Recombinant Slit2 suppresses neuroinflammation and Cdc42-mediated brain infiltration of peripheral immune cells via Robo1-srGAP1 pathway in a rat model of germinal matrix hemorrhage.

BACKGROUND: Germinal matrix hemorrhage (GMH) is a devastating neonatal stroke, in which neuroinflammation is a critical pathological contributor. Slit2, a secreted extracellular matrix protein, plays a repulsive role in axon guidance and leukocyte chemotaxis via the roundabout1 (Robo1) receptor. This study aimed to explore effects of recombinant Slit2 on neuroinflammation and the underlying mechanism in a rat model of GMH. METHODS: GMH was induced by stereotactically infusing 0.3 U of bacterial collagenase into the germinal matrix of 7-day-old Sprague Dawley rats. Recombinant Slit2 or its vehicle was administered intranasally at 1 h after GMH and daily for 3 consecutive days. A decoy receptor recombinant Robo1 was co-administered with recombinant Slit2 after GMH. Slit2 siRNA, srGAP1 siRNA or the scrambled sequences were administered intracerebroventricularly 24 h before GMH. Neurobehavior, brain water content, Western blotting, immunofluorescence staining and Cdc42 activity assays were performed. RESULTS: The endogenous brain Slit2 and Robo1 expressions were increased after GMH. Robo1 was expressed on neuron, astrocytes and infiltrated peripheral immune cells in the brain. Endogenous Slit2 knockdown by Slit2 siRNA exacerbated brain edema and neurological deficits following GMH. Recombinant Slit2 (rSlit2) reduced neurological deficits, proinflammatory cytokines, intercellular adhesion molecules, peripheral immune cell markers, neuronal apoptosis and Cdc42 activity in the brain tissue after GMH. The anti-neuroinflammation effects were reversed by recombinant Robo1 co-administration or srGAP1 siRNA. CONCLUSIONS: Recombinant Slit2 reduced neuroinflammation and neuron apoptosis after GMH. Its anti-neuroinflammation effects by suppressing onCdc42-mediated brain peripheral immune cells infiltration was at least in part via Robo1-srGAP1 pathway. These results imply that recombinant Slit2 may have potentials as a therapeutic option for neonatal brain injuries.

Genetic susceptibility to hereditary non-medullary thyroid cancer.

Non-medullary thyroid cancer (NMTC) is the most common type of thyroid cancer. With the increasing incidence of NMTC in recent years, the familial form of the disease has also become more common than previously reported, accounting for 5-15% of NMTC cases. Familial NMTC is further classified as non-syndromic and the less common syndromic FNMTC. Although syndromic NMTC has well-known genetic risk factors, the gene(s) responsible for the vast majority of non-syndromic FNMTC cases are yet to be identified. To date, several candidate genes have been identified as susceptibility genes in hereditary NMTC. This review summarizes genetic predisposition to non-medullary thyroid cancer and expands on the role of genetic variants in thyroid cancer tumorigenesis and the level of penetrance of NMTC-susceptibility genes.

SRGAP1 Controls Small Rho GTPases To Regulate Podocyte Foot Process Maintenance.

BACKGROUND: Previous research demonstrated that small Rho GTPases, modulators of the actin cytoskeleton, are drivers of podocyte foot-process effacement in glomerular diseases, such as FSGS. However, a comprehensive understanding of the regulatory networks of small Rho GTPases in podocytes is lacking. METHODS: We conducted an analysis of podocyte transcriptome and proteome datasets for Rho GTPases; mapped in vivo, podocyte-specific Rho GTPase affinity networks; and examined conditional knockout mice and murine disease models targeting Srgap1. To evaluate podocyte foot-process morphology, we used super-resolution microscopy and electron microscopy; in situ proximity ligation assays were used to determine the subcellular localization of the small GTPase-activating protein SRGAP1. We performed functional analysis of CRISPR/Cas9-generated SRGAP1 knockout podocytes in two-dimensional and three-dimensional cultures and quantitative interaction proteomics. RESULTS: We demonstrated SRGAP1 localization to podocyte foot processes in vivo and to cellular protrusions in vitro. Srgap1fl/fl*Six2Cre but not Srgap1fl/fl*hNPHS2Cre knockout mice developed an FSGS-like phenotype at adulthood. Podocyte-specific deletion of Srgap1 by hNPHS2Cre resulted in increased susceptibility to doxorubicin-induced nephropathy. Detailed analysis demonstrated significant effacement of podocyte foot processes. Furthermore, SRGAP1-knockout podocytes showed excessive protrusion formation and disinhibition of the small Rho GTPase machinery in vitro. Evaluation of a SRGAP1-dependent interactome revealed the involvement of SRGAP1 with protrusive and contractile actin networks. Analysis of glomerular biopsy specimens translated these findings toward human disease by displaying a pronounced redistribution of SRGAP1 in FSGS. CONCLUSIONS: SRGAP1, a podocyte-specific RhoGAP, controls podocyte foot-process architecture by limiting the activity of protrusive, branched actin networks. Therefore, elucidating the complex regulatory small Rho GTPase affinity network points to novel targets for potentially precise intervention in glomerular diseases.

Recombinant Slit2 attenuates neuroinflammation after surgical brain injury by inhibiting peripheral immune cell infiltration via Robo1-srGAP1 pathway in a rat model.

BACKGROUND AND PURPOSE: Peripheral immune cell infiltration to the brain tissue at the perisurgical site can promote neuroinflammation after surgical brain injury (SBI). Slit2, an extracellular matrix protein, has been reported to reduce leukocyte migration. This study evaluated the effect of recombinant Slit2 and the role of its receptor roundabout1 (Robo1) and its downstream mediator Slit-Robo GTPase activating protein 1 (srGAP1)-Cdc42 on peripheral immune cell infiltration after SBI in a rat model. METHODS: One hundred and fifty-three adult male Sprague-Dawley rats (280-350 g) were used. Partial resection of right frontal lobe was performed to induce SBI. Slit2 siRNA was administered by intracerebroventricular injection 24h before SBI. Recombinant Slit2 was injected intraperitoneally 1h before SBI. Recombinant Robo1 used as a decoy receptor was co-administered with recombinant Slit2. srGAP1 siRNA was administered by intracerebroventricular injection 24h before SBI. Post-assessments included brain water content measurement, neurological tests, ELISA, Western blot, immunohistochemistry, and Cdc42 activity assay. RESULTS: Endogenous Slit2 was increased after SBI. Robo1 was expressed by peripheral immune cells. Endogenous Slit2 knockdown worsened brain edema after SBI. Recombinant Slit2 administration reduced brain edema, neurological deficits, and pro-inflammatory cytokines after SBI. Recombinant Slit2 reduced peripheral immune cell markers cluster of differentiation 45 (CD45) and myeloperoxidase (MPO), as well as Cdc42 activity in the perisurgical brain tissue which was reversed by recombinant Robo1 co-administration and srGAP1 siRNA. CONCLUSIONS: Recombinant Slit2 improved outcomes by reducing neuroinflammation after SBI, possibly by decreasing peripheral immune cell infiltration to the perisurgical site through Robo1-srGAP1 mediated inhibition of Cdc42 activity. These results suggest that Slit2 may be beneficial to reduce SBI-induced neuroinflammation.

Hybrid de novo and haplotype-resolved genome assembly of Vechur cattle - elucidating genetic variation.

Cattle contribute to the nutritional needs and economy of a place. The performance and fitness of cattle depend on the response and adaptation to local climatic conditions. Genomic and genetic studies are important for advancing cattle breeding, and availability of relevant reference genomes is essential. In the present study, the genome of a Vechur calf was sequenced on both short-read Illumina and long-read Nanopore sequencing platforms. The hybrid de novo assembly approach was deployed to obtain an average contig length of 1.97 Mbp and an N50 of 4.94 Mbp. By using a short-read genome sequence of the corresponding sire and dam, a haplotype-resolved genome was also assembled. In comparison to the taurine reference genome, we found 28,982 autosomal structural variants and 16,926,990 SNVs, with 883,544 SNVs homozygous in the trio samples. Many of these SNPs have been reported to be associated with various QTLs including growth, milk yield, and milk fat content, which are crucial determinants of cattle production. Furthermore, population genotype data analysis indicated that the present sample belongs to an Indian cattle breed forming a unique cluster of Bos indicus. Subsequent FST analysis revealed differentiation of the Vechur cattle genome at multiple loci, especially those regions related to whole body growth and cell division, especially IGF1, HMGA2, RRM2, and CD68 loci, suggesting a possible role of these genes in its small stature and better disease resistance capabilities in comparison with the local crossbreeds. This provides an opportunity to select and engineer cattle breeds optimized for local conditions.

Familial nonmedullary thyroid cancer: a case series in Iranian patients with a meta-review of case series.

BACKGROUND: Nonmedullary thyroid cancer (NMTC) comprises approximately 90% of all thyroid cancers, and about 3% to 9% of NMTC cases have a familial origin. Familial NMTC (FNMTC) in the absence of a documented familial cancer syndrome such as Cowden syndrome is characterized by the occurrence of thyroid cancer of follicular cell origin in 2 or more first-degree relatives. METHODS: Whole-exome sequencing (WES) was used to identify pathogenic genetic variants in 2 Persian families with FNMTC. The purpose of this work is to assess the pathogenic status of these variants as well as the cosegregation status of the variants observed in the examined families. RESULTS: By analyzing WES data in the first family, SRGAP1: NM_020762: exon16: c.C1849T was identified as a pathogenic variant. This variant was confirmed by Sanger sequencing. In the second family, the variant FOXE1: NM_004473: exon1: c.531_532insCGCGA was identified but was not confirmed by Sanger sequencing. CONCLUSION: Based on the data, SRGAP1 can be a potential candidate gene for susceptibility to FNMTC in the first family. However, additional analyses like whole genome sequencing and copy number variations are required to ascertain the disease status in second family.

A proliferative to invasive switch is mediated by srGAP1 downregulation through the activation of TGF-ß2 signaling.

Many breast cancer (BC) patients suffer from complications of metastatic disease. To form metastases, cancer cells must become migratory and coordinate both invasive and proliferative programs at distant organs. Here, we identify srGAP1 as a regulator of a proliferative-to-invasive switch in BC cells. High-resolution light-sheet microscopy demonstrates that BC cells can form actin-rich protrusions during extravasation. srGAP1low cells display a motile and invasive phenotype that facilitates their extravasation from blood vessels, as shown in zebrafish and mouse models, while attenuating tumor growth. Interestingly, a population of srGAP1low cells remain as solitary disseminated tumor cells in the lungs of mice bearing BC tumors. Overall, srGAP1low cells have increased Smad2 activation and TGF-ß2 secretion, resulting in increased invasion and p27 levels to sustain quiescence. These findings identify srGAP1 as a mediator of a proliferative to invasive phenotypic switch in BC cells in vivo through a TGF-ß2-mediated signaling axis.

Recombinant Slit2 attenuates neuronal apoptosis via the Robo1-srGAP1 pathway in a rat model of neonatal HIE.

Apoptosis following hypoxic-ischemic injury to the brain plays a major role in neuronal cell death. The neonatal brain is more susceptible to injury as the cortical neurons are immature and there are lower levels of antioxidants. Slit2, an extracellular matrix protein, has been shown to be neuroprotective in various models of neurological diseases. However, there is no information about the role of Slit2 in neonatal hypoxia-ischemia. In this study, we evaluated the effect of Slit2 and its receptor Robo1 in a rat model with neonatal HIE. 10-day old rat pups were used to create the neonatal HIE model. The right common carotid artery was ligated followed by 2.5 h of hypoxia. Recombinant Slit2 was administered intranasally 1 h post HI, recombinant Robo1 was used as a decoy receptor and administered intranasally 1h before HI and srGAP1-siRNA was administered intracerebroventricularly 24 h before HI. Brain infarct area measurement, short-term and long-term neurological function tests, Western blot, immunofluorescence staining, Fluoro-Jade C staining, Nissl staining and TUNEL staining were the assessments done following drug administration. Recombinant Slit2 administration reduced neuronal apoptosis and neurological deficits after neonatal HIE which were reversed by co-administration of recombinant Robo1 and srGAP1-siRNA administration. Recombinant Slit2 showed improved outcomes possibly via the robo1-srGAP1 pathway which mediated the inhibition of RhoA. In this study, the results suggest that Slit2 may help in attenuation of apoptosis and could be a therapeutic agent for treatment of neonatal hypoxic ischemic encephalopathy.

Regulated recruitment of SRGAP1 modulates RhoA signaling for contractility during epithelial junction maturation.

Adherens junctions in epithelia are contractile structures, where coupling of adhesion to the actomyosin cytoskeleton generates mechanical tension for morphogenesis and homeostasis. In established monolayers, junctional contractility is supported by the interplay between cell signals and scaffolding proteins. However, less is known about how contractile junctions develop, especially during the establishment of epithelial monolayers. Here, we show that junctional tension increases concomitant with accumulation of actomyosin networks as Caco-2 epithelia become confluent. This is associated with development of a zone of RhoA signaling at junctions. Further, we find that the low levels of RhoA signaling and contractility found in subconfluent cultures reflect a mechanism for their active suppression. Specifically, the RhoA antagonist, SRGAP1, is present at subconfluent junctions to a greater extent than in confluent cultures and SRGAP1 RNAi restores RhoA signaling and contractility in subconfluent cultures to levels seen in confluent cells. Overall, these observations suggest that regulated changes in junctional contractility mediated by modulation of RhoA signaling occur as epithelial monolayers mature.

srGAP1 mediates the migration inhibition effect of Slit2-Robo1 in colorectal cancer.

BACKGROUND: The neuronal guidance molecule Slit2 plays suppressive role in tumorigenesis and progression. We previously showed that Slit2-Robo1 inhibit cell migration in colorectal cancer (CRC). However, little is known about its downstream effectors in CRC. This study tries to identify whether the Slit-Robo Rho GTPase activating protein 1 (srGAP1) could mediate the inhibitory effect of Slit2-Robo1 on CRC cell migration. METHODS: The protein expression of srGAP1 in clinical CRC tissues was tested by immunohistochemistry staining. Conditioned medium was prepared from HEK293 cells stably expressing Slit2-myc, Robo1-HA or RoboN (a soluble extracellular domain of Robo1). Immunoprecipitation (IP) was applied to check the interaction between Robo1 and srGAP1, and immunofluorescence (IF) was used to observe the subcellular localization of Robo1 and srGAP1. Small GTPase pull-down assay was used to determine the activity of Cdc42. A modified wound healing assay was performed to detect cell migration. RESULTS: The protein expression of srGAP1 was remarkably decreased in 47.5% of CRC tissues compared with adjacent noncancerous tissues, and the decreased srGAP1 expression was associated with lymphatic invasion, poor tumor differentiation, high TNM stage, and poor survival (P < 0.05). IP and IF assays revealed that srGAP1 was a Robo1-interacting protein and exhibited similar dynamic subcellular distribution after Slit2 treatment in CRC cells. Small GTPase pull-down assay and migration assay indicated that Slit2-Robo1 signaling inhibited Cdc42 activity and CRC cell motility through srGAP1. CONCLUSION: Downregulation of srGAP1 in CRC was associated with tumor progression and poor prognosis. srGAP1 is an important downstream molecule of Slit2 signalling in CRC, and mediates the anti-migration function of Slit2 by inhibiting Cdc42.