Acute gastroenteritis due to group A rotaviruses (RVAs) is the leading cause of infant and childhood morbidity and mortality particularly in developing countries including Pakistan. In this study we have characterized the whole genomes of five RVA strains  using the Illumina HiSeq platform. The strains PAK56 and PAK622 exhibited a typical Wa-like genotype constellation , whereas PAK419, PAK585, and PAK663 exhibited distinct DS-1-like genotype constellations . Despite their DS-1-like genotype constellation, strain PAK585 possessed the typical Wa-like G1P[8] genotypes, whereas both PAK419 and PAK663 possessed the G3 genotype. In addition, PAK419 also possessed the Wa-like NSP3 genotype T1, suggesting that multiple reassortments have occurred. On Phylogenetic analysis, all of the gene segments of the five strains examined in this study were genetically related to globally circulating human G1, G2, G3, G6, G8, G9, and G12 strains. Interestingly, the NSP2 gene of strain PAK419 showed closest relationship with Indian bovine strain (India/HR/B91), suggesting the occurrence of reassortment between human and bovine RVA strains. Furthermore, strains PAK419, PAK585, and PAK663 were closely related to one another in most of their gene segments, indicating that these strains might have been derived from a common ancestor. To our knowledge this is the first whole genome-based molecular characterization of human rotavirus strains in Pakistan. The results of our study will enhance our existing knowledge on the diversity and evolutionary dynamics of novel RVA strains including DS-1-like intergenogroup reassortant strains spreading in Asian countries including Pakistan, in the pre-vaccine era. Therefore, continuous surveillance is recommended to monitor the evolution, spread and genetic stability of novel reassortant rotavirus strains derived from such events. Reoviridae and are responsible for severe dehydrating diarrhea in children less than 5 years of age and in many animal species worldwide (Group A rotaviruses (RVAs) belong to the family orldwide . In spitorldwide .These non-enveloped viruses are triple layered with icosahedral symmetry and carry 11 segments of double stranded RNA encoding six structural proteins  and five to six non-structural proteins (NSP1-NSP6) . A classTwo major RVA genotype constellations are dominant in humans. The human Wa- like genotype constellation is characterized as I1-R1-C1-M1-A1-N1-T1-E1-H1, and is usually found in combination with the genotypes G1P[8], G3P[8], G4P[8], G9P[8], G12P[6], or G12P[8], whereas the human DS-1-like genotype constellation is characterized as I2-R2-C2-M2-A2-N2-T2-E2-H2 and tends to be combined with G2P[4], G8P[4] or G8P[6] . The humThe segmented nature and error-prone-RNA-dependent RNA polymerase, lacking proofreading activity, provide rotavirus with several evolutionary mechanisms including point mutation, recombination, reassortment and interspecies transmission . The emeThe first unusual DS-1-like G1P[8] strains  were detected in Japanese children suffering from severe gastroenteritis  and afteMost of these novel DS-1-like strains were not completely sequenced. Whole genome analysis is a reliable method for obtaining conclusive data on the origin of RVA strain, and for tracing its evolutionary pattern. During the last decade, research on RVA in Pakistan mainly focused on the detection of current RVA genotypes based on VP7 and VP4. To the best of our knowledge, this is the first time genomes of Pakistani RVA strains have been fully sequenced and characterized. In this study, the Illumina HiSeq platform was used to obtain the complete nucleotide sequences of the whole genomes of five Pakistani RVA strains.The study was approved by the Ethical Committee of PIMS, Benazir Bhutto Shaheed Hospital (BBH) and Internal Review Board (IRB) of COMSATS Institute of Information Technology, Islamabad. Written informed consent for molecular characterization of RVAs strains were obtained from the parents/guardians of the study participants.RVA strains, PAK56, PAK419, PAK585, PAK622, and PAK663, showed maximum diversity upon phylogenetic analysis were selected for whole genome sequencing. These strains were detected in five fecal samples from hospitalized children aged less than 10 years with severe gastroenteritis admitted to the emergency pediatrics department of BBH  in Rawalpindi, and PIMS  in Islamabad. These stool samples were collected during RVA surveillance program in Pakistan in 2015–2016 which involved a total of 639 samples . Of the g for 3 min. The supernatant was filtered through a 0.8 μm (PES) filter  at 17000 × g for 1 min. After filtration samples were treated for 2 h at 37°C with a cocktail of 7 μl of 20× homemade buffer , 2 μL of benzonase  and 1 μL of micrococcal nuclease . Reaction was stopped by adding 7 μl of 10 nM EDTA. Nucleic acid extraction was conducted by using the QIAamp® Viral RNA Mini kit from Qiagen  according to manufacturer instructions without adding carrier RNA.Each sample was enriched for viral particles by the NetoVIR (Novel Enrichment Technique Of VIRomes) protocol . BrieflyTM 2500 platform (Illumina) for 300 cycles (2 × 150 bp paired ends).cDNA synthesis was carried out and random PCR amplification for 17 cycles were performed using a Whole Transcriptome Amplification (WTA) Kit (Sigma-Aldrich) according to the manufacturer’s instructions. The PCR products were purified with the MSB Spin PCRapace spin columns . The libraries for Illumina sequencing were prepared using the Nextera XT Library Preparation Kit  according to the manufacturer’s guide. Library purification was performed using a 1.8 ratio of Agencourt AMPure XP beads . The quality check of the purified cDNA library was performed on 2100 Bioanalyzer (Agilent Technologies). The libraries were quantified with the KAPA Library Quantification kit (Kapa Biosystems) followed by sequencing on a HiSeqde novo using Trimmomatic and SPAdes, respectively  . Multipl(VP7)MH182443–MH182446 and MK370887, (VP4)MH182439–MH182442 and MK370888, (VP6)MH170022–MH170026, (VP1)MH166401–MH166404 and MK412569, (VP2)MH166405–MH166409, (VP3)MH170017–MH170021, (NSP1)MH182447–MH182451, (NSP2)MH182452–MH182456, (NSP3)MH182457–MH182461, (NSP4)MH182462–MH182466, and (NSP5)MH182467–MH182471.The nucleotide sequences were submitted to the GenBank under the following accession numbers: de novo assembled RVA genome contigs are given in the In order to gain insight into the genetic variability among strains PAK56, PAK419, PAK585, PAK622, and PAK663 and their genetic relatedness with other RVA strains worldwide, the full-genome sequences of these five strains were determined using the NetoVIR protocol in combination with NGS Illumina HiSeq sequencing. Full or nearly full length nucleotide sequences reads were obtained for all the 11 gene segments of the five human rotavirus strains via Illumina HiSeq platform. The contigs length and number of reads mapped against the Comparison of the complete genotype constellations of these strains with those of representative G1, G3, G9 and other RVA strains is shown in The strains PAK56, PAK419, PAK585, PAK622, and PAK663 were named as RVA/Human-wt/PAK/PAK56/2015/G9P[8], RVA/Human-wt/PAK/PAK419/2016/G3P[4], RVA/Human-wt/PAK/PAK585/2016/G1P[8], RVA/Human-wt/PAK/PAK622/2016/G3P[8], and RVA/Human-wt/PAK/PAK663/2016/G3P[4], respectively following instructions for uniformity of RVAs naming by the RCWG.Phylogenetic trees were constructed for each of the 11 segments of the five RVA strains obtained in this study, together with representative strains from GenBank.The VP7 gene of strain PAK56 clustered closely with G9-lineage three strains from all over the world , and exhibited maximum nucleotide (nt) identities (98.9–99.5%) with G9 strains in Korea (CAU08-350) and Russia (H508). The VP7 gene of strain PAK585 clustered closely with G1-lineage 1 strains from all over the world , and showed maximum nucleotide (nt) identities (99.3–99.4%) with G1 strain from Australia and India . On the other hand, the VP7 genes of strains PAK419, PAK622, and PAK663 clustered together with G3 strains belong to lineage three reported worldwide , and exhibited highest nucleotide identities (99.2–99.3%) with each other and with G3 strains from Russia (Nov11-N2317) and United States (SSCRTV_00017) .The VP4 genes of strains PAK419 and PAK663 clustered closely with other strains identified in P[4] lineage 5 from all over the world , and showed maximum nucleotide identities (98–99.2%) with each other and with DS-1-like strains in United States (LB1562) and Bangladesh (J331) In contrast, VP4 gene of strains PAK56, PAK622, and PAK585 clustered closely with other strains isolated in OP354-like P[8] lineage 4 isolated from other countries of the world, and showed maximum nucleotide identities (98–98.7%) with each other and with P[8] rotavirus strain from Russia (Nov10-N53) and Israel (R5751) .The NSP2 and NSP3 genes of strains PAK56 and PAK622 clustered closely together with other strains reported in NSP2 and NSP3 genotypes N1 and T1, respectively from all over the world . The above gene segments of these strains exhibited maximum nucleotide identities (99.4–99.9%) to each other and with Wa-like G1P[8] strains from Belgium (00045) and Korea (C1-81) and comparable identities (97.7–98%) to a cow rotavirus strain from Uganda found in a common branch. The NSP2 gene of strain PAK419 clustered closely together with other globally circulating human and animal rotavirus strains identified in NSP2 genotype N2, and showed maximum nucleotide (nt) identity (98.3%) with Indian bovine strain (HR/B91). While, NSP3 gene segment of PAK419 clustered closely together with strains reported worldwide in NSP3 genotype T1, and showed 98.9% nucleotide identity with Chinese Wa-like strain (E2484/G4P[8]). In contrast, the NSP2 and NSP3 genes of strains PAK585 and PAK663 clustered closely together with other strains identified in previously established genotypes of NSP2 and NSP3  from all over the world , and showed nucleotide identities of (96.4–97.3%) and (98–99.5%) with each other and with that of DS-1 like strains from the Philippines (TGO12-007), United States (LB1562), India (KOL-006), respectively , 5.The VP1, VP3, VP6, NSP1, NSP4, and NSP5 gene segments of three of our strains  phylogenetically clustered closely together with strains circulating globally  in the previously established genotypes R2, M2, I2, A2, E2, H2, respectively. These six genotypes of three strains showed maximum nucleotide similarities (99.3–99.9%) to each other and (98.2.8–99.5%) to the DS-1-like rotavirus strains detected in United States (LB1562), Bangladesh (MMC-88) Australia (CK20027), Thailand . In contrast the VP1, VP3, VP6, NSP1, and NSP4 gene segments of two of our strains (PAK56 and PAK622) clustered closely together with other globally reported strains  in the genotypes R1, M1, I1, and E1, respectively. These five genotypes of strains PAK56 and PAK622 showed maximum nucleotide similarities (98.9–100%) to each other and to the Wa-like strains from United States (Vanderbilt/VU-05-06-17), Belgium (BE00045), India (mcs72), South Africa (ZAF/MRC-DPRU1191) and Russia (Omsk08-351). While, gene segment NSP5 of PAK56 and PAK622 clustered with other rotavirus strains detected round the globe  in the genotype H1 and showed maximum nucleotide (nt) similarities of (99.4–99.8%) to rotavirus strains detected in Korea (CAU/09-371) and Africa (ZAF/MRC-DPRU1554) and comparable identities  with sheep (PER/SOV.4) and Alpaca (PER/AlpH31) rotavirus strains from Peru in the same cluster –11.The VP2 genes of strains PAK419, PAK585, and PAK663 clustered closely together with other strains identified in VP2 lineage C2 from all over the world , and exhibited maximum nucleotide similarities (99.1–99.2%) with co-circulating DS-1-like strain from Pakistan (PAK93) and comparable identities (98.5–98.7%) with Australian DS-1-like strain (CK20024/G2P[4]). On the other hand, the VP2 gene of strains PAK56 and PAK622 clustered with other strains identified in VP2 lineage C1 from all over the world  and showed maximum nucleotide identities (99.1–99.3%) with Wa-like strains from Australia (Victoria/CK00037) and Belgium (BE00031) and previously reported strain from Pakistan (HF66) .In the present study, the whole genomes of five human RVA strains  were characterized for the first time in children with severe gastroenteritis in Pakistan in 2015–2016. The two strains (PAK56 and PAK622) exhibited a typical Wa-like backbone while, three strains  showed genotype constellation involving genogroup 1 and genogroup 2 genes. The unique genotype constellation of  were found to be similar to DS-1-like intergenogroup reassortant strains. Pakistan has implemented RVA vaccine in national immunization program in January 2018, so the emergence of intergenogroup reassorted strains in the present study are not linked to RVA vaccine introduction.The DS-1-like G1P[8] double-gene reassortant strains were first detected in Japan followed by cases reported in Thailand, Vietnam, Malawi, and Brazil . The unuThe G3 genotype is documented as the third most predominant RVA genotype in humans mostly found in combination with P[8] . The G3 On the other hand, two Pakistani strains PAK56 and PAK622 having genotypes G9P[8] and G3P[8] detected in the present study were found to be very similar to a typical human Wa-like strains reported in several countries worldwide. The analysis of ten of the eleven gene segments  of strains PAK56 and PAK 622 showed a typical human Wa-like origin. While, gene segment NSP5 showed close relationship with human rotavirus strains detected in Korea and Africa and with sheep and Alpaca rotavirus strains from Peru in the same cluster. These observation suggests that interspecies transmission and reassortments might have occurred between human/caprine rotavirus strains. Also on the phylogenetic tree, these two strains were clustering closely in their maximum gene segments suggestive of their origin from a common ancestor.This is the first full genome characterization of human rotavirus strains circulating in Pakistan. The evolution of DS-1-like intergenogroup reassortant strains having G1P[8] and G3P[4] genotypes in Pakistan suggested the constant circulation of these unusual rotavirus strains and ongoing reassortment associated with them particularly in Asia. After WHO recommendation in 2009, the universal implementation of RVA vaccine is continue to increase globally. Whether or not the coinciding rise of strains with Wa-like VP4 and VP7 genes together with a DS-1-like backbone and mass vaccine introduction is consequential or purely random remains enigmatic. Therefore, further investigations via continuous surveillance of these strains is recommended to monitor the evolution, spread and genetic stability of novel reassortant rotavirus strains derived from such events. Pakistan has licensed RVA vaccine in national EPI program in January 2018. After the introduction of RVA vaccine in the country’s immunization program the careful monitoring is needed to estimate vaccine effectiveness and challenges to current vaccine strategies against such unusual strains.MH182443–MH182446 and MK370887, (VP4) MH182439–MH182442 and MK370888, (VP6) MH170022–MH170026, (VP1) MH166401–MH166404 and MK412569, (VP2) MH166405–MH166409, (VP3) MH170017–MH170021, (NSP1) MH182447–MH182451, (NSP2) MH182452–MH182456, (NSP3) MH182457–MH182461, (NSP4) MH182462–MH182466, (NSP5) MH182467–MH182471.The datasets generated for this study can be found in the nucleotide sequences were submitted to the GenBank under the following accession numbers: (VP7) The studies involving human participants were reviewed and approved by the Ethical Committee of PIMS, Benazir Bhutto Shaheed Hospital (BBH) and Internal Review Board (IRB) of COMSATS Institute of Information Technology. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. Written informed consent was obtained from the minor(s)’ legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.AS and NB conceived concept and designed the experiments. AS performed the experiments and wrote the manuscript. NB, HB, KY, and JM contributed the reagents and data analysis tools and helped in write-ups.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 G protein-coupled cannabinoid receptors type 1 (CB1R) and type 2 (CB2R), and their endocannabinoid (eCBs) ligands, have been implicated in several aspects of brain wiring during development. Here we aim to assess whether interfering with CB1R affects development, neuritogenesis and pathfinding of GnRH and AgRP neurons, forebrain neurons that control respectively reproduction and appetite. We pharmacologically and genetically interfered with CB1R in zebrafish strains with fluorescently labeled GnRH3 and the AgRP1 neurons. By applying CB1R antagonists we observed a reduced number of GnRH3 neurons, fiber misrouting and altered fasciculation. Similar phenotypes were observed by CB1R knockdown. Interfering with CB1R also resulted in a reduced number, misrouting and poor fasciculation of the AgRP1 neuron’s axonal projections. Using a bioinformatic approach followed by qPCR validation, we have attempted to link CB1R functions with known guidance and fasciculation proteins. The search identified stathmin-2, a protein controlling microtubule dynamics, previously demonstrated to be coexpressed with CB1R and now shown to be downregulated upon interference with CB1R in zebrafish. Together, these results raise the likely possibility that embryonic exposure to low doses of CB1R-interfering compounds could impact on the development of the neuroendocrine systems controlling sexual maturation, reproduction and food intake. Cannabis spp. have been used for thousands of years for their therapeutic and mood-altering/recreational properties. Cannabis-derived psychoactive compounds, mainly ∆9-tetrahydrocannabinol (∆9-THC) and cannabidiol (CBD), can pass the placental barrier and be transferred to the embryo [Cannabis consumption during pregnancy can exert adverse consequences on the progeny, including anxiety, cognitive and attention deficits, as well as depression [Marijuana and other derivatives of the plant e embryo . It is npression .In the central nervous system (CNS), ∆9-THC, CBD and various other synthetic cannabinoids bind the G protein-coupled receptors of the cannabinoid family, CB1R and CB2R . The cogFollowing binding of eCBs, phyto-CBs and synthetic CBs to CB1R, a variety of transduction pathways are activated in a time- and region-dependent way, depending on the cell type and receptor repertoires. In particular, eCBs have been proposed to activate Rho-class of guanosine tri-phosphate phosphatases (Rho-GTPases) and to control the neuronal actomyosin cytoskeleton . It has Daglα resulted in abnormal behaviors, characterized by stereotyped movement and altered motion perception [Data from the medical literature support the role of CB1R in brain wiring during early development. Fetuses exposed to cannabis during embryonic life have increased risk of cognitive deficit ,18, atterception .Few reports have linked the eCB/CB1R signaling functions to the maturation and physiology of the hypothalamic neuroendocrine systems controlling reproduction and appetite ,35,36. TAnother important prominent effect of the eCB system is the regulation of appetite and energy balance. The hypothalamic network of peptidergic neurons controlling food intake/energy metabolism, enhancing feeding behavior, includes mainly neurons expressing pro-opiomelanocortin (POMC), neuropeptide Y (NPY) and agouti-related peptide (AgRP) . This syWhile most previous works have focused on postnatal and adult hypothalamic functions, little is known on the role of CB1R and its misactivation for the correct development of hypothalamic neuroendocrine neurons and their connectivity needed to attain control of sexual maturation and/or reproduction and appetite/energy storage. This question is highly relevant, to fully assess the impact of exposure to these (and other molecules) during embryonic and early infancy on human health, and the possible link with adult pathologies, such as reduced fertility and altered food intake. Furthermore, these studies aimed to elucidate the role of eCB ligands and the mechanisms underlying axonal miswiring upon altered CB1R activation .We have investigated the possible role of CB1R altered activity on the extension, fasciculation and pathfinding of GnRH3 and AgRP axons during forebrain development in zebrafish. Using transgenic lines in which GnRH3 and AgRP neurons are fluorescently labeled, we show that pharmacological and genetic manipulation of CB1R resulted in abnormal guidance, fasciculation and routing of GnRH3 and AgRP1 axons during embryonic development, indicating that CB1R is a possible regulator of the development of reproduction- and food intake-related neurons.gnrh3::EGFP [agrp1::mCherry [gnrh3::EGFP embryos and also at 96 hpf for agrp1::mCherry embryos. The survival rate was examined at 24 hpf, 48 hpf and 72 hpf for gnrh3::EGFP embryos and at 24 hpf, 48 hpf, 72 hpf and 96 hpf for agrp1::mCherry embryos. Survival rate of agrp1::mCherry embryos at 96 hpf was analyzed as well because at 72 hpf the fluorescence signal of the AgRP1 fibers was still too weak to be reliably evaluated by fluorescence microscopy. The survival rate was calculated as the percentage of live embryos over the total number of embryos (30 embryos/well for each concentration); the hatching rate was calculated as the percentage of hatched embryos over the remaining living embryos.In order to select a range of non-toxic concentrations and non-teratogenic doses of CB1R agonists and antagonists, we initially determined their effects on the survival and hatching rates of zebrafish embryos. h3::EGFP ,46 and a:mCherry  zebrafisAs agonist of the CB1 receptor, we chose WIN55,212-2 (renamed WIN55 for simplicity), which has been reported to mimic endocannabinoids (eCBs) signaling in several studies ,48,49,50gnrh3::EGFP and agrp1::mCherry embryos  and the postoptic commissure. In addition, two tracts extend along the optic nerve and innervate the retina, a tract extends dorsally towards the region of the pineal gland and a tract extends posteriorly towards the hypothalamus and preoptic area. Between 3 and 15 days post-fertilization (dpf), soma migration takes place in an axonophilic manner along the terminal nerve and the ventral telencephalon towards the ventral hypothalamus . Some Gngnrh3::EGFP embryos at the 1-cell stage were treated with various concentrations of CB1R-ligands for 72 h after which the EGFP+ embryos were selected and examined by confocal microscopy. A proper view plane of the transgenic fish was adopted to allow the correct visualization of both GnRH3 neurons present in the olfactory region and GnRH3 fibers extending towards the anterior commissure  and for the control embryos . The results were then expressed as the number of embryos with the phenotype in the various conditions, as percentage over the total number of embryos examined, and reported in The number, neurite extension and commissural fasciculation of GnRH3 neurons and fibers were examined and compared to control embryos treated with DMSO only C. We fouAt higher magnification, we also counted the number of EGFP+ cells present in the olfactory region H. We obsgnrh3::EGFP embryos were injected at the 1-cell stage with a previously published z-cb1r morpholino, whose sequence was designed to bind to a region 5′ of the ATG start codon and was shown to block z-cb1r mRNA translation [In order to verify if down-modulation of CB1R could lead to phenotypes similar to the ones observed after treatment with CB1R ligands, we utilized a conventional antisense morpholino oligonucleotide (MO)-mediated strategy. nslation . Uninjecz-cb1r MO to deplete the endogenous CB1R protein was tested by Western blot analysis on total proteins, extracted from z-cb1r MO-injected embryos and from controls (uninjected embryos) at 72 hpf, using an anti-CB1R antibody. A significant dose-dependent reduction of the endogenous level of z-CB1R protein was observed in z-cb1r MO-injected embryos, with the most efficient concentration being 400 µM z-cb1r MO  or with scrambled (scrbl)-MO as control, and examined at 72 hpf to determine the number, neurite arborization and commissural organization by confocal microscopy  over the total number of embryos examined. As reported in z-cb1r MO-injected embryos vs. 14% in uninjected controls).Having proven the efficiency of croscopy C–E. Upongnrh3::EGFP embryos. CB1R immunoreactivity (red fluorescence) was detected in various regions of the basal forebrain, in particular in the anterior and postoptic commissures, in the optic chiasm and in the longitudinal tracts. In the same regions, EGFP+ axonal extensions are clearly seen in close proximity to the CB1R signal , N-deacetylase and N-sulfotransferase-3 , Neural growth regulator-1(NEGR1), Sodium voltage-gated channel alpha subunit-2 1.2), Seizure related gene-6 (SEZ6), HECT, C2 and WW domain containing E3 ubiquitin protein ligase-1 , Septin-6 (SEPT6) and Stathmin-2 .Among the genes appearing in relevant functional classes we noted the presence of semaphorins, cell adhesion molecules, kinesins, GABA and glutamate receptors. Finally, from the list of relevant genes we further selected them by a literature search and came up with a set of 8 genes putatively interacting with CB1R, or sharing a common expression pattern, and potentially involved in axon elongation–fasciculation-guidance: Glycoprotein M6A z-stmn2a, z-stmn2b, negr1, z-sez6a and z-sez6b, since they have been reported as regulator of axons growth and cell migration [For further analyses, we focused on igration ,61,62,63z-stmn2a, z-stmn2b, negr1, z-sez6a and z-sez6b mRNAs in samples extracted from embryos treated with WIN55 (1 nM), Rimonabant (1 nM) or AM251 (100 nM), or treated with DMSO only as control. All treatments resulted in a significant reduction in the abundance of z-stmn2a relative to the control  we tested the possible toxic effects of their chronic exposure on zebrafish embryos from 0 to 72 or 96 hpf using a wide range of concentrations. Embryo survival was affected only by the highest agonist concentration (1 μM WIN55), and therefore this concentration was excluded from subsequent experiments. The selected ligands concentrations did not interfere with the overall embryo development and the hatching rate. These results partly agree with a previous study , in whicgnrh3::EGFP and the agrp1::mCherry strains [gnrh3::EGFP embryos clearly showed abnormal fasciculation of GnRH3 axons in the anterior commissure, with some ectopic axons detaching from the commissure itself. Defective phenotypes resulted from exposures to all three tested drugs  even at the lowest concentrations, indicating that interfering with the endogenous eCB system, either through over-activation or inhibition, equally disrupts the modulatory control exerted by CB1R on axonal navigation and fasciculation. We obtained similar results by knocking down the CB1R with a morpholino strategy, confirming previous observations made on the anterior commissure [We analyzed the impact of chronic CB1R ligand exposure on the developing GnRH3 and AgRP1 systems, taking advantage of the fluorescently labelled neurons present in the  strains ,71. Pharmmissure . FurtherIn addition to the defects observed in fiber tracts, we found a significant decrease in the number of GnRH3 cell bodies  following both antagonist (AM251) exposure and CB1R knockdown. These results are consistent with the previously reported role of CB1R as a regulator of progenitor niches ,23. Alteagrp1::mCherry zebrafish embryos resulted in anterior commissure defects similar to those observed in gnrh3::EGFP embryos, suggesting that a common mechanism affected both GnRH3- and AgRP1-containing fibers. We found an increase in the number of AgRP1+ cell bodies in the hypothalamus in AM251-treated embryos, however this result could be affected by the methodology we chose for counting cells. It is possible that the apparent augmented number of AgRP1 cells depends on a decrease in fiber number, which would be in line with an axonal defect. Pharmacological interference of CB1R in In our experiments, we observed similar phenotypes when applying CB1R agonists or antagonist/inverse agonists. This is consistent with the fact that different studies reported opposite effects of CB1R activation on axonal growth. Indeed, in some cases CB1R-activation has been reported to induce collapse of the growth cone and axon retraction, while its inhibition resulted in axon elongation ,22,25,49Multiple mechanisms downstream of CB1R activation in developing neurons have been suggested. It has been reported that CB1R acts via the WAVE complex and Rac1-GTPases, and via Rho-GTPase and the Rho-dependent ROCK activation ,10; thesAnother reported mechanism involves the stathmin family of proteins and the fine modulation of microtubule dynamics at the growth cone. Stathmin-2, also known as SCG10  is a neuron-specific protein highly expressed in the developing forebrain, able to stabilize the plus ends both at steady state and early during polymerization by increasing the rate and extent of growth and facilitating microtubule extension into filopodia . OverexpDanio rerio) were authorized by the Ethical Committee of the University of Torino and the Italian Ministry of Health . The wild-type fish strain TL-AB was used. The fish strain Tg(gnrh3::EGFP) [agrp1::mCherry was obtained by breeding the TgBAC(agrp:GAL4-VP16) with the UAS::nfsB-mCherry strains, followed by selection of the reporter fishes by quick fluorescent examination at low magnification [All procedures using zebrafish (3::EGFP) ,46,71 wafication . Adult fTg(gnrh3::EGFP) embryos were grown in the presence of 0.003% 1-phenyl-2-thiourea (PTU) to prevent the formation of melanin pigment, which could interfere with the visualization of fluorescence in neurons and fibers; the agrp1::mCherry embryos were grown in fish water without PTU, because the pigmentation in this case did not affect the analysis under microscope.The The endpoints used to assess developmental toxicity comprised embryos survival  and hatching rate (percentage of hatching embryos/living embryos), observed every 24 h during the whole exposure period (72 or 96 hpf). Dead embryos were removed without solution renewal.z-cb1r MO was designed as previously published [z-cb1r MO:5′ CTAGAGGAAACCTGTCGGAGGAAAT 3′Mismatch_PPFIA scrambled MO (control):5′ TCGTGGCCATCAACTCGAACA 3′.To down-modulate CB1R we utilized a conventional antisense morpholino oligonucleotide (MO)-mediated strategy . The ublished . The MOsz-cb1r MO (or the scrambled MO as control), in the presence of Phenol Red for subsequent selection of the injected embryos. Uninjected embryos were also analyzed.Zygotes were collected at the 1-cell stage and injected under stereological examination with 50 µM, 200 µM or 400 µM of Zebrafish embryonic heads at 72 hpf were lysed manually in modified RIPA buffer composed as follows: 150 mM NaCl, 50 mM Tris pH 7.5 with 1% Triton X-100, 0.5% Na deoxycholate, 0.1% SDS, 2 mM EDTA, 1 mM sodium orthovanadate, 1 mM sodium fluoride and protease inhibitors. Homogenates were centrifuged at 3000 rpm for 5 min at 4 °C, extracts were quantified with BCA method (Bradford). A total of 30 µg of proteins/lane were separated in 10% acrylamide gel and transferred to a PVDF membrane . Membranes were saturated with 5% BSA in 0.3% TBS-Tween-20, washed in TBS-Tween, incubated overnight at 4 °C with an anti-CB1R primary antibody (diluted 1:800), then washed in TBS-Tween, incubated with an anti-rabbit IgG HRP-conjugated secondary antibody , washed and developed with chemo-luminescence reagent Luminata TM Forte Western HRP Substrate (Millipore). Images were acquired with the ChemiDoc system (Bio-Rad), exported and analyzed.gnrh3::EGFP fish embryos were collected at 72 hpf and processed as described above. Embryos were sacrificed with a tricaine overdose, fixed in 4% PFA overnight, then washed in PBS. Solution was replaced with 0.1M PB and 7% sucrose at 4 °C for 12 h, then with 0.1M PB and 30% sucrose at 4 °C overnight. Samples were then embedded in OCT  and sectioned at 14 μm thickness. Sections were washed in 0.01M PBS pH 7.4 for 10 min and incubated in normal goat serum (diluted 1:100 in PBS-Triton-X 100), then incubated with the anti-CB1R antibody  O/N. Slices were then washed in 0.01M PBS pH 7.4 for 10 min and incubated with anti-rabbit IgG CY3-conjugated secondary antibody  for 45 min. For cellular nuclei visualization, slices were incubated with DAPI , then washed in PBS and mounted in DABCO. The fluorescent signal was visualized using a Leica TCS SP5 .http://www.hprd.org). Only those which appear in the list of at least two entry genes were further considered. Conserved coexpression in transcriptomic datasets was searched for using FuncPred (https://www.mbc.unito.it/en/notizie/funcpred) [http://www.biomart.org).Known and putative protein::protein interactions were searched for using the Human Protein Reference Database (uncpred) ,58. Convhttp://www.ncbi.nlm.nih.gov/geo/) [As a reference dataset of differentially expressed genes in the hypothalamus, we used the dataset GSE21278 present in Gene Expression Omnibus (ov/geo/) . This dahttp://geneontology.org/page/go-enrichment-analysis and at http://amp.pharm.mssm.edu/Enrichr/.Functional annotation and definition of the over-represented categories was done using the tools available at All data were statistically evaluated using commercially available software . Significance was calculated using a t-test and one-way ANOVA followed by Bonferroni’s post hoc test for multiple comparisons.g at 4 °C, RNA was precipitated in isopropanol. Samples were centrifuged at 12,000× g at 4 °C for 15 min and the RNA was then washed with 75% ethanol. The RNA pellet was briefly air-dried, resuspended in 20 µL sterile water and stored at −20 °C. Samples were quantified using a NanoDrop1000 spectrophotometer . Real-time qPCR was performed using Superscript III Platinum One-step qRT-PCR system  and the thermal cycler Rotor Gene Q . Each RNA sample was analyzed in three technical replicates containing 50 ng of total RNA. Primers sequences werez-β-actinFCCCACATAGGAGTCTTTCTGRTCCCCTTGTTCACAATAACCz-cb1rFCAGAACAGATCATACACCATGARTGGTCTTATTCATCATCTACGCz-hoxb7aFGCTGGCGATCTCTGTAAAGCRTTTTGATGGTAGCCCCTCTGz-hoxa10bFGAGCTAAGGGGGTCCACTGRCACTTTTGGAATCTCCTGCTTTz-hox11aFCAGCAAACAGTCGACACCACRCGGTCGCTCCTTTCCTTCz-stmn2aFTCCATGCTCTCGCTTATTTGRGGAGGATGTAGAGATGTGCTz-stmn2bFATGGAGCAGATCAAGGAGAARGGAGGATGTAGAGATGTGCTz-sez6aFAGTTTAGCAGTGAACGAACCRGAAGACCAGTGACTGAAACCz-sez6bFGTCCATTTTCATCCCTGTGGRGGCTGCTTTGAATCATAGGTz-negr1FTTTTCGAGTGGTACAAGGGARGGGTTGGATGGATTCAGAGGTotal RNA was extracted from 72 hpf embryo heads with the TRIzol reagent , following the manufacturer’s instructions. Genomic DNA was destroyed by passing each sample into a 22G needle connected to a 1 mL syringe. Chloroform was added and, after 15–20 min of centrifugation at 12,000× z-β-actin housekeeping mRNA was used for normalization. The mRNA expression was calculated relative to the one of control embryos (=1).Quantification of mRNA abundance in each sample was done using a standard curve, built with several dilution of the samples. The abundance of the gnrh3::EGFP) or 96 h (agrp1::mCherry) without solution replacement for the entire exposure period. At 24 hpf 0.003% PTU was added to the water for gnrh3::EGFP fish embryos. Embryos were grown in 6-well Petri dishes, 30 embryos/well. Negative control embryos were treated with 0.1% DMSO alone. Upon fertilization, at the 1-cell stage, embryos were treated with the following ligands : CB1R-agonist WIN 55,212-2 ; CB1R-inverse agonist Rimonabant (SR141716A) ; CB1R-antagonist AM251 . All these compounds were dissolved in 0.1% dimethyl-sulfoxide (DMSO) and added to the fish water for 72 h  at 4 °C O/N, washed in PBS and embedded in 4% low melting agarose. The apical portion of the head was manually dissected from the rest of the embryo. Confocal microscopy examination was carried out at 20× and 40× magnification with a Leica TCS SP5 or Leica TCS SP8 (Leica Microsystems). Images were acquired as Z-stacks of 5 μm-thick optical sections. For counting, stacks of Z-slices at 40× were used, images were processed and analyzed with the assistance of ImageJ software  after adjustment of contrast and brightness.For whole mount visualization, the p ≤ 0.05 was taken as minimum level of significance. Data are expressed as mean ± standard error of the mean (SEM) from three independent experiments. The results of pharmacological treatments/morpholino injections were compared to control (DMSO-treated/uninjected embryos). Statistical analyses were performed using commercially available software (SPSS version 18.0 for Windows and Microsoft Excel 2011). Significance was calculated using Fisher’s exact test for the analysis of altered phenotypes and one-way analysis of variance (ANOVA) for all other experiments, followed by Bonferroni’s and Tuckey’s post hoc tests for multiple comparisons. The impact of exogenous cannabinoids on the gonadotropic neurons and the orexigenic/anorexigenic neurons has long been investigated ,75,76. M


I249/M280 during EAE. We hypothesize that dysregulated microglial responses due to impaired CX3CR1 signaling enhance neuronal/axonal damage. We generated an animal model replacing the mouse CX3CR1 locus for the hCX3CR1I249/M280 variant. Upon EAE induction, these mice exhibited exacerbated EAE correlating with severe inflammation and neuronal loss. We also observed that mice with aberrant CX3CR1 signaling are unable to produce FKN and ciliary neurotrophic factor during EAE in contrast to wild type mice. Our results provide validation of defective function of the hCX3CR1I249/M280 variant and the foundation to broaden the understanding of microglia dysfunction during neuroinflammation.Multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system (CNS) is the leading cause of non-traumatic neurological disability in young adults. Immune mediated destruction of myelin and oligodendrocytes is considered the primary pathology of MS, but progressive axonal loss is the major cause of neurological disability. In an effort to understand microglia function during CNS inflammation, our laboratory focuses on the fractalkine/CX3CR1 signaling as a regulator of microglia neurotoxicity in various models of neurodegeneration. Fractalkine (FKN) is a transmembrane chemokine expressed in the CNS by neurons and signals through its unique receptor CX3CR1 present in microglia. During experimental autoimmune encephalomyelitis (EAE), CX3CR1 deficiency confers exacerbated disease defined by severe inflammation and neuronal loss. The CX3CR1 human polymorphism I249/M280 present in ∼20% of the population exhibits reduced adhesion for FKN conferring defective signaling whose role in microglia function and influence on neurons during MS remains unsolved. The aim of this study is to assess the effect of weaker signaling through hCX3CR1 V 249/T280 allele, while the variant CX3CR1I249/M280 is present in 25–30% of the population. Cells expressing CX3CR1I249/M280 are less responsive to FKN  inflammation, and demyelination in MS models has not been directly addressed.Inflammatory demyelination is a hallmark of the pathology of multiple sclerosis (MS) and microglia are considered responsible for exacerbating myelin loss. Although the link between microglia mediated inflammation and neurodegeneration is being increasingly recognized, the mechanisms that connect microglia, inflammation, and demyelination are unclear. Our previous studies have shown that the neuronal derived chemokine, fractalkine , and its unique receptor, CX3CR1, expressed by microglia, are highly abundant in the healthy brain and spinal cord and have direct roles in inhibiting microglia inflammatory behavior . Notablye to FKN  and in a lesions . HoweverI249/M280 variant in signaling to FKN, we sought to determine how closely the expression of the human variant recapitulates the phenotype observed in CX3CR1-deficient mice. For this, we generated a model in which mice in the C57BL6 background express the I249/M280 variant of the human CX3CR1 receptor in lieu of the murine counterpart. In this study, we report the phenotype of CX3CR1I249/M280 expressing mice under active disease in myelin oligodendrocyte glycoprotein (MOG35-55) induced experimental autoimmune encephalomyelitis (EAE). We describe disease progression, CNS cellular infiltration, demyelination, neuronal loss, and expression of inflammation-associated genes. Our results show that EAE progression is more severe in CX3CR1I249/M280 mice than WT mice and closely resembles the disease course observed in CX3CR1-KO mice. More severe demyelination and neuronal loss was also observed in CX3CR1I249/M280 mice, particularly in cerebellar regions. Differences in disease severity did not appear to be associated with increased frequencies of IL-17 or IFN-γ producing MOG35-55 specific T cells, as revealed by comparable proportions of IFN-γ and IL-17 producing T cells in WT, CX3CR1-KO and CX3CR1I249/M280 draining lymph nodes at 11 days post-immunization. CD4+ T cells represented the predominant population expressing IL-17 or IFN-γ, with negligible detection of IL-17/IFN-γ double positive cells in intracellular cytokine image flow cytometry assays. In CNS tissues, analyses of inflammatory cells revealed comparable levels of infiltration of dendritic cells (DCs), neutrophils and CD4+ T cells, however, CX3CR1I249/M280 mice displayed increased frequencies of CD45Lo/CD11b+ microglia and CD8+ T cells. Transcriptional analyses revealed several genes highly upregulated in all genotypes but of particular interest was lipocalin 2, and immunostaining confirmed its significant upregulation in WT tissues compared to CX3CR1-KO and CX3CR1I249/M280 brains. Overall these results indicate that CX3CR1I249/M280 expressing mice exhibit an EAE phenotype that closely resembles the phenotype of CX3CR1-KO mice. These findings highlight the neuroprotective effects of FKN during EAE and provide a potential novel pathway to understand how LCN2 could be targeted to control inflammation and promote repair, based on its regulation by CX3CR1/FKN signaling.Due to the proposed defect of the human CX3CR1in vitro using bone marrow derived macrophages grown on poly-D-lysin coated flasks in complete RPMI media  for 4 days and electroporated with 2 μg of pIRES2 expression plasmid containing the human CX3CR1 ORF for the reference hCX3CR1V 249/TM280 or hCX3CR1I249/M280 genes. Cells were expanded for 3 days in complete media and the number of cells that mobilized across a millicell PCF 8 μm insert were quantified by virtue of RFP expression from expressing vector and DAPI nuclear staining. Five images at 20x were acquired per insert and cells were quantified. Data was reported as number of cells per mm2.Responsiveness of human CX3CR1 receptors to mouse fractalkine was tested I249/M280 complete open reading frame (ORF), a neomycin gene within lox P sites, and the 3′ untranslated region of mouse CX3CR1 [long arm (LA)]. A genomic fragment containing the hCX3CR1I249/M280 ORF was subcloned from plasmid pSC59 (I249/M280 mice that were backcrossed (>N6) to C57BL/6 mice. Cx3cr1 transcript levels were assessed in microglia sorted from WT, KO, and hCX3CR1I249/M280 mice. Mice were genotyped by polymerase chain reaction (PCR) using specific primers to identify not only insertion of the human cx3cr1 gene but also locus specific insertion: SA-13: 5′-CCAAGACAGAATTTCACTGTGTAGC-3′; SA-14: 5′-CAACAAATTTCCCACCAGGCCAATG-3′; SA-15: 5′-TACCTGGCCATCGTCCTGGCCGCCA-3′; SA-16: 5′-ACGAGACTAGTGAGACGTGCTACTT-3′. Crossing of these mice with B6.FVB-TG (EIIa-cre)C5379Lmgd/J  resulted in cre-mediated recombination of the floxed neomycin resistance gene, removal of the stop cassette, and concomitant loss of the SA-15/SA-16 1,004 bp fragment and subsequent expression of the hCX3CR1 unit. Validation of the introduction of the human gene and respective I249/M280 mutation was further confirmed by sequencing.Genomic fragments of the murine CX3CR1 locus were isolated from the pEasyFlox-CX3CR1-eGFP targeting vector  by PCR tid pSC59 . The homGFP/GFPCX3CR1 mice were purchased from The Jackson laboratory , hCX3CR1I249/M280 were backcrossed to C57BL/6J background for seven generations. Mice were maintained at the Laboratory Animal Resource Center at the University of Texas at San Antonio. All experiments were performed in accordance with NIH guidelines and approved by UTSA-Institutional Animal Care and Use Committee.C57BL/6J wild type (WT) and 35-55 peptide in CFA, and 200 ng of pertussis toxin on days 0 and 2 post-immunization as previously described , inflammatory monocytes , cerebellar neurons , pro-inflammatory cytokine interleukin-1 beta , myelin basic protein (MBP) , and astrocytes . Secondary antibodies were purchased from Jackson ImmunoResearch Laboratory as follows: Cy3-goat anti rabbit, Cy5-donkey anti rat, Cy2-goat anti mouse, biotinylated-goat anti rabbit, biotinylated-goat anti mouse, and biotinylated-rabbit anti rat. Biotinylated antibodies are detected by using stable DAB (Thermo Fisher Scientific) containing diaminobenzidine and hydrogen peroxidase.++, Mg++, Lonza) followed by 4% PFA. Dissected brains were post-fixed overnight in 4% PFA and cryoprotected in 20% glycerol in 80mM phosphate buffer (pH 7.6) for at least 24 h. Free floating sections (30 μm) were generated using a freezing microtome and stored at -20°C until use. For immunostaining, tissues were blocked and permeabilized with 10% normal goat or donkey serum (Jackson ImmunoResearch) containing 0.3% Triton X-100 (Sigma) for 1 h at room temperature. Primary antibodies were incubated overnight at 4°C. Tissues were washed in PBS supplemented with 0.1% Triton X-100 and incubated with host-appropriate secondary antibodies for 2 h at room temperature. Tissues were then mounted onto superfrost plus slides, allowed to air dry and baked at 50°C for 20 min. For quantification of myelin content and calbindin and IBA-1 immunoreactivity three tissue sections per mouse were stained and eight random images per sections were acquired. Individual cells were counted using the count analysis tool in Adobe Extended CS6v13 (Adobe) and immunoreactive area obtained in Image J as previously described .Mononuclear cells were isolated from brain and spinal cord of CX3CR1-WT, CX3CR1-KO, and hCX3CR1escribed . Cellula2 under the following conditions: cRMPI . For intracellular cytokine staining, the following reagents were used: Mouse Fc block ; fixation/permeabilization buffer (Invitrogen); perm/wash buffer (BD Biosciences); cell staining buffer (BioLegend) and the following antibody cocktail: TCR-b V450 , CD4-APC-Cy7 , CD8a-PerCP/Cy5.5 , IL-17-PE  and IFN-γ-APC  Samples were acquired using an ImageStreamX-Imaging Flow Cytometer-ISX-MKII (EMD Millipore) at the Cell Analysis Core, UTSA. Data analysis was performed using IDEAS software version 6.2.To assess cytokine production on T cells infiltrating the CNS, immunized mice were sacrificed at peak disease and mononuclear cells from brain tissues were isolated as previously described . T cells35-55 and sacrificed 10 days post-immunization. Inguinal lymph nodes were harvested and cells were plated at a density of 5 × 105 per well. Assay was performed as previously described . Spinal cord, cerebellum, and forebrain were dissected and disrupted manually using dounce homogenizers. Suspensions were made in lysis buffer containing 150 mM NaCl, 10 mM Tris-Base, 1 mM EDTA pH 8.0 and 1X protein inhibitor cocktail (Roche). Lysates were centrifuged at 12,000 rpm for 15 min at 4°C, aliquoted and stored at -80°C. Total protein concentrations were determined using the Bio-Rad protein reagent assay (Bio-Rad) and quantities of soluble factors were measured by enzyme-linked immunosorbent assay (ELISA) using the following kits: DuoSet ELISA development system (R&D Systems) for mouse CX3CL1/Fractalkine, DuoSet ELISA development system (R&D Systems) for mouse IL-1b/IL-1F2 and ELISA kit (CUSABIO) for the mouse CNTF. Samples were run in duplicate. Results were normalized to total protein and reported in picogram (pg) amounts per milligram (mg) of protein as previously described  in CNS tissues, mice were anesthetized with 5% isoflurane in an induction chamber, followed by perfusion with ice cold Hanks balanced salt solution , libraries were obtained from the genome sequencing facility . Raw reads were imported into CLC Genomics Workbench v10.1.1. Initially, low quality reads were trimmed; remaining reads for each sample were mapped to the annotated mouse genome (GRCm38), followed by differential gene expression (DGE) analysis of EAE samples from CX3CR1-WT, CX3CR1-KO, and hCX3CR1I249/M280 mice against their respective naïve controls using the RNA-seq tool within CLC Genomics Workbench software. Genes were deemed to be differentially expressed if the false discovery rate (FDR) was 0.05 or less.Total RNA (100 ng) from cerebellar extracts at chronic EAE was used to analyze gene expression changes using nCounter and nSolver 3.0 software (NanoString Technologies). Transcript levels of 582 genes were assessed with the nCounter GE-Mouse Immunology v1 Kit (NanoString Technologies) performed by the Genomic and RNA Profiling Core Baylor College of Medicine (Department of Advanced Technology Cores). Data were initially normalized using positive and negative controls and housekeeping specific probes . SamplesHomo sapiens CX3CR1 is reported in the Uniprot database in FASTA format and was used for structural prediction model  and variant CX3CR1ficients  which asficients . Since Cficients . The theficients .t-test or One-way ANOVA followed by Tukey’s post-test when comparing multiple groups. p-Values < 0.05 considered statistically significant. RNA seq data was subjected to exact test comparison of EAE tissues against their naïve counterparts. Bonferroni and FDR corrections were applied. FDR p-values < 0.05 were considered statistically significant.Data are expressed as mean ± SEM in graphs and plots. In scattered plots, each dot represents data from an individual mouse. Data were plotted in GraphPad Prism 5 and statistical comparisons performed by non-paired two-tail Student’s I249/M280) was introduced into the mouse genome replacing the mouse counterpart . Comparison of protein sequence between the WT and variant receptors revealed that the T280M substitution contributes to an increase in the hydrophobicity of the region between amino acids 275 and 290 . The hydropathic index is important to understand how the mutations in the variant receptor affect the protein regions potentially involved in the interaction ligand–receptor. A tridimensional model of the variant receptor proposes an alteration in the interaction with the cellular membrane resulting in a shift toward the intracellular space of the alpha-helix containing the V249I substitution . This shift has a significant effect in the extracellular portion of the protein as it results in the disruption of disulfide bridges (C21–C265), thereby generating conformational changes in the protein structure that alter CX3CR1-FKN recognition domain which could potentially negatively affect protein function . Results indicate that after active immunization with MOG35-55 peptide, clinical disease was more severe in mice expressing the human CX3CR1 variant when compared to WT mice . Interestingly, the time at which the hCX3CR1I249/M280 mice reached their highest clinical disability  resembled the observations of the CX3CR1-KO mice. Despite the fact that the maximum score observed in the hCX3CR1I249/M280 mice showed an intermediate value between the WT and CX3CR1-KO mice , the cumulative disease score, a measure of clinical disease severity closely resembled and reproduced previous observations from our laboratory reporting an exacerbated phenotype in mice lacking CX3CR1 . These data support the notion of impaired fractalkine signaling as a result of the expression of the adhesive deficient human I249/M280 polymorphic variant.Data from our laboratory has shown that fractalkine/CX3CR1 signaling regulates microglia neurotoxicity in selected mouse models of neurodegeneration . To compFigure 4A, top). Severe inflammation in mice expressing the hCX3CR1I249/M280 receptor correlated with significant decrease in calbindin+ cells in the cerebellum  based on the quantification of neurons in the Purkinje cell layer and its comparison to immunized WT mice. Neuronal loss in CX3CR1-KO and hCX3CR1I249/M280 mice correlated with worsened demyelination identified by the loss of MBP staining in the cerebellar white matter when compared with myelin staining in naïve mice . These observations recapitulate the cerebellar pathology observed in the CX3CR1-KO mice, suggesting a crucial role for defective CX3CR1 signaling in the inflammatory mediated events that lead to the development of pathological features during MOG35-55 induced neurodegeneration.To investigate the association between clinical manifestation and CNS pathology, we assessed infiltration and neuronal damage in brain sections of mice sacrificed at the chronic phase of EAE. Higher clinical scores during EAE were associated with severe infiltration of blood-derived inflammatory cells to the CNS and their actions on axonal fibers that lead to the ataxia and paraplegia observed in the time course of the disease. Persistent extravasation of inflammatory cells, particularly into the cerebellum of immunized mice, are depicted by the increase in CD45 staining upon EAE induction  reveals a higher proportion of CD45Hi cells at peak disease  compared to chronic stages when CD45Lo/CD11b+ microglia appear as the predominant cell population . Interestingly, the proportion of microglia at peak EAE in hCX3CR1I249/M280 mice slightly exceeded what was observed in CX3CR1-KO mice . Assessment of myeloid populations revealed CD45HiCD11b+Ly6C+Ly6G-CD11c- macrophages  as the most abundant myeloid cells in the brain of EAE-induced mice both at peak and chronic EAE , regardless of the CX3CR1 genotype. Also, comparable levels of CD45HiCD11b+Ly6G-Ly6C-CD11c+ myeloid derived dendritic cells (mDCs)  were observed during chronic EAE  across all genotypes. Flow cytometry analyses were also conducted in spinal cord tissues to classify the components of the inflammatory response in hCX3CR1I249/M280 mice . Severe infiltration, reflected by the close to 70% of the total CD45Hi population, was observed in the spinal cord of mice at peak disease . Similarly to our observations in the brain, the severe inflammatory reaction appears to be the result of macrophages mobilization , and excessive extravasation of CD4+ T cells . Flow cytometry data from brains of EAE mice at chronic disease  show a diminished inflammatory state by a reduction on the overall CD45Hi population across all the genotypes. In contrast, WT mice exhibit a persistent state of cellular infiltration into the spinal cord when compared to hCX3CR1I249/M280 mice (and to CX3CR1-KO to a lesser degree) . Previous studies revealed that autoreactive T cells against myelin proteins play a critical role on disease development during EAE. More specifically, T cell responses that result in the generation of IFN-γ secreting Th1 and IL-17 secreting Th17 CD4+ cells are involved in inflammatory-mediated damage to myelin and neurons in EAE. The activation of CD4+ T cells is initiated in peripheral lymph nodes with the binding to the antigenic peptide on major histocompatibility complex (MHC) class II and co-stimulatory molecules on antigen presenting cells . Cytokine-producing T cells specific for MOG35-55 antigen were quantified as spot forming units and compared among WT, CX3CR1-KO and hCX3CR1I249/M280 genotypes . Peripheral deficiency or defective CX3CR1-signaling did not have a negative effect in the priming and generation of pathogenic CD4+ T cells. Similar to the ELISpot results, IFN-γ and IL-17 producing CD4+ T cells were detected in the CNS  of mice at peak disease . These cells were harvested and, upon activation in vitro, showed no evident difference in cytokine production due to CX3CR1 signaling, supporting the notion that differential manifestation of EAE symptoms and CNS pathology does not seem to be the result of altered T cells responses in CX3CR1-KO or hCX3CR1I249/M280 mice. The abundance of T cells in the spinal cord of EAE mice was consistent with the notion that CD4+ T cells are the primary drivers of pathological alteration in this murine model . Pathological data from MS patients shows that CD8+ T cells may play an important role in the propagation of the inflammation and tissue damage  providing a relevant foundational argument for the utilization of these mice as a valid model for human inflammatory diseases of the CNS.Analysis of the inflammatory response in EAE affected brains by flow cytometry analysis . LCN2 was the most upregulated gene in hCX3CR1M280 mice displaying a 26.19 fold increase, accompanied by higher levels in CX3CR1-KO mice (↑91.61) and WT mice (↑100.64). Increased LCN2 mRNA was validated at the protein level by immune-detection of LCN2 protein in brain and spinal cord tissues of EAE mice . The immunopositive area was quantified in brain tisues  and LCN2+ cells were counted in spinal cord . LCN2 levels in naïve tissues are represented in dotted blue line in respective graphs . Other major upregulated genes include Ccl5, Cxcl9, Cxcl10, cytochrome b beta (Cybb), complement component C3 (C3), and CD74. Inflammatory related genes upregulated in KO mice and with lesser abundance in hCX3CR1M280 mice include H2-Aa, H2-Ab1, and H2-Eb1.A fundamental concept underlying MS and EAE pathology is the associated inflammatory reaction, and the interplay between hematogenous derived cells and resident brain cells that contribute directly and/or indirectly to demyelination and axonal damage. In our efforts to further assess differences and similarities between the response to EAE in WT, CX3CR1-KO and hCX3CR1Figures 8A,B) accompanied by changes in cell morphology, reflected by retraction of cellular processes and enlargement of the cell body . Microglia activation is calculated by measurements of the roundness of individual cells as depicted by the transformation index . Assessment of activation markers by microglia show an increased expression of MHC-II molecules during peak EAE, which were found ameliorated during chronic stages of the disease . Negligible changes in the median fluorescence intensity (MFI) for CD11b  or CD86  were observed in microglia in diseased mice, suggesting that antigen presentation through MHC-II is one of the most prominent features of microglia during EAE. Basal level for activation markers on naïve microglia are noted in dotted lines . Infiltrating monocytes (CD45HiCD11b+Ly6C+) showed increased MFI for MHC-II, CD11b, and CD86 only during peak EAE, with values comparable to naïve microglia by later stages of the disease. Activation markers on naïve blood macrophages did not show statistical differences across the genotypes . Further RNA-seq analysis from microglia sorted from WT and CX3CR1-KO mice provided evidence for a role of microglia in myelination, iron trafficking, anti-inflammation, and neurogenesis driven by the expression of CX3CR1  as noted by the upregulation of genes involved in these physiological process only in WT microglia. In contrast, microglia from mice lacking the CX3CR1 receptor show upregulation of genes involved in inflammatory processes. Fold change and FDR p-value for significant genes (upregulated and downregulated) in CX3CR1-WT and CX3CR1-KO microglia during EAE are found in Supplementary Tables S2.Our laboratory and others have shown that FKN regulates microglia function by inhibiting microglia activation and the release of inflammatory mediators such as IL-1β. Induction of EAE results in increased microglia numbers in cerebellar tissues . A sevenfold increase in fractalkine was observed in naïve CX3CR1-KO mice in comparison to naïve WT mice, supporting a role for CX3CR1 as a scavenger receptor . Similar to the effects on fractalkine production, we observed impaired production of CNTF in the CNS of both mouse strains with defective CX3CR1 signaling, CX3CR1-KO and hCX3CR1I249/M280 mice at chronic EAE . Also, the defect on CNTF production correlated with the significant neuronal cell loss observed as a result of CX3CR1 dysfunction in CX3CR1-KO and hCX3CR1I249/M280 mice. To address if IL-1β levels were differentially regulated in the diseased brain, cytokine ELISA was performed , and combined with IL-1β immunostaining, the data indicate that soluble and cell-associated IL-1β is increased more abundantly in WT mice .Within the CNS, fractalkine has been proposed to act through the interaction with its unique CX3CR1 receptor expressed on microglia to maintain their homeostatic resting state. We found that basal fractalkine levels detected in soluble brain and spinal cord extracts of WT mice have a fivefold increase during EAE-induced disease , and abundance of microglia and CD8+ T cells in hCX3CR1I249/M280 mice . Although this report does not include a direct comparison to the reference hCX3CR1V 249/TM280, this humanized system serves as a proof of principle to validate alternative interventions/therapies to modulate the inflammatory reaction in the EAE brain with direct translational relevance to human disease and will provide valuable information on the function of these polymorphic receptors in various immune cell types.Chemokines and their receptors play an important role during CNS autoimmune inflammation, not only by guiding immune cell infiltration within demyelinating lesions but also by promoting a communication system for resident CNS cells. As an example, FKN and CX3CR1 are abundantly expressed in the naïve CNS by neurons and microglia respectively. FKN and CX3CR1 play an important role in brain homeostasis and the severe phenotype of mice lacking FKN or CX3CR1 in selective models of neurodegeneration poses a question about the impact of polymorphic CX3CR1 variants during neuroinflammatory insults. In this study, we describe the phenotype of mice expressing the human CX3CR1in vitro that the reference human receptor was able to signal in response to mouse FKN and induce cell migration in a dose dependent manner, whereas the variant hCX3CR1I249/M280 displayed blunted responses, correlating with published studies that established the adhesive defective properties of the hCX3CR1I249/M280  assessing chemotactic responses toward the mouse FKN in transmigration assays, (2) confirming homologous recombination of the target construct by Southern blots using 3′ and 5′ probes, and (3) comparing CX3CR1 expression at the transcript level in CNS tissues  across the genotypes, showing that mice expressing the hCX3CR1I249/M280 variant receptor exhibit CX3CR1 mRNA levels comparable to WT mice.We showed 249/M280 . Protein35-55 antigen specific T cells in draining lymph nodes. Similarly, myeloid cell infiltration and abundance of CD4+ T cells did not reveal significant differences among CX3CR1-WT, CX3CR1-KO, and hCX3CR1I249/M280 groups. However, CD8+ T cell infiltration appeared to be increased in mice expressing the variant receptor. Similarly, microglial frequency quantified by flow cytometry appear increased in hCX3CR1I249/M280 mice. Of interest was the distinct transcriptional profile of WT versus KO CNS tissues during the chronic phase of EAE. Using the inflammatory code set from nanostring technologies, lipocalin 2 (LCN2) was the top upregulated gene with a higher abundance in both CX3CR1-WT and CX3CR1-KO tissues compared to hCX3CR1I249/M280. Validation at the protein level revealed a similar pattern of expression in brain tissues in agreement with other EAE studies  also revealed several significant differentially regulated genes, with particular relevance in WT microglia transcripts involved in myelination , iron trafficking , neurogenesis (Bst1), and anti-inflammatory pathways . Due to the expression of IL-1 related transcripts and the association of IL-1β with microglial neurotoxicity (I249/M280 mice most IL-1β+ cells colocalized with GFAP+ staining. Also, of relevance is the observation of decreased CNTF protein levels in CX3CR1-KO and CX3CR1I249/M280 mice, in particular because CNTF is widely recognized as a key survival factor not only for neurons but also for oligodendrocytes (Analyses of cellular infiltration revealed comparable frequencies of IFN-γ and IL-17 MOG studies . LCN2 is studies , inflamm studies , neuroge studies , iron tr studies , and red studies . The phe studies  and anotin vitro . The datin vitro , understtoxicity  we assesdrocytes . Similar+CD4+ T cells were enriched in cerebrospinal fluid of relapsing-remitting MS patients (Hi NK cells in peripheral blood which correlated with disease activity (HiNK cells via elimination of autoreactive T cells is proposed (I249/M280 variant show higher disease scores, significant demyelination, decreased cerebellar neuronal counts, higher CD45LoCD11b+, and CD8 T cell densities. Using nanostring analyses from the top significantly upregulated genes among WT, KO and hCX3CR1I249/M280 mice, LCN2, Ccl5, Cxcl9 and genes related to MHC-II antigen processing and presentation were in lesser abundance in hCX3CR1I249/M280 mice. Previously published observations support the relevance of FKN/CX3CR1 pathway in CNS pathology, the results in the current study establish the potential risk of worsening disease severity in a hCX3CR1I249/M280 expressing host. Future studies guided to compare microglia, and CD8 T cells responses in mice alternatives models engineered to express the human reference and variant CX3CR1 receptors will be critical to validate the data presented here and will overall provide a better understanding of the impact of this polymorphic variants in optic nerve pathology and models of demyelinating disease.The combined actions of genetic and environmental factors influence susceptibility to autoimmune disease . In the patients  and CX3Cpatients . It was patients . MS patiactivity , and CX3proposed . EAE-indproposed . OverallSC designed experiments, performed research, analyzed data, and wrote the article. SK performed research, analyzed data, and revised the manuscript critically. VT, KC, IC, SS, and AM performed research. SW revised the work critically and provided intellectual resources. JP-T performed research and revised the work critically. AO and PA analyzed and interpreted data. RR conceived the study. AC conceived the study, designed experiments, performed research, analyzed data, and wrote the article.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.


Arabidopsis. Loss-of-function mutation of BLI results in chronic up-regulation of a set of both canonical UPR genes and non-canonical UPR downstream genes, leading to cell death and growth retardation. Genetic analysis indicates that BLI-regulated vegetative growth phenotype is dependent on IRE1A/IRE1B but not their canonical splicing target bZIP60. Genetic complementation with mutation analysis suggests that the D570/K572 residues in the ATP-binding pocket and N780 residue in the RNase domain of IRE1A are required for the activation of canonical UPR gene expression, in contrast, the D570/K572 residues and D590 residue in the protein kinase domain of IRE1A are important for the induction of non-canonical UPR downstream genes in the BLI mutant background, which correlates with the shoot growth phenotype. Hence, our results reveal the important role of IRE1A in plant growth and development, and BLI negatively controls IRE1A’s function under normal growth condition in plants.The unfolded protein response (UPR) is required for protein homeostasis in the endoplasmic reticulum (ER) when plants are challenged by adverse environmental conditions. Inositol-requiring enzyme 1 (IRE1), the bifunctional protein kinase / ribonuclease, is an important UPR regulator in plants mediating cytoplasmic splicing of the mRNA encoding the transcription factor bZIP60. This activates the UPR signaling pathway and regulates canonical UPR genes. However, how the protein activity of IRE1 is controlled during plant growth and development is largely unknown. In the present study, we demonstrate that the nuclear and Golgi-localized protein BLISTER (BLI) negatively controls the activity of IRE1A/IRE1B under normal growth condition in  Arabidopsis. Through genetic analysis, our results also provide novel insights into how the protein kinase domain and ribonuclease domain contribute to the function of IRE1A in downstream gene expression.When unfolded or misfolded proteins are accumulated in the ER, a much conserved response, called the unfolded protein response (UPR), is elicited to lighten the load of unfolded proteins in the ER by bringing the protein-folding and degradation capacities into alignment with the protein folding demands. However, over-activation of the UPR pathways under normal growth conditions affects plant growth and development. The bifunctional protein kinase / ribonuclease protein IRE1 is important for UPR gene regulation, but how IRE1’ protein activity is tightly controlled in plants is currently unknown. Here we report that BLISTER (BLI) negatively controls the IRE1’s function under normal growth condition in  Protein folding demands on the secretory pathway escalate constantly during different developmental stages and environmental conditions while protein folding capacity is limited depending on ER chaperones, oxidoreductases, N-glycosylation etc. ,2. When bidopsis . The UPRbidopsis . Recentlbidopsis . HoweverBLI results in the activation of IRE1A and up-regulation of both canonical and non-canonical UPR genes under normal growth conditions, however, the IRE1-bZIP60 pathway is not responsible for the vegetative growth retardation phenotype in the BLI mutant plants. Further studies demonstrated that the residues D570/K572 and D590 in the protein kinase domain of IRE1A are important for non-canonical UPR gene expression and growth inhibition, while the D570/K572 residues and N780 residue of IRE1A are required for its ribonuclease activity to induce canonical UPR downstream genes. Together, these results demonstrated the important role of IRE1A in growth and development, and revealed BLI as a new negative regulator of IRE1 in Arabidopsis.In the current study, we found that BLISTER (BLI) negatively regulates IRE1A function in Arabidopsis. Mutation of Arabidopsis thaliana [BLI in two loss-of-function Arabidopsis mutant alleles (bli-1 and bli-11) conferred growth retardation [BiP3, NSF, ERDJ3A, ERDJ3B and SARA1A  epidermal leaves [BLI-GFP was co-expressed with the ER marker WAK2-RFP, Golgi marker SYP32-RFP or the trans-Golgi/early endosome marker VTI12-RFP, or the nucleus marker H2B-RFP [Arabidopsis plants. Confocal microscopy revealed that BLI-GFP not only localized to nucleus but also co-localized with SYP32-RFP but not with VTI12-RFP or WAK2-RFP in root cells (Previously we identified a plant specific protein BLISTER (BLI), which interacts with the Polycomb-group (Pc-G) histone methyltransferase CURLY LEAF (CLF) and controls the expression of Pc-G target genes for cellular differentiation in thaliana . Mutatioardation ,27 and ud SARA1A . The splegulated , suggestt plants . These rl leaves . Our prel leaves –7, so we H2B-RFP  in Arabiot cells . We concBLI mutant plants depends on ER stress response modulators, we crossed bli (bli-1 hereafter) to either bzip28 or bzip60 and generated bli bzip28 and bli bzip60 double mutants. The bzip28 and bzip60 single mutants grew as normally as the WT  comparing to that in WT plants under normal growth conditions , an N-glycosylation inhibitor specifically inducing misfolded protein accumulation in ER, in the growth medium, the bli-1 mutant plants were very sensitive to ER stress in terms of shoot growth, leaf chlorosis and root growth, when compared to the WT, which could be partially rescued in the bli ire1a and bli ire1b double mutant plants  is widely used to eliminate the phosphate group from phosphorylated proteins, and molecular weight shift after CIAP treatment has been successfully used for detection the phosphorylation status of mammalian IRE1 [IRE1A native promoter in the bli ire1a double mutant background (bli ire1a phenotype to the bli phenotype (bli ire1a phenotype (MRN1 and LTP4) was restored when IRE1A and N780A were expressed  is one of the most important ER transmembrane sensors that activates the UPR for protein homeostasis under ER stress conditions through its specific splicing RNA target or non-specific RNA substrates in a process called RIDD . Howeverpanicles . Howeverantibody  did not m events . HoweverY2 cells . RecentlY2 cells . Given tArabidopsis IRE1B  in the nucleotide-binding pocket abolished IRE1B’s autophosphorylation and reduced its RNase activity to splice bZIP60 mRNA, while the D628A mutation (corresponding to D828A mutation of yeast IRE1) within the conserved DFG kinase motif only abolished IRE1B’s autophosphorylation; the N820A mutation (corresponding to N1057A mutation of yeast IRE1) in the RNase domain only abolished IRE1B’s RNase activity in Arabidopsis during ER stress response [bli mutant background under normal growth conditions. Our results showed that D570A K572A mutations and N780A mutation of IRE1A reduced its ribonuclease activity for canonical UPR downstream gene expression while D570A K572A mutations and D590A mutation in the protein kinase domain of IRE1A affected its protein kinase activity for non-canonical UPR gene expression. Thus, the regulatory effect of protein kinase activity on its RNase activity is common for these two IRE1 proteins in Arabidopsis plants, which is different from yeast cells in which 1KR32 mutation in the nucleotide-binding pocket retains the RNase activity [in vitro, however, detection of the autophosphorylation of these two proteins in vivo has not yet been successful, most probably because of the low level of these proteins in plants. Given that D590A mutation in IRE1A and D628A mutation in IRE1B do not affect the RNase activity of IRE1 in Arabidopsis, these results suggest that the ATP binding site of IRE1 rather than the protein kinase activity is more important for the activation of RNase activity in plants.The N-terminal part of IRE1 is in the ER lumen while the C-terminus is facing the cytoplasm which contains a protein kinase domain and an RNase domain. Previously, D608N K610N mutations of response . Howeveractivity  and IRE1ire1a ire1b double mutant plants under unstressed conditions suggests that IRE1 also have other function in integrating physiological signals to maintain specific secretory activity [ire1a ire1b double mutant in the absence of stress was dependent on IRE1B [bli mutant under normal growth conditions were dependent on IRE1A by analyzing the advance mutants in the bli mutant background. The root growth phenotype and reproductive development defects in bli mutant were not rescued by IRE1A or IRE1B mutation, suggesting that BLI also has IRE1-independent roles in plant growth and development, or IRE1A and IRE1B are too important for reproductive development so that the bli ire1a and bli ire1b double mutants could not recover. Nevertheless, our data demonstrated that the function of IRE1A in shoot growth regulation in bli mutant dependents on neither its RNase activity, nor its splicing target bZIP60. The protein kinase domain of IRE1A is required for the regulation of growth and development in bli mutant plants, probably through regulating of non-canonical UPR downstream genes.Besides the important roles of IRE1 in plant ER stress responses, the root-specific phenotype of Arabidopsis activity . Mutant on IRE1B . Our resArabidopsis  background. Seeds of WT, bli-1 (SAIL_107_D04), bli-11 (GABI-Kat_663H12), bzip28 (SALK_132285), bzip60 (SALK_050203), ire1a (SALK_018112), ire1b (SAIL_238_F07), double mutants and transgenic plants were sterilized  and sown on germination medium . Seeds were stratified for two days at 4°C and grown under long day (LD) conditions, (16/8 h light / dark cycle at 22°C) in the medium or in the soil. For ER stress treatment, plants were grown on MS medium for 6 days and then transferred to either MS medium or MS plus 0.3 μg/ml tunicamycin  for additional 6 days. For silique length measurement, two longest siliques from each of the five main inflorescences were selected and measured for each genotype. For statistical analysis of gene expression, student’s t test was used. For multiple comparisons of phenotypes, LSD (Least Significant Difference) test was carried out following the ANOVA  analysis.All of the bli, bli ire1a and bli ire1b were boiled for 1 min in trypan blue staining buffer containing 12.5% phenol, 12.5% glycerol, 12.5% lactic acid, 48% ethanol, and 0.025% trypan blue (Sangon Biotech), incubated for 10 min at room temperature, followed by destaining five times in 70% chloral hydrate. For DAB staining, leaves of 14-day-old wild-type, bli, bli ire1a and bli ire1b leaves were incubated in 1 mg/ mL DAB (Sigma) solution (pH 3.8) for 5 h at room temperature in darkness and then boiled in 95% boiling ethanol for 10 min before photograph.Trypan blue staining and DAB staining were performed as described in previous literature . For tryIRE1A:IRE1A construct. The site-specific-mutated PCR products were amplified by overlapping PCR based on the IRE1A:IRE1A construct. All the error-free constructs were introduced into the bli+/- ire1a-/- double mutant plants via Agrobacterium-mediated transformation. The T1 seeds were grown on selective MS medium plus 30 mg/L hygromycin (Roche) and genotyped to select transgenic plants in bli-/- ire1a-/- background. The T3 seeds were obtained and grown on MS medium for phenotypic analysis along with genotyping and gene expression analysis. At least five independent lines of each construct were selected and examined. All the primers used are listed in The IRE1A genomic sequences including 2.5 kb upstream promoter region were amplified from wild-type and inserted into pCambia 1300 to obtain the https://github.com/jstjohn/SeqPrep) and Sickle (https://github.com/najoshi/sickle). Totally ~5.5–10 GB clean reads for each sample were obtained. Differential gene expression was assessed using Cuffdiff (http://cole-trapnell-lab.github.io/cufflinks/cuffdiff/index.html). The cutoff for significant differential expression was set as fold change (FC) ≥2 or FC≤0.5 and p-value p≤0.05. Parameters for sequencing quality control are listed in For RNA-Seq analyses, 14-days-old seeding were collected and immediately frozen in liquid nitrogen. Total RNA were extracted with Trizol (Invitrogen) and sequenced on the Illumina HiSeq 4000 platform by commercial company (Major Bio) following the standard Illumina protocols . RNA-SeqBLI, containing the BLI coding region and 1.7 kb upstream of the transcriptional start site, was amplified from genomic DNA, and cloned into pGKGWG. The BLI:BLI-GFP construct was introduced to the bli-11 mutant background and stable transformed plants were obtained. Confocal laser scanning microscopy was performed with root tissues using LSM 780 and LSM 510 microscopes (Zeiss). Image acquisition was carried out sequentially to prevent crosstalk between channels. GFP was excited at 488 nm, and emission was detected at 510–550 nm. RFP was excited at 561 nm and emission was detected at 575–630 nm. All the primers used are listed in Genomic anti-His antibody (Abmart). All the primers used are listed in For de-phosphorylation assay, partial IRE1A sequences (AA345-841) were amplified from the wild-type Arabidopsis cDNA and cloned into pET28 to express the His-tag IRE1A truncation protein. Various mutated forms of IRE1A were amplified by overlapping PCR based on the wild-type form. All purified proteins (2 μg) were treated with or without CIAP (Takara) for 2 min at 37°C. The reaction were stopped by adding 4 μL 5X SDS buffer and boiling at 95°C for 5 min. All the proteins were resolved in Phos-tag gels and then immunoblotted with bZIP28), AT1G42990 (bZIP60), AT2G17520 (IRE1A), AT5G24360 (IRE1B), AT3G23980 (BLI), AT1G09080 (BiP3), AT4G21730 (NSF), AT3G08970, (ERDJ3A), AT3G62600, (ERDJ3B), AT1G09180 (SARA1), AT5G64060 (NAC103), AT5G42600 (MRN1) and AT5G59310 (LTP4). The RNA-Seq data in this article can be found in the Gene Expression Omnibus (GEO) under the accession number GSE124235.Sequence data from this article can be found in the Arabidopsis Genome Initiative under the following accession numbers: At3g10800  normalized to that in the wild-type plants (WT), both of which were normalized to the expression of ACTIN. Error bars represent SE (n = 3). bZIP60U, unspliced bZIP60; bZIP60S, spliced bZIP60. Asterisks indicate significance levels when comparing to the WT control in t-test. . B, Venn diagrams showing the overlapping regulated genes between BLI-dependent genes and canonical UPR genes. Canonical UPR genes were obtained by comparing gene expression profiles of WT plants treated with or without ER stress inducer tunicamycin (5 μg/ml) for 12 hours followed by RNA-Seq analysis. Up-regulation: fold change ≥2, p<0.05; down-regulation: fold change ≤0.5, p<0.05.(PDF)Click here for additional data file.S2 FigA-B, Phenotypic analysis. T-DNA mutants of IRE1A (ire1a), or IRE1B (ire1b) or bZIP28 (bzip28) or bZIP60 (bzip60) were grown together with wild-type plants (WT) in standard MS growth medium. 2-week-old plant seedlings were photographed (A) and siliques lengths were measured at reproductive stage (B). Error bars represent SD (n = 10). Letters above the bars indicate significant differences as determined by LSD test following ANOVA analysis (p<0.05). Bar = 5 mm. C, UPR gene expression analysis. Total RNA was exacted from 2-week-old plants for qRT-PCR analysis. Error bars represent SE (n = 3). Asterisks indicate significance levels when comparing to the WT control in t-test. . bZIP60U, unspliced bZIP60; bZIP60S, spliced bZIP60.(PDF)Click here for additional data file.S3 FigBLI, IRE1A or IRE1B was checked by RT-PCR. UBQ5 was used as a loading control.Total RNA was extracted from various plant materials grown under normal growth conditions and the expression of (PDF)Click here for additional data file.S4 FigBLI (bli-1) was crossed to either IRE1A mutant (ire1a) or IRE1B mutant (ire1b) to generate the respective double mutant plants. Wild-type (WT), single mutants and double mutants were vertically grown for 6 days on MS medium, transferred to either MS or MS plus 0.3 μg/ml tunicamycin (TM) plates and grown for additional 6 days, then photographed (A). Primary root length was measured (B). There were five plants in each of the three replicates. Error bars represent SE (n = 3). Letters above the bars indicate significant differences as determined by LSD test following ANOVA analysis (p<0.05). Bar = 10 mm.(A-B) ER stress related phenotype of mutant plants. T-DNA mutant of (PDF)Click here for additional data file.S5 Figire1a ire1b bli+/- plants were grown in soils in well-controlled growth chamber and their siliques were examined after self-crossing. Arrow heads point to the aborted embryos.WT and (PDF)Click here for additional data file.S6 FigProtein sequences of the Arabidopsis IRE1s , rice IRE1 , yeast IRE1  and human IRE1  were subjected to sequence alignment using CLUSTAL 2.1 with the default setting. Only the C-terminal regions beyond the transmembrane domains are shown. The protein kinase domain is shaded in grey while the endonuclease domain is shaded in yellow. Amino acids predicted to be important for the protein kinase activity or endonuclease activity are highlighted in red.(PDF)Click here for additional data file.S7 Figbli ire1a double mutant background. Total RNA was extracted from various plant materials grown under normal growth conditions and the expression of total IRE1A was checked by RT-PCR. UBQ5 was used as a loading control.The wild-type form and various mutated forms of IRE1A were expressed the (PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.


Staphylococcus aureus monofunctional PGT enzyme, revealing electron density for a second MoeA bound to the jaw subdomain as well as the PGT donor site. Isothermal titration calorimetry confirms two drug-binding sites with markedly different affinities and positive cooperativity. Hydrophobic cluster analysis suggests that the membrane-interacting surface of the jaw subdomain has structural and physicochemical properties similar to amphipathic cationic Bacterial peptidoglycan glycosyltransferases (PGT) catalyse the essential polymerization of lipid II into linear glycan chains required for peptidoglycan biosynthesis. The PGT domain is composed of a large head subdomain and a smaller jaw subdomain and can be potently inhibited by the antibiotic moenomycin A (MoeA). We present an X-ray structure of the MoeA-bound  PG is s domains . The PGT domains . Multipl domains . Crystal domains A and/or  domains  confirm  domains .Fig. 1  (S. aureus penicillin-binding protein 2 (SaPBP2) (Escherichia coli penicillin-binding protein 1b (EcPBP1b)  . The extyme (51) . Anotheryme (51) , which i (SaMGT) , S. aure(SaPBP2)  and EschEcPBP1b) . The flalipid II . The flalipid II . The schlipid II ; however3HZS] originally solved by a research team at Pfizer  ID code  t Pfizer . In addi22.1SaMGT (residues Q28-R269) was cloned, expressed and purified as described previously . Further2.2Calorimetric titrations of SaMGT with MoeA were performed on a VP-ITC microcalorimeter  at 25 °C and measured in duplicate. The gel filtration purified SaMGT was concentrated and dialyzed overnight against buffer A  was performed using the program BUSTER 2.10.2  plots  using th-helices . Usually2.53VMT) was used to model the N-terminal transmembrane S. aureus cell membrane containing 57% phosphatidylglycerol (PG), 38% lysyl-phosphatidylglycerol (Lys-PG), and 5% diphosphatidylglycerol . The lipid composition of this model membrane was based on experimentally determined values  (6FTB) with improved overall statistics from the original as shown in The authors of the high-resolution crystal structure of the MoeA-bound SaMGT E100Q mutant (PDB ID code  3HZS)  reportedphenix.polder tool in PHENIX (Aquifex aeolicus PBP1A (PDB ID code  2OQO) (The difference map produced by the n PHENIX  shows fotructure B. The sa density  and this density . None of density . Additioolaurate  used durolaurate . The pololaurate . Structue  2OQO) C (Yuan ee  2OQO) .3VMT) onto 6FTB showed that the A and B rings of Our analyses also revealed a large positive electron density extending from the bound phosphate ion at the base of the jaw subdomain (His107 to Leu112), along the external surface, connecting to an additional area of positive electron density at the mouth of a deep pocket just below the catalytic site B. This d MoeAJAW C. Superitic site C. The ri3HZS) and re-refined (PDB ID code  6FTB) SaMGT-MoeA structures yield a root-mean-square deviation (RMSD) of 0.092 Å over the No other changes were found in the protein chain overall except for extension of the map at the N-terminus by one residue so that Glu59 is now included. Superimposition of the original  and cooperativity factor  (3HZS) revealed the solution structures of cysteine deleted protegrin-1  (1VM4) (3HZS) revealed the solution structures of the cationic antimicrobial peptide (AMP) nisin-lipid II complex (PDB ID code  1WCO) (1MQZ) (3E7R) (The hydrogen bond interactions between the phosphate ion of the rch.php)  to scan e  2MQ2)  and a no) (1VM4)  as the be  1WCO) , mersaci) (1MQZ)  and the ) (3E7R)  as the t) (3E7R) .Fig. 3St3VMT  and 3HZS show that the PP moiety of the lipid II analog comes very close to binding the PP cage-like motif at the “tip” of the flap  adopts a unique nest-like motif called “PP cage” to bind the PP moiety of lipid II by hydrogen bonds from the amide groups of the nisin backbone A Hsu et. Deletinthe flap C.3.43HZS, 3DWK  and EcPWK (5HLB ) have sh) method  has succsferases  and catasferases . Here, wA 2D HCA plot showed that nine hydrophobic clusters (C1 to C9) cover the entire PGT domain . ClusterClusters C3 and C5 belong to the flap segment and motif III respectively and form the base of the jaw subdomain that makes direct contacts with the membrane-anchored lipid II substrate. A notable feature of these two clusters is the presence of charged and polar residues in the vicinity of the hydrophobic residue clusters. Additionally, the shapes of the hydrophobic clusters C3 and C5 indicate a strong preference for operties B, making3.5The structural similarities between the PP-binding motifs in the flap of SaMGT and AMP nisin prompted us to analyse and compare the physicochemical properties of the membrane-interacting surface of the jaw subdomain with the amphipathic cationic CAH AMPs A. The baCAH AMPs .A 2D HCA of Magainin revealed a distribution pattern of hydrophobic and positively charged residues describing the conserved structural and physicochemical properties of ACAH AMPs A. Remark3.63.6.1The flexibility of the full-length SaMGT within the membrane was analyzed by measuring the root-mean-square fluctuation (RMSF) of the ubdomain .Part of the flap region (residues Phe120 to Gln129), however, was significantly more flexible than the rest of the jaw subdomain, with the RMSF values reaching 4 Å . These r3.6.2The flap region, in particular the soluble and structurally disordered part (residues Phe120 to Gln129), moved in a stochastic manner, i.e. the flap either flipped towards or away from motif III. In one of the 500 ns simulations, we found a significant motion towards motif III, such that some of its residues shifted as far as 10 Å from the crystal structure . We expl3.6.3d Asn141 . The pol44.1In vitro experiments by Thierry Vernet and co-workers on the PGT domain of Streptococcus pneumoniae PBP1b (6FTB), residues Asp101 (motif I) and Gln136 (motif II) make hydrogen bond interactions with The schematic models describing PGT processive catalytic mechanism for glycan strand synthesis  generallae PBP1b  and PBP2ae PBP1b  showed tactivity . Our HCAactivity  revealedactivity . This poactivity , Fig. 8.in vitro PGT assay  (N-oxide (LDAO) stabilized by hydrophobic interactions from residues in the SaPBP2 flap Our discovery of lography B stronglhy (3HZS . Hydroge binding . In the chain A)  suggestichain A)  because chain A) . This suchain A) , which f α-helix B (Loveri α-helix . The bac α-helix  and a do α-helix .4.26FTB), we performed ITC of MoeA binding to full-length SaMGT. Our ITC data has shown two MoeA binding events with positive cooperativity in the SaMGT active site (3DWK chains B and C) and in this conformation the acceptor site is not available for lipid II binding. In vitro experiments using the synthetic lipid II and lipid IV derivatives have shown that the formation of first lipid IV is the rate-limiting step in PG synthesis, probably because lipid II is not an optimal substrate in the donor site for the initiation step and that the rate-limiting step can be changed using longer glycan strands such as lipid IV (in vitro PGT polymerisation assay (6FTB), specially the The interaction of MoeA and other potential PGT inhibitors to full-length and truncated variants of class A PBPs and MGT has been extensively studied using various techniques: nuclear magnetic resonance , surfaceive site . SPR dat 0.44 μM . Interes 0.44 μM . The ITChiometry . To resoive site . Further in PGTs . In contlipid IV . Moreovelipid IV . This sulipid IV . MD anallipid IV  would pron assay . Interes≈20-fold . Taken t4.3Based on the above observations (including the amino acid mutations within the active site and jaw subdomain ), we nownor site B and C, http://hecbiosim.ac.uk), supported by EPSRC (Grant No. EP/L000253/1).This work was supported by the BBSRC grants BB/M029573/1 (to S.K.) and BB/N003241/1 (to D.I.R.). The computing in this project made use of time on ARCHER granted via the UK High-End Computing Consortium for Biomolecular Simulation, HECBioSim ( A.S.P. and D.I.R. designed research; A.S.P. and F.S. performed research; A.S.P., F.S., A.J.L., C.G.D., D.J.S., S.K. and D.I.R. analyzed data; and A.S.P., F.S., S.K. and D.I.R. wrote the manuscript.6FTBPDB:  None.