 5.0–6.0, 6.0–7.0, or ≥ 7.0 mg/dL, irrespective of allocation. Changes in the estimated glomerular filtration rate (eGFR) after 24 months were compared among the groups in relation to baseline or mean UA levels.2, respectively. A negative correlation between changes in eGFR after 24 months and baseline UA level was observed with adjustment for confounding factors. The relationship between changes in eGFR and mean UA levels during trial period showed a similar trend.For baseline UA levels , the change in eGFR after 24 months was − 1.32 ± 10.3, − 1.74 ± 8.94, − 2.53 ± 7.34, and − 3.51 ± 9.10 mL/min/1.73 mIn CKD patients with dyslipidemia, hyperuricemia was an independent risk factor for CKD progression. An ongoing clinical trial  may reveal the significance of strict UA-lowering therapy in CKD patients.The online version of this article (10.1007/s10157-019-01840-4) contains supplementary material, which is available to authorized users. Our understanding and interpretation of the significance of the relationship between uric acid (UA) and renal function have changed with time. Although it was widely known in the mid-nineteenth century that gout was associated with chronic kidney disease (CKD) , the conFor almost 2 decades, the use of animal models of hyperuricemia without urate crystals has been reported –5. StudiIn humans, the association between hyperuricemia and renal dysfunction has been studied primarily through prospective observational research. Iseki and colleagues conducted research on 48,177 healthy Japanese living in Okinawa Prefecture, which revealed that the presence of hyperuricemia  is associated with increased risk of the requirement of initiation of dialysis within 7 years . BellomoSome interventional studies have reported that lowering UA levels through drug therapy has a beneficial effect on renal function. In a study of 54 patients with hyperuricemia combined with CKD, deterioration of renal function was alleviated following administration of allopurinol for 12 months . AllopurOn the other hand, however, some studies in CKD patients reported that hyperuricemia was not an independent risk factor for CKD progression , 17. TheTo further investigate the relationship between UA level and renal function, we analyzed data from the Assessment of Clinical Usefulness in CKD Patients with Atorvastatin (ASUCA) trial, which examined the reno-protective effects of lipid-lowering therapy with atorvastatin in Japanese CKD patients with hyperlipidemia. Using the data of this trial, we determined the relationship between renal function evaluated by eGFR and UA levels at baseline and during the trial period.This is a post hoc sub-analysis of data from the ASUCA trial (UMIN000001778)—a randomized, open-label, parallel-group comparison trial that examined the effectiveness of lipid-lowering therapy using statin for CKD patients with hyperlipidemia. The protocol and main results have already been reported , 19.2. After screening, 334 patients were randomized into the control group or the atorvastatin group. The treatment period was 24 months and the target low-density lipoprotein cholesterol (LDL-C) level was 100 mg/dL. In the control group, treatment began with diet therapy followed by the administration of drugs other than statin if LDL-C was not sufficiently reduced. The atorvastatin group was treated by administration of atorvastatin in addition to diet therapy. Laboratory tests during a study period were scheduled to be done just before the start of treatment protocol, and 1, 3, 6, 9, 12, 18, and 24 months after the start of treatment protocol.The ASUCA trial enrolled 344 Japanese CKD patients with dyslipidemia. The main eligibility criteria at enrollment were as follows: (1) 40–75 years old, (2) not being treated with statin, and (3) urinary protein positive or an eGFR of < 60 mL/min/1.73 mIn this sub-analysis, of 344 participants of the ASUCA trial, 279 participants whose data were available at baseline and after 24 months of treatment were examined, irrespective of allocation. For analysis of relationship between baseline UA levels and eGFR changes, we divided the participants into four groups according to baseline serum UA level: < 5.0 mg/dL, 5.0–6.0 mg/dL, 6.0–7.0 mg/dL, or ≥ 7.0 mg/dL. Next, we analyzed the relationship of renal function with mean UA level by dividing the participants into four groups according to their mean serum UA level during the trial period: < 5.0 mg/dL, 5.0–6.0 mg/dL, 6.0–7.0 mg/dL, and ≥ 7.0 mg/dL.p < 0.05 were considered significant. The primary endpoint was the change in eGFR at 24 months from baseline. One-way analysis of variance (ANOVA) was applied to compare average values among the groups, and linear multiple regression analysis was also performed. In conducting multiple regression analysis, for comparison between four groups based on baseline UA level, sex, age, baseline eGFR, log-transformed urinary albumin excretion (UACR), presence of hypertension, diabetes, use of RAAS inhibitor, use of diuretics, smoking habit, systolic blood pressure level, and allocation were adjusted, and for groups based on mean UA level during the trial period, sex, age, baseline eGFR, log-transformed UACR, baseline UA, presence of hypertension, diabetes, use of RAAS inhibitor, use of diuretics, smoking habit, systolic blood pressure level, and allocation were adjusted. All statistical analyses were performed with EZR , which is a modified version of R commander designed to incorporate statistical functions frequently used for biostatistics [Continuous variables are expressed as mean values ± standard deviation. Categorical variables are described as percent. Values of two-tailed atistics .2 for the < 5.0 mg/dL, 5.0–6.0 mg/dL, 6.0–7.0 mg/dL, and ≥ 7.0 mg/dL baseline serum UA groups, respectively. When comparing between groups without adjustment, the decrease in eGFR tended to be larger when the baseline serum UA level was higher, although this was not statistically significant. Linear multiple regression analysis . Linear multiple regression analysis .This study revealed a negative correlation between baseline serum UA levels and renal function after 24 months of lipid-lowering treatment in Japanese CKD patients with hyperlipidemia. When baseline UA levels were below 5.0 mg/dL, reduced deterioration of renal function was observed throughout the trial period compared with patients whose baseline levels were ≥ 7.0 mg/dL, even after adjustment for age, sex, baseline eGFR, and systolic blood pressure. Mean serum UA levels during the trial period were also found to be associated with renal function. When mean UA levels were < 5.0 mg/dL, renal function was preserved compared with that of patients whose mean UA levels were ≥ 7.0 mg/dL, even after adjustment for possible confounders.Several studies have used animal models to estimate the mechanisms in which low UA levels protect renal function. Sanchez-Lozada et al. reported that increased UA levels lead to increased oxidative stress, impairment of vascular endothelial function, and increased renal vascular resistance resulting in systemic or glomerular hypertension –23. NakaUA is also known to have antioxidant activity and to be involved in the type 2 immune response, and recent attention has been paid to its potential protective activity toward living bodies and organs , 26. TheIn a report by Hayashino et al., low UA levels  were associated with a higher risk of developing overt proteinuria. Our data showed that, in the group with lowest UA, an average baseline UA level was 4.1 mg/dL (interquartile range = 4.0–4.9), and an average UA value during the trial period was 4.2 (interquartile range = 3.9–4.7). Therefore, the effect of UA levels < 4.0 mg/dL on renal function has not been sufficiently examined. Extremely low UA levels may be related to poor renal function, but further studies are needed to confirm this.A randomized-controlled trial of topiroxostat in Japanese CKD patients with hyperuricemia is currently ongoing  to investigate whether the administration of UA-lowering therapy, adapted to achieve a strict therapeutic management target level, could represent an approach with which to improve renal-function protection. It is expected that the optimal method of UA management will be clarified for patients with CKD based on the results of these studies.Our research has several limitations. First, it is a post hoc analysis, and therefore, the possibility of uncontrolled confounding factors remains. For example, the status of using uric acid-lowering drugs was unknown. Second, an intervention study is necessary to demonstrate whether the target value presented at this time is useful for therapeutic intervention in CKD patients. Third, the participants of this study were CKD patients with hyperlipidemia, and the underlying diseases of CKD were mainly diabetes and hypertension. It is, therefore, unclear whether our results will apply to general CKD patients. Finally, because the sample size in this study was relatively small, the relationship between changes in renal function and serum uric acid levels may not be fully accurately assessed.In CKD patients with dyslipidemia, increased baseline serum UA levels and mean levels throughout the trial period were associated with prognosis of renal function, respectively. Further studies are needed to determine whether UA level lower than that currently targeted in general clinical practice is beneficial for the renal function.Supplementary file1 (DOCX 22 kb)Below is the link to the electronic supplementary material.