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Results for “Slit Homolog 2 Protein”

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PubMed2005

Netrin-1 and slit-2 regulate and direct neurite growth of ventral midbrain dopaminergic neurons.

We investigated the roles of netrin-1 and slit-2 in regulation and navigation of dopamine (DA) axon growth using an explant culture preparation of embryonic ventral midbrain (embryonic day 14) and a co-culture system. We found that netrin-1 protein significantly enhanced DA axonal outgrowth and promoted DA axonal outgrowth in a co-culture system of netrin-1 expressing cells. Such effects were mediated by the receptor DCC as demonstrated by antibody perturbation of the DCC receptor. In contrast, slit-2 inhibited DA neuron extensions and repelled DA neurite growth. These slit-2 activities required robo receptors since the reduced neurite extension was abolished by addition of excess robo receptors. In this system, netrin-1 stimulated and slit-2 opposed DA neurite growth. Such regulation may be important for DA axonal maintenance, regeneration, and phenotypic target recognition.

Animals↗

PubMed2001

Characterization of Slit protein interactions with glypican-1.

We have demonstrated previously that the Slit proteins, which are involved in axonal guidance and related developmental processes in nervous tissue, are ligands of the glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan glypican-1 in brain (Liang, Y., Annan, R. S., Carr, S. A., Popp, S., Mevissen, M., Margolis, R. K., and Margolis, R. U. (1999) J. Biol. Chem. 274, 17885--17892). To characterize these interactions in more detail, recombinant human Slit-2 protein and the N- and C-terminal portions generated by in vivo proteolytic processing were used in an enzyme-linked immunosorbent assay to measure the binding of a glypican-Fc fusion protein. Saturable and reversible high affinity binding to the full-length protein and to the C-terminal portion that is released from the cell membrane was seen, with dissociation constants in the 80-110 nm range, whereas only a relatively low level of binding to the larger N-terminal segment was detected. Co-transfection of 293 cells with Slit and glypican-1 cDNAs followed by immunoprecipitation demonstrated that these interactions also occur in vivo, and immunocytochemical studies showed colocalization in the embryonic and adult central nervous system. The binding affinity of the glypican core protein to Slit is an order of magnitude lower than that of the glycanated proteoglycan. Glypican binding to Slit was also decreased 80--90% by heparin (2 microg/ml), enzymatic removal of the heparan sulfate chains, and by chlorate inhibition of glypican sulfation. The differential effects of N- or O-desulfated heparin on glypican binding also indicate that O-sulfate groups on the heparan sulfate chains play a critical role in heparin interactions with Slit. Our data suggest that glypican binding to the releasable C-terminal portion of Slit may serve as a mechanism for regulating the biological activity of Slit and/or the proteoglycan.

Amino Acid Sequence↗

PubMed2025

A Multi-omics Exploration Revealing SLIT2 as a Prime Therapeutic Target for Peripheral Facial Paralysis: Integrating Single-Cell Transcriptomics and Plasma Proteome Data.

Peripheral facial paralysis (PFP) is a common neurological disorder characterized by facial-nerve dysfunction. Identifying therapeutic targets and understanding the molecular and cellular mechanisms underlying PFP are crucial for developing effective treatment strategies. This study combined Mendelian randomization (MR) analysis and single-cell RNA sequencing (scRNA-seq) to explore potential therapeutic candidates and their roles in PFP pathophysiology. The MR analysis included 1925 publicly available plasma protein cis-heritability instruments. Instrumental variables were selected for MR analysis to identify plasma proteins associated with PFP, followed by colocalization analysis to evaluate shared genetic variants between the identified proteins and PFP. After the initial identification of plasma proteins associated with Bell's palsy using MR analysis, a rat model of facial-nerve injury was established to further dissect underlying mechanisms at cellular and molecular levels. Using scRNA-seq technology, we delved deeply into cellular Heterogeneity and dynamic changes in gene expression in the facial-nerve nucleus tissues under both injured and control conditions, thereby achieving a systematic study ranging from macroscopic genetic associations to microscopic cellular functions. Finally, expression patterns were preliminarily validated by performing in vitro immunofluorescence analysis on the facial-nerve nucleus samples of SD rats. The MR analysis results identified 30 plasma proteins significantly associated with PFP, with nine target genes showing differential expression in the scRNA-seq data. Colocalization analysis demonstrated that slit guidance Ligand 2 (SLIT2), semaphorin 4D (SEMA4D), EGF containing fibulin extracellular matrix protein 1 (EFEMP1), and sprouty related EVH1 domain containing 2 (SPRED2) shared causal variants with PFP. SLIT2 was highly expressed in the microglia and inhibitory neurons in the experimental group, whereas SEMA4D showed elevated expression across multiple glial cell types in the same group. In contrast, EFEMP1 and SPRED2 showed distinct expression patterns in fibroblasts and oligodendrocytes. The role of SLIT2 has been previously well-documented in many central nervous system diseases. However, for the first time, this study detected SLIT2 alteration after facial-nerve injury. Altered intercellular signaling, particularly enhanced SLIT2-ROBO signaling between neurons and glial cells, was observed in the PFP group. Pseudotime analysis revealed dynamic SLIT2 expression during microglia and inhibitory neuron differentiation, mirroring changes in ROBO1 expression. Immunofluorescence analysis of rat facial-nerve nucleus samples verified that SLIT2 protein levels were significantly increased in the facial-nerve nuclei of injured samples. In conclusion, despite the fact that this study is primarily founded on animal models and despite notable differences existing between animals and humans in terms of the facial motor nucleus, this study successfully identified SLIT2 as potential therapeutic targets for PFP. The SLIT2-ROBO axis stands out as a particularly promising candidate. SLIT2 may play a role in modulating neuroimmune interactions and promoting nerve repair. These findings provide a foundation for future clinical studies and targeted interventions to enhance recovery from PFP. Future research should focus on human sample validation to enhance clinical translation.

Animals↗

PubMed2004

Local inhibition guides the trajectory of early longitudinal tracts in the developing chick brain.

During development of the chick central nervous system, the trajectories of the descending medial and lateral longitudinal fascicles (MLF and LLF) are pioneered by axons originating from the interstitial nucleus of Cajal (INC) and the mesencephalic trigeminal nucleus (MTN), respectively. Both tracts cross rhombomere 1 at two specific locations in the basal plate. In this study, we have investigated the molecular properties of these crossing points and find that they are permissive regions situated in an otherwise inhibitory boundary region. We show that the dorsal part of rhombomere 1 is inhibitory for the growth of both MTN and INC axons. Ventrally, MLF and LLF axons are repelled from the midline by Slit proteins. Our results reveal the existence of a new repulsive/inhibitory mechanism for axons in the alar plate in addition to the ventral repulsion by Slit proteins. This suggests a model where MLF and LLF axons are channeled longitudinally within the neural tube by both dorsal and ventral constraints.

Animals↗

PubMed2004

Calcium signaling in chemorepellant Slit2-dependent regulation of neuronal migration.

Migration of neuronal precursor cells in the developing brain is guided by extracellular cues, but intracellular signaling processes underlying the guidance of neuronal migration are largely unknown. By examining the migration of cerebellar granule neurons along the surface of cocultured astroglial cells, we found that an extracellular gradient of Slit2, a chemorepellant for neuronal migration in vivo, caused a reversal in the direction of migration without affecting the migration speed. A Slit2 gradient elevated the intracellular concentration of Ca2+, probably due to calcium release from the internal store, led to a reversal of the preexisting asymmetric intracellular Ca2+ distribution in the soma of migrating neurons, and this reversal was closely related with its action of reversing the migrating direction. Asymmetric Ca2+ distribution in the soma was both necessary and sufficient for directing neuronal migration. These results have demonstrated an important role for Ca2+ in mediating neuronal responses to Slit2 and suggest a general mechanism for neuronal guidance.

Animals↗

PubMed2002

Growth-associated protein-43 is required for commissural axon guidance in the developing vertebrate nervous system.

Growth-associated protein-43 (GAP-43) is a major growth cone protein whose phosphorylation by PKC in response to extracellular guidance cues can regulate F-actin behavior. Here we show that 100% of homozygote GAP-43 (-/-) mice failed to form the anterior commissure (AC), hippocampal commissure (HC), and corpus callosum (CC) in vivo. Instead, although midline fusion was normal, selective fasciculation between commissural axons was inhibited, and TAG-1-labeled axons tangled bilaterally into Probst's bundles. Moreover, their growth cones had significantly smaller lamellas and reduced levels of F-actin in vitro. Likewise, 100% of GAP-43 (+/-) mice with one disrupted allele also showed defects in HC and CC, whereas the AC was unaffected. Individual GAP-43 (+/-) mice could be assigned to two groups based on the amount that PKC phosphorylation of GAP-43 was reduced in neocortical neurons. In mice with approximately 1% phosphorylation, the HC and CC were absent, whereas in mice with approximately 10% phosphorylation, the HC and CC were smaller. Both results suggest that PKC-mediated signaling in commissural axons may be defective. However, although Probst's bundles formed consistently at the location of the glial wedge, both GAP-43 (-/-) and GAP-43 (+/+) cortical axons were still repulsed by Slit-2 in vitro, precluding failure of this deflective signal from the glial wedge as the source of the phenotype. Nonetheless, the data show that a functional threshold of GAP-43 is required for commissure formation and suggests that failure to regulate F-actin in commissural growth cones may be related to inhibited PKC phosphorylation of GAP-43.

Agenesis of Corpus Callosum↗

PubMed2002

Conserved modularity and potential for alternate splicing in mouse and human Slit genes.

The vertebrate Slit gene family currently consists of three members; Slit1, Slit2 and Slit3. Each gene encodes a protein containing multiple epidermal growth factor and leucine rich repeat motifs, which are likely to have importance in cell-cell interactions. In this study, we sought to fully define and characterise the vertebrate Slit gene family. Using long distance PCR coupled with in silico mapping, we determined the genomic structure of all three Slit genes in mouse and man. Analysis of EST and genomic databases revealed no evidence of further Slit family members in either organism. All three Slit genes were encoded by 36 (Slit3) or 37 (Slit1 and Slit2) exons covering at least 143 kb or 183 kb of mouse or human genomic DNA respectively. Two additional potential leucine-rich repeat encoding exons were identified within intron 12 of Slit2. These could be inserted in frame, suggesting that alternate splicing may occur in Slit2. A search for STS sequences within human Slit3 anchored this gene to D5S2075 at the 5' end (exon 4) and SGC32449 within the 3' UTR, suggesting that Slit3 may cover greater than 693 kb. The genomic structure of all Slit genes demonstrated considerable modularity in the placement of exon-intron boundaries such that individual leucine-rich repeat motifs were encoded by individual 72 bp exons. This further implies the potential generation of multiple Slit protein isoforms varying in their number of repeat units. cDNA library screening and EST database searching verified that such alternate splicing does occur.

3' Untranslated Regions↗

PubMed2002

SLIT2, a human homologue of the Drosophila Slit2 gene, has tumor suppressor activity and is frequently inactivated in lung and breast cancers.

Slit2 plays a vital role in axon guidance by signaling through Robo receptors. Recent evidence suggests that Slit2 protein may function in other settings because human and Xenopus Slit2 has been shown to inhibit leukocyte chemotaxis. SLIT2 protein is a putative ligand for the ROBO receptors. We recently demonstrated that ROBO1 is inactivated by promoter region hypermethylation in <20% of human cancers; furthermore, tumor suppressor activity has not been shown. Thus, the importance of ROBO1 inactivation in human cancer is uncertain. Therefore, we investigated the status of SLIT2 located at 4p15.2 in lung and breast cancers. Although somatic SLIT2 mutations were not detected, epigenetic inactivation was common. SLIT2 promoter methylation was detected in 59% of breast cancer, 77% of non-small cell lung cancer, and 55% of small cell lung cancer cell lines. In these tumor lines, SLIT2 expression was restored by treatment with a demethylating agent. SLIT2 promoter methylation was detected in 43% of breast cancer, 53% of non-small cell lung cancer, and 36% of small cell lung cancer primary tumors. The majority of methylated tumors demonstrated allelic loss at 4p15.2. In addition, SLIT2 expression was down-regulated in methylated breast tumors, relative to normal control, as demonstrated by quantitative real-time reverse transcription-PCR. Overexpression of SLIT2 suppressed >70% of colony growth in each of three breast tumor lines (with either absent or low SLIT2 expression). Because SLIT2 is primarily a secreted protein, SLIT2-conditioned medium suppressed the growth of several breast cancer lines (with absent or weak SLIT2expression) by 26-51% but had no significant effect on a breast tumor cell line that expresses normal levels of SLIT2. These findings demonstrate that SLIT2 is frequently inactivated in lung and breast cancer by promoter region hypermethylation and allele loss and is an excellent candidate for the lung and breast tumor suppressor gene previously mapped to 4p15.2.

Animals↗

PubMed2006

Potential role of the Slit/Robo signal pathway in angiogenesis.

Intensive investigations on angiogenesis and vasculogenesis have increased our understanding of molecular mechanisms of blood vessel formation during pathologic and developmental conditions. However, endothelial cells (ECs), the main component of vasculature, are heterogeneous, as revealed by our phenotypic and molecular biological studies in the laboratory, and it is still hard to adequately understand the molecular mechanisms of angiogenesis and vasculogenesis. Indeed, there are several major ligand/receptor signal pathways: VEGF/VEGFR, Jagged-1/Notch, Wnt ligand/frizzled receptor, and ephrin/Eph; each of which having distinct and independent roles during vascular formation. In this review, we focus on the angiogenic effect of the Slit and Robo signal pathway that was formally known as neuronal axon guidance. Among the existing vascular signals, this pathway is the most recently found ligand/receptor vascular signal, and may play important physiological roles as other major receptor/ligand signals do. Here, we briefly address: (1) the background of Slit and Robo families; (2) expression patterns of Slit and Robo; (3) functional roles of the Slit/Robo pathway in vascular formation; and (4) confronting tasks of this novel vascular pathway in the near future. Together, a summary of these data suggest the essential role of the Slit/Robo pathway in angiogenesis, and may explain why multiple vascular signals exist in heterogenic endothelial cells.

Angiogenesis Inhibitors↗

PubMed2003

Slit and glypican-1 mRNAs are coexpressed in the reactive astrocytes of the injured adult brain.

The slit family serves as a repellent for growing axons toward correct targets during neural development. A recent report describes slit mRNAs expressed in various brain regions in adult rats. However, their functions in the adult nervous system remain unknown. In the present study, we investigated whether slit mRNAs were expressed in the cryo-injured brain, using in situ hybridization. All slit family members were expressed at the lesion. Slit2 mRNA was the most intensely expressed in the cells surrounding the necrotic tissue. A double-labeling study showed that slit2 mRNA was expressed in the glial fibrillary acidic protein (GFAP)-positive reactive astrocytes. In addition, glypican-1, a heparan sulfate proteoglycan that serves as a high-affinity receptor for Slit protein, was coexpressed with slit2 mRNA in the reactive astrocytes. These findings suggested that slit2 might prevent regenerating axons from entering into the lesion in concert with glypican-1.

Animals↗

PubMed2006

Slits contribute to the guidance of retinal ganglion cell axons in the mammalian optic tract.

RGC axons extend in the optic tracts in a manner that correlates with the expression in the hypothalamus and epithalamus of a soluble factor inhibitory to RGC axon outgrowth. Additionally, although the RGC axons extend adjacent to the telencephalon, they do not normally grow into this tissue. Here, we show that slit1 and slit2, known chemorepellents for RGC axons expressed in specific regions of the diencephalon and telencephalon, help regulate optic tract development. In mice lacking slit1 and slit2, a subset of RGC axons extend into the telencephalon and grow along the pial surface but not more deeply into this tissue. Surprisingly, distinct guidance errors occur in the telencephalon of slit1 -/-; slit2 +/- and slit1/2 -/- embryos, suggesting that the precise level of Slits is critical for determining the path followed by individual axons. In mice lacking both slit1 and slit2, a subset of RGC axons also project aberrantly into the epithalamus, pineal and across the dorsal midline. However, many axons reach their primary target, the superior colliculus. This demonstrates that Slits play an important role in directing the guidance of post-crossing RGC axons within the optic tracts but are not required for target innervation.

Animals↗

PubMed2004

SLIT2-mediated ROBO2 signaling restricts kidney induction to a single site.

Kidney development occurs in a stereotypic position along the body axis. It begins when a single ureteric bud emerges from the nephric duct in response to GDNF secreted by the adjacent nephrogenic mesenchyme. Posterior restriction of Gdnf expression is considered critical for correct positioning of ureteric bud development. Here we show that mouse mutants lacking either SLIT2 or its receptor ROBO2, molecules known primarily for their function in axon guidance and cell migration, develop supernumerary ureteric buds that remain inappropriately connected to the nephric duct, and that the SLIT2/ROBO2 signal is transduced in the nephrogenic mesenchyme. Furthermore, we show that Gdnf expression is inappropriately maintained in anterior nephrogenic mesenchyme in these mutants. Thus our data identify an intercellular signaling system that restricts, directly or indirectly, the extent of the Gdnf expression domain, thereby precisely positioning the site of kidney induction.

Animals↗

PubMed2003

Frequent epigenetic inactivation of the SLIT2 gene in gliomas.

The SLIT family of genes consists of large extracellular matrix-secreted and membrane-associated glycoproteins. The Slits (Slit1-3) are ligands for the repulsive guidance receptors, the robo gene family. The Slit-Robo interactions mediate the repulsive cues on axons and growth cones during neural development. In a recent report, we demonstrated that promoter region CpG island of human SLIT2 was frequently hypermethylated in lung, breast and colorectal tumours and the silenced gene transcript suppressed the malignant phenotype in in vitro assays. In this report we undertook epigenetic, genetic and expression analysis of SLIT2 gene in a large series of gliomas and glioma cell lines. Promoter region CpG island of SLIT2 was found to be methylated in 71% (5/7) of glioma cell lines and was unmethylated in five DNA samples from normal brain tissues. The hypermethylation of the SLIT2 promoter region in glioma cell lines correlated with loss of expression and treatment with the demethylating agent 5-aza-2'-deoxycytidine reactivated SLIT2 gene expression. In primary gliomas, SLIT2 was methylated in 59% (37/63) of tumours analysed. In addition, SLIT2 expression was downregulated in methylated gliomas relative to unmethylated tumour samples, as demonstrated by quantitative real-time RT-PCR. Loss of heterozygosity analysis revealed that SLIT2 methylated gliomas retained both alleles of a microsatellite marker within 100 kb of the SLIT2 gene at 4p15.2. Exogenous expression of SLIT2 in a glioma cell line that was heavily methylated for SLIT2 decreased in vitro colony formation. Our data indicate that SLIT2 is frequently inactivated by promoter region CpG island hypermethylation in gliomas and may be a good candidate for a glioma tumour suppressor gene (TSG) located at 4p15.2. Furthermore, our data suggest that a detailed analysis of both the cancer genome and epigenome will be required to identify key TSGs involved in glioma development.

Antimetabolites, Antineoplastic↗

PubMed2002

Patterning of the basal telencephalon and hypothalamus is essential for guidance of cortical projections.

We have investigated the mechanisms that control the guidance of corticofugal projections as they extend along different subdivisions of the forebrain. To this aim, we analyzed the development of cortical projections in mice that lack Nkx2-1, a homeobox gene whose expression is restricted to two domains within the forebrain: the basal telencephalon and the hypothalamus. Molecular respecification of the basal telencephalon and hypothalamus in Nkx2-1-deficient mice causes a severe defect in the guidance of layer 5 cortical projections and ascending fibers of the cerebral peduncle. These axon tracts take an abnormal path when coursing through both the basal telencephalon and hypothalamus. By contrast, loss of Nkx2-1 function does not impair guidance of corticothalamic or thalamocortical axons. In vitro experiments demonstrate that the basal telencephalon and the hypothalamus contain an activity that repels the growth of cortical axons, suggesting that loss of this activity is the cause of the defects observed in Nkx2-1 mutants. Furthermore, analysis of the expression of candidate molecules in the basal telencephalon and hypothalamus of Nkx2-1 mutants suggests that Slit2 contributes to this activity.

Animals↗

PubMed2004

Cutting edge: bone morphogenetic protein antagonists Drm/Gremlin and Dan interact with Slits and act as negative regulators of monocyte chemotaxis.

Drm/Gremlin and Dan, two homologous secreted antagonists of bone morphogenic proteins, have been shown to regulate early development, tumorigenesis, and renal pathophysiology. In this study, we report that Drm and Dan physically and functionally interact with Slit1 and Slit2 proteins. Drm binding to Slits depends on its glycosylation and is not interfered with by bone morphogenic proteins. Importantly, Drm and Dan function as inhibitors for monocyte migration induced by stromal cell-derived factor 1alpha (SDF-1alpha) or fMLP. The inhibition of SDF-1alpha-induced monocyte chemotaxis by Dan is not due to blocking the binding of SDF-1alpha to its receptor. Thus, the results identify that Drm and Dan can interact with Slit proteins and act as inhibitors of monocyte chemotaxis, demonstrating a previously unidentified biological role for these proteins.

Animals↗

PubMed2000

Squeezing axons out of the gray matter: a role for slit and semaphorin proteins from midline and ventral spinal cord.

Commissural axons cross the nervous system midline and then turn to grow alongside it, neither recrossing nor projecting back into ventral regions. In Drosophila, the midline repellent Slit prevents recrossing: axons cross once because they are initially unresponsive to Slit, becoming responsive only upon crossing. We show that commissural axons in mammals similarly acquire responsiveness to a midline repellent activity upon crossing. Remarkably, they also become responsive to a repellent activity from ventral spinal cord, helping explain why they never reenter that region. Several Slit and Semaphorin proteins, expressed in midline and/or ventral tissues, mimic these repellent activities, and midline guidance defects are observed in mice lacking neuropilin-2, a Semaphorin receptor. Thus, Slit and Semaphorin repellents from midline and nonmidline tissues may help prevent crossing axons from reentering gray matter, squeezing them into surrounding fiber tracts.

Animals↗

PubMed2002

Slit1 and slit2 proteins control the development of the lateral olfactory tract.

The development of olfactory bulb projections that form the lateral olfactory tract (LOT) is still poorly understood. The septum and the olfactory cortex have been shown to secrete diffusible factors repelling olfactory axons in vitro and are likely to cause the axons to avoid the septum region in vivo. Slit2, a member of the Slit gene family, has been proposed to be this septal factor based on its expression in the embryonic septum and its ability to repel and collapse olfactory axons. However, this issue is still controversial, and recent in vitro studies have questioned the role of the septum and Slit proteins in organizing LOT projections. We therefore decided to examine directly the role of Slit proteins in mediating olfactory axon guidance in vivo using mice with targeted deletions in the Slit1 and Slit2 genes. When olfactory bulb explants are cocultured with septum from Slit1- and/or Slit2-deficient mice, the septum repulsive activity for olfactory bulb axons is progressively abolished in a gene dose-dependent manner. Anterograde tracing of olfactory bulb axons showed that the LOT develops normally in Slit1 or Slit2 single-deficient mice but is completely disorganized in Slit1/Slit2 double-deficient embryos, with many axons reaching the midline and entering the septum region. Therefore, our study showed that the septum chemorepellent is a combination of Slit1 and Slit2 and that these molecules play a significant role in olfactory bulb axon guidance in vivo.

Animals↗

PubMed2001

Sensory axon response to substrate-bound Slit2 is modulated by laminin and cyclic GMP.

In vertebrates, Slit2 is a chemorepellent for some developing axons but stimulates axonal elongation and branching of sensory axons. In vivo, Slit2 is cleaved into 140-kDa N-terminal (Slit2-N) and 55- to 60-kDa C-terminal fragments, but the uncleaved/full-length form can also be isolated from brain extracts. As Slit2-N and full-length Slit2 bind tightly to cell membranes, we decided to explore the response of rat dorsal root ganglia (DRG) axons to substrate-bound Slit2 fragments in the stripe assay. Slit2 fragments were avoided by DRG axons when expressed on membranes or coated as stripes on laminin. However, when the Slit2 stripes were coated on fibronectin, DRG axons still avoided full-length Slit2 but grew preferentially on Slit2-N. DRG axon response to Slit2 fragments could be modulated by cGMP and by a laminin-1 peptide. These results strongly support the idea that extracellular matrix proteins modulate the response of growth cones to chemotropic molecules by modulating cyclic nucleotide levels.

Age Factors↗