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    Alomone Labs anti sema3a
    Presymptomatic elevation in the levels of <t>Sema3A</t> and NRP1 in ALS models. A , B , Western blot analysis of P30 and P60 GC muscle extracts revealed that the levels of Sema3A are elevated in presymptomatic SOD1 G93A muscles compared with their corresponding LM control wherein at earlier stages we found no significant difference . Tubulin was used as a loading control. P30 (Student's t test, n = 3, * p = 0.042). P60 (Student's t test, n = 4, * p = 0.038). C , qPCR analysis of presymptomatic P60 and P30 GC muscle extracts also shows an elevation in the mRNA levels of Sema3A in SOD1 G93A (Student's t test, SOD1 G93A , n = 5, LM, n = 4, * p = 0.049). D , Immunostaining of primary myocytes after 7 d in culture shows elevated levels of Sema3A in primary myocytes of SOD1 G93A . White represents Sema3A. Blue represents nuclear DAPI staining. Scale bars, 5 μm. E , Image analysis reveals an increase in Sema3A intensity in SOD1 G93A primary myocytes (Student's t test, n = 3, ∼80 cells, *** p = 0.00001). F , Western blot analysis of primary myocyte-CM revealed a higher level of Sema3A in the CM of SOD1 G93A . Cultures were lysed after CM collection, and equal loading volumes of lysates were immunoblotted for ERK to validate CM, which was produced from a similar mass of myocytes (Student's t test, n = 3, * p = 0.018). G , H , Immunostaining of fixed whole P60 GC muscles shows distinct Sema3A expression in the NMJs of SOD1 G93A mice. White represents Sema3A. Red represents TMR-BTX labeling of acetylcholine receptors on postsynapse. Blue represents presynaptic NFH in neurons. The percentage of muscle fibers expressing Sema3A in their NMJs in P60 SOD1 G93A mice is higher (∼100 NMJ per 1 biological repeat; Student's t test, SOD1 G93A , n = 4; WT, n = 3; * p = 0.011). Scale bars, 10 μm. We also examined Sema3A expression in later stages of the disease . I , Western blot analysis of GC muscle extracts from P60 mice revealed elevated NRP1 levels in the muscles of SOD1 G93A . Tubulin was used as a loading control (Student's t test, n = 3, * p = 0.048). J , Western blot analysis of SN extract from P60 mice shows an elevation in the levels of NRP1 in the SNs of SOD1 G93A mice ( n = 3). K , Western blot analysis of primary MN lysates after 3 d in culture reveals an elevation in the NRP1 levels in SOD1 G93A MNs, which are not regulated by Sema3A binding . ERK was used as a loading control (Student's t test, n = 3, * p = 0.031). L–N , Immunostaining of primary MNs after 3 d in culture shows an elevation in the levels of NRP1 in both axons (inset, ∼4.1-fold) and somata (∼1.9-fold) of SOD1 G93A MNs. White represents NRP1. Blue represents NFH. Somata (Student's t test, n = 3, ∼40 cells, *** p = 0.00021); axons (Student's t test, n = 3, ∼40 cells, * p = 0.012). Scale bars, 10 μm. O , P , Immunostaining of fixed whole P60 GC muscles shows distinct NRP1 expression in the NMJs of SOD1 G93A mice. White represents NRP1. Red represents BTX. Blue represents NFH. The percentage of muscle fibers expressing NRP1 in their NMJs in P60 SOD1 G93A mice is higher (Student's t test, SOD1 G93A , n = 4; WT, n = 3; * p = 0.042). Scale bars, 5 μm. We further examined NRP1 expression in later stages of the disease . Elevations in Sema3A and its coreceptor were found also in human sALS samples . A–C , E , F , I–K , M , N , Data represent the mean fold difference over the LM control ± SEM.
    Anti Sema3a, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "miR126-5p Downregulation Facilitates Axon Degeneration and NMJ Disruption via a Non–Cell-Autonomous Mechanism in ALS"

    Article Title: miR126-5p Downregulation Facilitates Axon Degeneration and NMJ Disruption via a Non–Cell-Autonomous Mechanism in ALS

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.3037-17.2018

    Presymptomatic elevation in the levels of Sema3A and NRP1 in ALS models. A , B , Western blot analysis of P30 and P60 GC muscle extracts revealed that the levels of Sema3A are elevated in presymptomatic SOD1 G93A muscles compared with their corresponding LM control wherein at earlier stages we found no significant difference . Tubulin was used as a loading control. P30 (Student's t test, n = 3, * p = 0.042). P60 (Student's t test, n = 4, * p = 0.038). C , qPCR analysis of presymptomatic P60 and P30 GC muscle extracts also shows an elevation in the mRNA levels of Sema3A in SOD1 G93A (Student's t test, SOD1 G93A , n = 5, LM, n = 4, * p = 0.049). D , Immunostaining of primary myocytes after 7 d in culture shows elevated levels of Sema3A in primary myocytes of SOD1 G93A . White represents Sema3A. Blue represents nuclear DAPI staining. Scale bars, 5 μm. E , Image analysis reveals an increase in Sema3A intensity in SOD1 G93A primary myocytes (Student's t test, n = 3, ∼80 cells, *** p = 0.00001). F , Western blot analysis of primary myocyte-CM revealed a higher level of Sema3A in the CM of SOD1 G93A . Cultures were lysed after CM collection, and equal loading volumes of lysates were immunoblotted for ERK to validate CM, which was produced from a similar mass of myocytes (Student's t test, n = 3, * p = 0.018). G , H , Immunostaining of fixed whole P60 GC muscles shows distinct Sema3A expression in the NMJs of SOD1 G93A mice. White represents Sema3A. Red represents TMR-BTX labeling of acetylcholine receptors on postsynapse. Blue represents presynaptic NFH in neurons. The percentage of muscle fibers expressing Sema3A in their NMJs in P60 SOD1 G93A mice is higher (∼100 NMJ per 1 biological repeat; Student's t test, SOD1 G93A , n = 4; WT, n = 3; * p = 0.011). Scale bars, 10 μm. We also examined Sema3A expression in later stages of the disease . I , Western blot analysis of GC muscle extracts from P60 mice revealed elevated NRP1 levels in the muscles of SOD1 G93A . Tubulin was used as a loading control (Student's t test, n = 3, * p = 0.048). J , Western blot analysis of SN extract from P60 mice shows an elevation in the levels of NRP1 in the SNs of SOD1 G93A mice ( n = 3). K , Western blot analysis of primary MN lysates after 3 d in culture reveals an elevation in the NRP1 levels in SOD1 G93A MNs, which are not regulated by Sema3A binding . ERK was used as a loading control (Student's t test, n = 3, * p = 0.031). L–N , Immunostaining of primary MNs after 3 d in culture shows an elevation in the levels of NRP1 in both axons (inset, ∼4.1-fold) and somata (∼1.9-fold) of SOD1 G93A MNs. White represents NRP1. Blue represents NFH. Somata (Student's t test, n = 3, ∼40 cells, *** p = 0.00021); axons (Student's t test, n = 3, ∼40 cells, * p = 0.012). Scale bars, 10 μm. O , P , Immunostaining of fixed whole P60 GC muscles shows distinct NRP1 expression in the NMJs of SOD1 G93A mice. White represents NRP1. Red represents BTX. Blue represents NFH. The percentage of muscle fibers expressing NRP1 in their NMJs in P60 SOD1 G93A mice is higher (Student's t test, SOD1 G93A , n = 4; WT, n = 3; * p = 0.042). Scale bars, 5 μm. We further examined NRP1 expression in later stages of the disease . Elevations in Sema3A and its coreceptor were found also in human sALS samples . A–C , E , F , I–K , M , N , Data represent the mean fold difference over the LM control ± SEM.
    Figure Legend Snippet: Presymptomatic elevation in the levels of Sema3A and NRP1 in ALS models. A , B , Western blot analysis of P30 and P60 GC muscle extracts revealed that the levels of Sema3A are elevated in presymptomatic SOD1 G93A muscles compared with their corresponding LM control wherein at earlier stages we found no significant difference . Tubulin was used as a loading control. P30 (Student's t test, n = 3, * p = 0.042). P60 (Student's t test, n = 4, * p = 0.038). C , qPCR analysis of presymptomatic P60 and P30 GC muscle extracts also shows an elevation in the mRNA levels of Sema3A in SOD1 G93A (Student's t test, SOD1 G93A , n = 5, LM, n = 4, * p = 0.049). D , Immunostaining of primary myocytes after 7 d in culture shows elevated levels of Sema3A in primary myocytes of SOD1 G93A . White represents Sema3A. Blue represents nuclear DAPI staining. Scale bars, 5 μm. E , Image analysis reveals an increase in Sema3A intensity in SOD1 G93A primary myocytes (Student's t test, n = 3, ∼80 cells, *** p = 0.00001). F , Western blot analysis of primary myocyte-CM revealed a higher level of Sema3A in the CM of SOD1 G93A . Cultures were lysed after CM collection, and equal loading volumes of lysates were immunoblotted for ERK to validate CM, which was produced from a similar mass of myocytes (Student's t test, n = 3, * p = 0.018). G , H , Immunostaining of fixed whole P60 GC muscles shows distinct Sema3A expression in the NMJs of SOD1 G93A mice. White represents Sema3A. Red represents TMR-BTX labeling of acetylcholine receptors on postsynapse. Blue represents presynaptic NFH in neurons. The percentage of muscle fibers expressing Sema3A in their NMJs in P60 SOD1 G93A mice is higher (∼100 NMJ per 1 biological repeat; Student's t test, SOD1 G93A , n = 4; WT, n = 3; * p = 0.011). Scale bars, 10 μm. We also examined Sema3A expression in later stages of the disease . I , Western blot analysis of GC muscle extracts from P60 mice revealed elevated NRP1 levels in the muscles of SOD1 G93A . Tubulin was used as a loading control (Student's t test, n = 3, * p = 0.048). J , Western blot analysis of SN extract from P60 mice shows an elevation in the levels of NRP1 in the SNs of SOD1 G93A mice ( n = 3). K , Western blot analysis of primary MN lysates after 3 d in culture reveals an elevation in the NRP1 levels in SOD1 G93A MNs, which are not regulated by Sema3A binding . ERK was used as a loading control (Student's t test, n = 3, * p = 0.031). L–N , Immunostaining of primary MNs after 3 d in culture shows an elevation in the levels of NRP1 in both axons (inset, ∼4.1-fold) and somata (∼1.9-fold) of SOD1 G93A MNs. White represents NRP1. Blue represents NFH. Somata (Student's t test, n = 3, ∼40 cells, *** p = 0.00021); axons (Student's t test, n = 3, ∼40 cells, * p = 0.012). Scale bars, 10 μm. O , P , Immunostaining of fixed whole P60 GC muscles shows distinct NRP1 expression in the NMJs of SOD1 G93A mice. White represents NRP1. Red represents BTX. Blue represents NFH. The percentage of muscle fibers expressing NRP1 in their NMJs in P60 SOD1 G93A mice is higher (Student's t test, SOD1 G93A , n = 4; WT, n = 3; * p = 0.042). Scale bars, 5 μm. We further examined NRP1 expression in later stages of the disease . Elevations in Sema3A and its coreceptor were found also in human sALS samples . A–C , E , F , I–K , M , N , Data represent the mean fold difference over the LM control ± SEM.

    Techniques Used: Western Blot, Immunostaining, Staining, Produced, Expressing, Labeling, Binding Assay

    Sema3A as well as primary myocytes expressing diverse ALS-causing mutations impair the growth of wild-type HB9::GFP motor axons and enhance their retraction and degeneration. A , Experimental procedure illustration and representative time-lapse images of HB9::GFP motor axons in the distal compartment of an MFC with no muscles after applying Sema3A to the distal compartment. After 6 h, axons in the distal compartment of chambers that were treated with Sema3A undergo degeneration, whereas axons in the control chamber or axons cotreated with NRP1 antibody and Sema3A continue growing. Scale bar, 20 μm. B , Quantification of the rate of degraded axons in the distal compartment revealed a higher percentage of degradation in chambers that were exposed to Sema3A compared with either control or coapplication of Sema3A and NRP1 antibody (∼60 axons for Sema3A treatment, ∼70 axons for Control; Student's t test; n = 4; mean ± SEM, *** p = 0.00022). C , Schematic view of the experimental procedure in D–F . HB9::GFP SC explants and primary myocytes of SOD1 G93A , TDP43 A315T , C9orf72-PR 50 , C9orf72-GR 50 , or LM, GFP, and SOD1 wt as controls were cocultured in an MFC , and the growth of HB9::GFP axons was assessed by time-lapse imaging of the microgroove compartment. D , Representative time-lapse images of the HB9::GFP axon growth when cocultured with (left to right) LM, SOD1 G93A , and SOD1 G93A + NRP1 antibody. The presence of SOD1 G93A myocytes in the distal compartment triggers the retraction and degeneration of HB9::GFP motor axons growing in the groove compartment and prevents their traversing. When NRP1 antibody is applied to the distal compartment, together with SOD1 G93A -expressing myocytes, axons are less prone to degenerate. Scale bar, 5 μm. E , Quantification of the rate of axons traversing the distal compartment in B shows the mean percentage of axons that traversed the distal compartment out of the total axons in each field ( n = 3; NRP1 antibody experiment, n = 4; Student's t test, * p = 0.025, * p = 0.0433). F , Quantification of the rate of axons traversing the distal compartment shows the mean percentage of axons that traversed the distal compartment out of the total number of axons in each field in coculture with TDP43 A315T , C9orf72-PR 50 , C9orf72-GR 50 myocytes, or GFP as a control. The traversing rate of HB9::GFP motor axons into the distal compartment in each of the cocultures with muscle-expressing ALS mutations is significantly reduced ( n = 3; Student's t test, GR 50 , * p = 0.0137; PR 50 , * p = 0.0374; TDP, * p = 0.0304). G , Representative images of fixed and immunostained SOD1 G93A motor axons in the distal compartment of an MFC after applying LM or SOD1 G93A myocyte CM to the distal compartment. After 48 h, axons in the distal compartment of chambers that were treated with SOD1 G93A CM underwent degeneration, whereas axons that were treated with LM CM remained intact. WT MN axons remained intact after application of either CM . When NRP1 antibody is applied to the distal compartment, together with SOD1 G93A CM, axons are less prone to degenerate, suggesting the involvement of multiple factors . Green represents acetylated tubulin. Scale bar, 20 μm. H , Quantification of the rate of degenerated SOD1 G93A axons in the distal compartment treated with control CM, SOD1 G93A CM, or SOD1 G93A CM that was coincubated with anti-NRP1 antibody (Student's t test; n = 3; *** p = 5 × 10 −7 , * p = 0.018). ** p < 0.01.
    Figure Legend Snippet: Sema3A as well as primary myocytes expressing diverse ALS-causing mutations impair the growth of wild-type HB9::GFP motor axons and enhance their retraction and degeneration. A , Experimental procedure illustration and representative time-lapse images of HB9::GFP motor axons in the distal compartment of an MFC with no muscles after applying Sema3A to the distal compartment. After 6 h, axons in the distal compartment of chambers that were treated with Sema3A undergo degeneration, whereas axons in the control chamber or axons cotreated with NRP1 antibody and Sema3A continue growing. Scale bar, 20 μm. B , Quantification of the rate of degraded axons in the distal compartment revealed a higher percentage of degradation in chambers that were exposed to Sema3A compared with either control or coapplication of Sema3A and NRP1 antibody (∼60 axons for Sema3A treatment, ∼70 axons for Control; Student's t test; n = 4; mean ± SEM, *** p = 0.00022). C , Schematic view of the experimental procedure in D–F . HB9::GFP SC explants and primary myocytes of SOD1 G93A , TDP43 A315T , C9orf72-PR 50 , C9orf72-GR 50 , or LM, GFP, and SOD1 wt as controls were cocultured in an MFC , and the growth of HB9::GFP axons was assessed by time-lapse imaging of the microgroove compartment. D , Representative time-lapse images of the HB9::GFP axon growth when cocultured with (left to right) LM, SOD1 G93A , and SOD1 G93A + NRP1 antibody. The presence of SOD1 G93A myocytes in the distal compartment triggers the retraction and degeneration of HB9::GFP motor axons growing in the groove compartment and prevents their traversing. When NRP1 antibody is applied to the distal compartment, together with SOD1 G93A -expressing myocytes, axons are less prone to degenerate. Scale bar, 5 μm. E , Quantification of the rate of axons traversing the distal compartment in B shows the mean percentage of axons that traversed the distal compartment out of the total axons in each field ( n = 3; NRP1 antibody experiment, n = 4; Student's t test, * p = 0.025, * p = 0.0433). F , Quantification of the rate of axons traversing the distal compartment shows the mean percentage of axons that traversed the distal compartment out of the total number of axons in each field in coculture with TDP43 A315T , C9orf72-PR 50 , C9orf72-GR 50 myocytes, or GFP as a control. The traversing rate of HB9::GFP motor axons into the distal compartment in each of the cocultures with muscle-expressing ALS mutations is significantly reduced ( n = 3; Student's t test, GR 50 , * p = 0.0137; PR 50 , * p = 0.0374; TDP, * p = 0.0304). G , Representative images of fixed and immunostained SOD1 G93A motor axons in the distal compartment of an MFC after applying LM or SOD1 G93A myocyte CM to the distal compartment. After 48 h, axons in the distal compartment of chambers that were treated with SOD1 G93A CM underwent degeneration, whereas axons that were treated with LM CM remained intact. WT MN axons remained intact after application of either CM . When NRP1 antibody is applied to the distal compartment, together with SOD1 G93A CM, axons are less prone to degenerate, suggesting the involvement of multiple factors . Green represents acetylated tubulin. Scale bar, 20 μm. H , Quantification of the rate of degenerated SOD1 G93A axons in the distal compartment treated with control CM, SOD1 G93A CM, or SOD1 G93A CM that was coincubated with anti-NRP1 antibody (Student's t test; n = 3; *** p = 5 × 10 −7 , * p = 0.018). ** p < 0.01.

    Techniques Used: Expressing, Imaging

    miR126-5p is depleted in SOD1 G93A muscles and regulates Sema3 and NRP expression. A , NanoString chip screen heat map of significantly altered miRs in P60 muscles of SOD1 G93A compared with LM mice (extended table under ). Red and green represent a high or low abundance of miRs, respectively. * p < 0.05 (Student's t test; n = 3). B , miR126-5p was the most significantly downregulated miRNA in SOD1 G93A muscles (Student's t test; n = 3, ** p = 0.003). C , qPCR analysis of P60 GC muscle extracts further validates the decrease in miR 126-5p in SOD1 G93A ( n = 3). D–G , qPCR analysis of Sema3A, NRP1, Sema3B, and NRP2 transcript levels in HeLa cells overexpressing either miR126-5p or miR142 demonstrates a reduction in their expression levels specifically under miR126-5p overexpression (Student's t test; n = 3, * p = 0.0438, * p = 0.034, * p = 0.031, and * p = 0.0434, respectively). H , Representative TIRF images of U87MG cells reveal a detachment of the cell membrane from the culture dish surface after Sema3A is added to the culture medium. Scale bar, 10 μm. I , Impedance recording of live cells over time shows that U87MG cells overexpressing miR126-5p are unresponsive to Sema3A added to the culture medium because their impedance continuously increases, whereas the impedance of U87MG cells overexpressing miR142 decreases after treatment .
    Figure Legend Snippet: miR126-5p is depleted in SOD1 G93A muscles and regulates Sema3 and NRP expression. A , NanoString chip screen heat map of significantly altered miRs in P60 muscles of SOD1 G93A compared with LM mice (extended table under ). Red and green represent a high or low abundance of miRs, respectively. * p < 0.05 (Student's t test; n = 3). B , miR126-5p was the most significantly downregulated miRNA in SOD1 G93A muscles (Student's t test; n = 3, ** p = 0.003). C , qPCR analysis of P60 GC muscle extracts further validates the decrease in miR 126-5p in SOD1 G93A ( n = 3). D–G , qPCR analysis of Sema3A, NRP1, Sema3B, and NRP2 transcript levels in HeLa cells overexpressing either miR126-5p or miR142 demonstrates a reduction in their expression levels specifically under miR126-5p overexpression (Student's t test; n = 3, * p = 0.0438, * p = 0.034, * p = 0.031, and * p = 0.0434, respectively). H , Representative TIRF images of U87MG cells reveal a detachment of the cell membrane from the culture dish surface after Sema3A is added to the culture medium. Scale bar, 10 μm. I , Impedance recording of live cells over time shows that U87MG cells overexpressing miR126-5p are unresponsive to Sema3A added to the culture medium because their impedance continuously increases, whereas the impedance of U87MG cells overexpressing miR142 decreases after treatment .

    Techniques Used: Expressing, Over Expression

    Overexpression of miR126-5p in primary SOD1 G93A myocytes blocks motor axon degeneration and preserves NMJ activity in a compartmental coculture. A , B , Western blot analysis of transfected myocyte extract overexpressing miR126-5p or miR142 and their CM validates miR126-5p as a regulator of Sema3A specifically in muscles. ERK was used as a loading control (Student's t test, n = 3, * p = 0.0499 and * p = 0.05, respectively). C , Schematic view of the experimental procedure in D , E . HB9::GFP SC explants and primary myocytes of SOD1 G93A mice were cocultured in a MFC. The growth of the HB9::GFP axons was assessed both by time-lapse imaging of the microgroove compartment and by imaging axons that traversed the distal compartment. D , Representative time-lapse images and quantification of HB9::GFP axon growth when cocultured with SOD1 miR126 myocytes (top) or SOD1 miR142 myocytes (bottom). SOD1 miR126 myocytes in the distal compartment enhanced the axonal traversal of the distal compartment compared with the SOD1 miR142 myocytes. The data are shown as the mean rate of axons that traversed the distal compartment out of the total number of axons in each field ± SEM (Student's t test, n = 3, * p = 0.04216). E , Representative time-lapse images and quantification of HB9::GFP axon growth when cocultured with PR 50 miR126 myocytes (top) or PR 50 miR142 myocytes (bottom). PR 50 miR126 myocytes in the distal compartment enhanced the axonal traversal of the distal compartment compared with PR 50 miR142 myocytes. The data are shown as the mean rate of axons that traversed the distal compartment out of the total number of axons in each field ± SEM (Student's t test, n = 3, *** p = 0.0039). F , Representative myocyte contraction plot showing the bursting contractile behavior of innervated myocytes in vitro . G , Quantification of the percentage of innervated myocytes that contract in a bursting pattern shows a diminished rate of bursting behavior in SOD1 G93A myocytes compared with LM controls. SOD1 miR126 myocytes show an increase in the rate of bursting myocytes back to the LM levels. The data are shown as the mean percentage of bursting myocytes ± SEM (Student's t test, n = 3, * p = 0.0291, * p = 0.0156, ** p = 0.005656). *** p < 0.001.
    Figure Legend Snippet: Overexpression of miR126-5p in primary SOD1 G93A myocytes blocks motor axon degeneration and preserves NMJ activity in a compartmental coculture. A , B , Western blot analysis of transfected myocyte extract overexpressing miR126-5p or miR142 and their CM validates miR126-5p as a regulator of Sema3A specifically in muscles. ERK was used as a loading control (Student's t test, n = 3, * p = 0.0499 and * p = 0.05, respectively). C , Schematic view of the experimental procedure in D , E . HB9::GFP SC explants and primary myocytes of SOD1 G93A mice were cocultured in a MFC. The growth of the HB9::GFP axons was assessed both by time-lapse imaging of the microgroove compartment and by imaging axons that traversed the distal compartment. D , Representative time-lapse images and quantification of HB9::GFP axon growth when cocultured with SOD1 miR126 myocytes (top) or SOD1 miR142 myocytes (bottom). SOD1 miR126 myocytes in the distal compartment enhanced the axonal traversal of the distal compartment compared with the SOD1 miR142 myocytes. The data are shown as the mean rate of axons that traversed the distal compartment out of the total number of axons in each field ± SEM (Student's t test, n = 3, * p = 0.04216). E , Representative time-lapse images and quantification of HB9::GFP axon growth when cocultured with PR 50 miR126 myocytes (top) or PR 50 miR142 myocytes (bottom). PR 50 miR126 myocytes in the distal compartment enhanced the axonal traversal of the distal compartment compared with PR 50 miR142 myocytes. The data are shown as the mean rate of axons that traversed the distal compartment out of the total number of axons in each field ± SEM (Student's t test, n = 3, *** p = 0.0039). F , Representative myocyte contraction plot showing the bursting contractile behavior of innervated myocytes in vitro . G , Quantification of the percentage of innervated myocytes that contract in a bursting pattern shows a diminished rate of bursting behavior in SOD1 G93A myocytes compared with LM controls. SOD1 miR126 myocytes show an increase in the rate of bursting myocytes back to the LM levels. The data are shown as the mean percentage of bursting myocytes ± SEM (Student's t test, n = 3, * p = 0.0291, * p = 0.0156, ** p = 0.005656). *** p < 0.001.

    Techniques Used: Over Expression, Activity Assay, Western Blot, Transfection, Imaging, In Vitro

    Alterations in Semaphorin3A regulation by miR126-5p trigger MN degeneration in ALS. miR126-5p is a negative regulator of Sema3 signaling in skeletal muscles. Downregulation of miR126-5p in ALS disease drives the overexpression and secretion of Sema3A and potentially other NMJ-destabilizing factors in skeletal muscles. The downregulation in miR126-5p in diseased MNs drives the overexpression of NRP1 specifically in axons. The excess binding and activation of the NRP1 receptor by its overexpressed ligand Sema3A as a result of miR126-5p alteration promote NMJ disruption and axon degeneration in a spatially confined process.
    Figure Legend Snippet: Alterations in Semaphorin3A regulation by miR126-5p trigger MN degeneration in ALS. miR126-5p is a negative regulator of Sema3 signaling in skeletal muscles. Downregulation of miR126-5p in ALS disease drives the overexpression and secretion of Sema3A and potentially other NMJ-destabilizing factors in skeletal muscles. The downregulation in miR126-5p in diseased MNs drives the overexpression of NRP1 specifically in axons. The excess binding and activation of the NRP1 receptor by its overexpressed ligand Sema3A as a result of miR126-5p alteration promote NMJ disruption and axon degeneration in a spatially confined process.

    Techniques Used: Over Expression, Binding Assay, Activation Assay

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  • 90

    Structured Review

    Alomone Labs anti sema3a
    Presymptomatic elevation in the levels of <t>Sema3A</t> and NRP1 in ALS models. A , B , Western blot analysis of P30 and P60 GC muscle extracts revealed that the levels of Sema3A are elevated in presymptomatic SOD1 G93A muscles compared with their corresponding LM control wherein at earlier stages we found no significant difference . Tubulin was used as a loading control. P30 (Student's t test, n = 3, * p = 0.042). P60 (Student's t test, n = 4, * p = 0.038). C , qPCR analysis of presymptomatic P60 and P30 GC muscle extracts also shows an elevation in the mRNA levels of Sema3A in SOD1 G93A (Student's t test, SOD1 G93A , n = 5, LM, n = 4, * p = 0.049). D , Immunostaining of primary myocytes after 7 d in culture shows elevated levels of Sema3A in primary myocytes of SOD1 G93A . White represents Sema3A. Blue represents nuclear DAPI staining. Scale bars, 5 μm. E , Image analysis reveals an increase in Sema3A intensity in SOD1 G93A primary myocytes (Student's t test, n = 3, ∼80 cells, *** p = 0.00001). F , Western blot analysis of primary myocyte-CM revealed a higher level of Sema3A in the CM of SOD1 G93A . Cultures were lysed after CM collection, and equal loading volumes of lysates were immunoblotted for ERK to validate CM, which was produced from a similar mass of myocytes (Student's t test, n = 3, * p = 0.018). G , H , Immunostaining of fixed whole P60 GC muscles shows distinct Sema3A expression in the NMJs of SOD1 G93A mice. White represents Sema3A. Red represents TMR-BTX labeling of acetylcholine receptors on postsynapse. Blue represents presynaptic NFH in neurons. The percentage of muscle fibers expressing Sema3A in their NMJs in P60 SOD1 G93A mice is higher (∼100 NMJ per 1 biological repeat; Student's t test, SOD1 G93A , n = 4; WT, n = 3; * p = 0.011). Scale bars, 10 μm. We also examined Sema3A expression in later stages of the disease . I , Western blot analysis of GC muscle extracts from P60 mice revealed elevated NRP1 levels in the muscles of SOD1 G93A . Tubulin was used as a loading control (Student's t test, n = 3, * p = 0.048). J , Western blot analysis of SN extract from P60 mice shows an elevation in the levels of NRP1 in the SNs of SOD1 G93A mice ( n = 3). K , Western blot analysis of primary MN lysates after 3 d in culture reveals an elevation in the NRP1 levels in SOD1 G93A MNs, which are not regulated by Sema3A binding . ERK was used as a loading control (Student's t test, n = 3, * p = 0.031). L–N , Immunostaining of primary MNs after 3 d in culture shows an elevation in the levels of NRP1 in both axons (inset, ∼4.1-fold) and somata (∼1.9-fold) of SOD1 G93A MNs. White represents NRP1. Blue represents NFH. Somata (Student's t test, n = 3, ∼40 cells, *** p = 0.00021); axons (Student's t test, n = 3, ∼40 cells, * p = 0.012). Scale bars, 10 μm. O , P , Immunostaining of fixed whole P60 GC muscles shows distinct NRP1 expression in the NMJs of SOD1 G93A mice. White represents NRP1. Red represents BTX. Blue represents NFH. The percentage of muscle fibers expressing NRP1 in their NMJs in P60 SOD1 G93A mice is higher (Student's t test, SOD1 G93A , n = 4; WT, n = 3; * p = 0.042). Scale bars, 5 μm. We further examined NRP1 expression in later stages of the disease . Elevations in Sema3A and its coreceptor were found also in human sALS samples . A–C , E , F , I–K , M , N , Data represent the mean fold difference over the LM control ± SEM.
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    1) Product Images from "miR126-5p Downregulation Facilitates Axon Degeneration and NMJ Disruption via a Non–Cell-Autonomous Mechanism in ALS"

    Article Title: miR126-5p Downregulation Facilitates Axon Degeneration and NMJ Disruption via a Non–Cell-Autonomous Mechanism in ALS

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.3037-17.2018

    Presymptomatic elevation in the levels of Sema3A and NRP1 in ALS models. A , B , Western blot analysis of P30 and P60 GC muscle extracts revealed that the levels of Sema3A are elevated in presymptomatic SOD1 G93A muscles compared with their corresponding LM control wherein at earlier stages we found no significant difference . Tubulin was used as a loading control. P30 (Student's t test, n = 3, * p = 0.042). P60 (Student's t test, n = 4, * p = 0.038). C , qPCR analysis of presymptomatic P60 and P30 GC muscle extracts also shows an elevation in the mRNA levels of Sema3A in SOD1 G93A (Student's t test, SOD1 G93A , n = 5, LM, n = 4, * p = 0.049). D , Immunostaining of primary myocytes after 7 d in culture shows elevated levels of Sema3A in primary myocytes of SOD1 G93A . White represents Sema3A. Blue represents nuclear DAPI staining. Scale bars, 5 μm. E , Image analysis reveals an increase in Sema3A intensity in SOD1 G93A primary myocytes (Student's t test, n = 3, ∼80 cells, *** p = 0.00001). F , Western blot analysis of primary myocyte-CM revealed a higher level of Sema3A in the CM of SOD1 G93A . Cultures were lysed after CM collection, and equal loading volumes of lysates were immunoblotted for ERK to validate CM, which was produced from a similar mass of myocytes (Student's t test, n = 3, * p = 0.018). G , H , Immunostaining of fixed whole P60 GC muscles shows distinct Sema3A expression in the NMJs of SOD1 G93A mice. White represents Sema3A. Red represents TMR-BTX labeling of acetylcholine receptors on postsynapse. Blue represents presynaptic NFH in neurons. The percentage of muscle fibers expressing Sema3A in their NMJs in P60 SOD1 G93A mice is higher (∼100 NMJ per 1 biological repeat; Student's t test, SOD1 G93A , n = 4; WT, n = 3; * p = 0.011). Scale bars, 10 μm. We also examined Sema3A expression in later stages of the disease . I , Western blot analysis of GC muscle extracts from P60 mice revealed elevated NRP1 levels in the muscles of SOD1 G93A . Tubulin was used as a loading control (Student's t test, n = 3, * p = 0.048). J , Western blot analysis of SN extract from P60 mice shows an elevation in the levels of NRP1 in the SNs of SOD1 G93A mice ( n = 3). K , Western blot analysis of primary MN lysates after 3 d in culture reveals an elevation in the NRP1 levels in SOD1 G93A MNs, which are not regulated by Sema3A binding . ERK was used as a loading control (Student's t test, n = 3, * p = 0.031). L–N , Immunostaining of primary MNs after 3 d in culture shows an elevation in the levels of NRP1 in both axons (inset, ∼4.1-fold) and somata (∼1.9-fold) of SOD1 G93A MNs. White represents NRP1. Blue represents NFH. Somata (Student's t test, n = 3, ∼40 cells, *** p = 0.00021); axons (Student's t test, n = 3, ∼40 cells, * p = 0.012). Scale bars, 10 μm. O , P , Immunostaining of fixed whole P60 GC muscles shows distinct NRP1 expression in the NMJs of SOD1 G93A mice. White represents NRP1. Red represents BTX. Blue represents NFH. The percentage of muscle fibers expressing NRP1 in their NMJs in P60 SOD1 G93A mice is higher (Student's t test, SOD1 G93A , n = 4; WT, n = 3; * p = 0.042). Scale bars, 5 μm. We further examined NRP1 expression in later stages of the disease . Elevations in Sema3A and its coreceptor were found also in human sALS samples . A–C , E , F , I–K , M , N , Data represent the mean fold difference over the LM control ± SEM.
    Figure Legend Snippet: Presymptomatic elevation in the levels of Sema3A and NRP1 in ALS models. A , B , Western blot analysis of P30 and P60 GC muscle extracts revealed that the levels of Sema3A are elevated in presymptomatic SOD1 G93A muscles compared with their corresponding LM control wherein at earlier stages we found no significant difference . Tubulin was used as a loading control. P30 (Student's t test, n = 3, * p = 0.042). P60 (Student's t test, n = 4, * p = 0.038). C , qPCR analysis of presymptomatic P60 and P30 GC muscle extracts also shows an elevation in the mRNA levels of Sema3A in SOD1 G93A (Student's t test, SOD1 G93A , n = 5, LM, n = 4, * p = 0.049). D , Immunostaining of primary myocytes after 7 d in culture shows elevated levels of Sema3A in primary myocytes of SOD1 G93A . White represents Sema3A. Blue represents nuclear DAPI staining. Scale bars, 5 μm. E , Image analysis reveals an increase in Sema3A intensity in SOD1 G93A primary myocytes (Student's t test, n = 3, ∼80 cells, *** p = 0.00001). F , Western blot analysis of primary myocyte-CM revealed a higher level of Sema3A in the CM of SOD1 G93A . Cultures were lysed after CM collection, and equal loading volumes of lysates were immunoblotted for ERK to validate CM, which was produced from a similar mass of myocytes (Student's t test, n = 3, * p = 0.018). G , H , Immunostaining of fixed whole P60 GC muscles shows distinct Sema3A expression in the NMJs of SOD1 G93A mice. White represents Sema3A. Red represents TMR-BTX labeling of acetylcholine receptors on postsynapse. Blue represents presynaptic NFH in neurons. The percentage of muscle fibers expressing Sema3A in their NMJs in P60 SOD1 G93A mice is higher (∼100 NMJ per 1 biological repeat; Student's t test, SOD1 G93A , n = 4; WT, n = 3; * p = 0.011). Scale bars, 10 μm. We also examined Sema3A expression in later stages of the disease . I , Western blot analysis of GC muscle extracts from P60 mice revealed elevated NRP1 levels in the muscles of SOD1 G93A . Tubulin was used as a loading control (Student's t test, n = 3, * p = 0.048). J , Western blot analysis of SN extract from P60 mice shows an elevation in the levels of NRP1 in the SNs of SOD1 G93A mice ( n = 3). K , Western blot analysis of primary MN lysates after 3 d in culture reveals an elevation in the NRP1 levels in SOD1 G93A MNs, which are not regulated by Sema3A binding . ERK was used as a loading control (Student's t test, n = 3, * p = 0.031). L–N , Immunostaining of primary MNs after 3 d in culture shows an elevation in the levels of NRP1 in both axons (inset, ∼4.1-fold) and somata (∼1.9-fold) of SOD1 G93A MNs. White represents NRP1. Blue represents NFH. Somata (Student's t test, n = 3, ∼40 cells, *** p = 0.00021); axons (Student's t test, n = 3, ∼40 cells, * p = 0.012). Scale bars, 10 μm. O , P , Immunostaining of fixed whole P60 GC muscles shows distinct NRP1 expression in the NMJs of SOD1 G93A mice. White represents NRP1. Red represents BTX. Blue represents NFH. The percentage of muscle fibers expressing NRP1 in their NMJs in P60 SOD1 G93A mice is higher (Student's t test, SOD1 G93A , n = 4; WT, n = 3; * p = 0.042). Scale bars, 5 μm. We further examined NRP1 expression in later stages of the disease . Elevations in Sema3A and its coreceptor were found also in human sALS samples . A–C , E , F , I–K , M , N , Data represent the mean fold difference over the LM control ± SEM.

    Techniques Used: Western Blot, Immunostaining, Staining, Produced, Expressing, Labeling, Binding Assay

    Sema3A as well as primary myocytes expressing diverse ALS-causing mutations impair the growth of wild-type HB9::GFP motor axons and enhance their retraction and degeneration. A , Experimental procedure illustration and representative time-lapse images of HB9::GFP motor axons in the distal compartment of an MFC with no muscles after applying Sema3A to the distal compartment. After 6 h, axons in the distal compartment of chambers that were treated with Sema3A undergo degeneration, whereas axons in the control chamber or axons cotreated with NRP1 antibody and Sema3A continue growing. Scale bar, 20 μm. B , Quantification of the rate of degraded axons in the distal compartment revealed a higher percentage of degradation in chambers that were exposed to Sema3A compared with either control or coapplication of Sema3A and NRP1 antibody (∼60 axons for Sema3A treatment, ∼70 axons for Control; Student's t test; n = 4; mean ± SEM, *** p = 0.00022). C , Schematic view of the experimental procedure in D–F . HB9::GFP SC explants and primary myocytes of SOD1 G93A , TDP43 A315T , C9orf72-PR 50 , C9orf72-GR 50 , or LM, GFP, and SOD1 wt as controls were cocultured in an MFC , and the growth of HB9::GFP axons was assessed by time-lapse imaging of the microgroove compartment. D , Representative time-lapse images of the HB9::GFP axon growth when cocultured with (left to right) LM, SOD1 G93A , and SOD1 G93A + NRP1 antibody. The presence of SOD1 G93A myocytes in the distal compartment triggers the retraction and degeneration of HB9::GFP motor axons growing in the groove compartment and prevents their traversing. When NRP1 antibody is applied to the distal compartment, together with SOD1 G93A -expressing myocytes, axons are less prone to degenerate. Scale bar, 5 μm. E , Quantification of the rate of axons traversing the distal compartment in B shows the mean percentage of axons that traversed the distal compartment out of the total axons in each field ( n = 3; NRP1 antibody experiment, n = 4; Student's t test, * p = 0.025, * p = 0.0433). F , Quantification of the rate of axons traversing the distal compartment shows the mean percentage of axons that traversed the distal compartment out of the total number of axons in each field in coculture with TDP43 A315T , C9orf72-PR 50 , C9orf72-GR 50 myocytes, or GFP as a control. The traversing rate of HB9::GFP motor axons into the distal compartment in each of the cocultures with muscle-expressing ALS mutations is significantly reduced ( n = 3; Student's t test, GR 50 , * p = 0.0137; PR 50 , * p = 0.0374; TDP, * p = 0.0304). G , Representative images of fixed and immunostained SOD1 G93A motor axons in the distal compartment of an MFC after applying LM or SOD1 G93A myocyte CM to the distal compartment. After 48 h, axons in the distal compartment of chambers that were treated with SOD1 G93A CM underwent degeneration, whereas axons that were treated with LM CM remained intact. WT MN axons remained intact after application of either CM . When NRP1 antibody is applied to the distal compartment, together with SOD1 G93A CM, axons are less prone to degenerate, suggesting the involvement of multiple factors . Green represents acetylated tubulin. Scale bar, 20 μm. H , Quantification of the rate of degenerated SOD1 G93A axons in the distal compartment treated with control CM, SOD1 G93A CM, or SOD1 G93A CM that was coincubated with anti-NRP1 antibody (Student's t test; n = 3; *** p = 5 × 10 −7 , * p = 0.018). ** p < 0.01.
    Figure Legend Snippet: Sema3A as well as primary myocytes expressing diverse ALS-causing mutations impair the growth of wild-type HB9::GFP motor axons and enhance their retraction and degeneration. A , Experimental procedure illustration and representative time-lapse images of HB9::GFP motor axons in the distal compartment of an MFC with no muscles after applying Sema3A to the distal compartment. After 6 h, axons in the distal compartment of chambers that were treated with Sema3A undergo degeneration, whereas axons in the control chamber or axons cotreated with NRP1 antibody and Sema3A continue growing. Scale bar, 20 μm. B , Quantification of the rate of degraded axons in the distal compartment revealed a higher percentage of degradation in chambers that were exposed to Sema3A compared with either control or coapplication of Sema3A and NRP1 antibody (∼60 axons for Sema3A treatment, ∼70 axons for Control; Student's t test; n = 4; mean ± SEM, *** p = 0.00022). C , Schematic view of the experimental procedure in D–F . HB9::GFP SC explants and primary myocytes of SOD1 G93A , TDP43 A315T , C9orf72-PR 50 , C9orf72-GR 50 , or LM, GFP, and SOD1 wt as controls were cocultured in an MFC , and the growth of HB9::GFP axons was assessed by time-lapse imaging of the microgroove compartment. D , Representative time-lapse images of the HB9::GFP axon growth when cocultured with (left to right) LM, SOD1 G93A , and SOD1 G93A + NRP1 antibody. The presence of SOD1 G93A myocytes in the distal compartment triggers the retraction and degeneration of HB9::GFP motor axons growing in the groove compartment and prevents their traversing. When NRP1 antibody is applied to the distal compartment, together with SOD1 G93A -expressing myocytes, axons are less prone to degenerate. Scale bar, 5 μm. E , Quantification of the rate of axons traversing the distal compartment in B shows the mean percentage of axons that traversed the distal compartment out of the total axons in each field ( n = 3; NRP1 antibody experiment, n = 4; Student's t test, * p = 0.025, * p = 0.0433). F , Quantification of the rate of axons traversing the distal compartment shows the mean percentage of axons that traversed the distal compartment out of the total number of axons in each field in coculture with TDP43 A315T , C9orf72-PR 50 , C9orf72-GR 50 myocytes, or GFP as a control. The traversing rate of HB9::GFP motor axons into the distal compartment in each of the cocultures with muscle-expressing ALS mutations is significantly reduced ( n = 3; Student's t test, GR 50 , * p = 0.0137; PR 50 , * p = 0.0374; TDP, * p = 0.0304). G , Representative images of fixed and immunostained SOD1 G93A motor axons in the distal compartment of an MFC after applying LM or SOD1 G93A myocyte CM to the distal compartment. After 48 h, axons in the distal compartment of chambers that were treated with SOD1 G93A CM underwent degeneration, whereas axons that were treated with LM CM remained intact. WT MN axons remained intact after application of either CM . When NRP1 antibody is applied to the distal compartment, together with SOD1 G93A CM, axons are less prone to degenerate, suggesting the involvement of multiple factors . Green represents acetylated tubulin. Scale bar, 20 μm. H , Quantification of the rate of degenerated SOD1 G93A axons in the distal compartment treated with control CM, SOD1 G93A CM, or SOD1 G93A CM that was coincubated with anti-NRP1 antibody (Student's t test; n = 3; *** p = 5 × 10 −7 , * p = 0.018). ** p < 0.01.

    Techniques Used: Expressing, Imaging

    miR126-5p is depleted in SOD1 G93A muscles and regulates Sema3 and NRP expression. A , NanoString chip screen heat map of significantly altered miRs in P60 muscles of SOD1 G93A compared with LM mice (extended table under ). Red and green represent a high or low abundance of miRs, respectively. * p < 0.05 (Student's t test; n = 3). B , miR126-5p was the most significantly downregulated miRNA in SOD1 G93A muscles (Student's t test; n = 3, ** p = 0.003). C , qPCR analysis of P60 GC muscle extracts further validates the decrease in miR 126-5p in SOD1 G93A ( n = 3). D–G , qPCR analysis of Sema3A, NRP1, Sema3B, and NRP2 transcript levels in HeLa cells overexpressing either miR126-5p or miR142 demonstrates a reduction in their expression levels specifically under miR126-5p overexpression (Student's t test; n = 3, * p = 0.0438, * p = 0.034, * p = 0.031, and * p = 0.0434, respectively). H , Representative TIRF images of U87MG cells reveal a detachment of the cell membrane from the culture dish surface after Sema3A is added to the culture medium. Scale bar, 10 μm. I , Impedance recording of live cells over time shows that U87MG cells overexpressing miR126-5p are unresponsive to Sema3A added to the culture medium because their impedance continuously increases, whereas the impedance of U87MG cells overexpressing miR142 decreases after treatment .
    Figure Legend Snippet: miR126-5p is depleted in SOD1 G93A muscles and regulates Sema3 and NRP expression. A , NanoString chip screen heat map of significantly altered miRs in P60 muscles of SOD1 G93A compared with LM mice (extended table under ). Red and green represent a high or low abundance of miRs, respectively. * p < 0.05 (Student's t test; n = 3). B , miR126-5p was the most significantly downregulated miRNA in SOD1 G93A muscles (Student's t test; n = 3, ** p = 0.003). C , qPCR analysis of P60 GC muscle extracts further validates the decrease in miR 126-5p in SOD1 G93A ( n = 3). D–G , qPCR analysis of Sema3A, NRP1, Sema3B, and NRP2 transcript levels in HeLa cells overexpressing either miR126-5p or miR142 demonstrates a reduction in their expression levels specifically under miR126-5p overexpression (Student's t test; n = 3, * p = 0.0438, * p = 0.034, * p = 0.031, and * p = 0.0434, respectively). H , Representative TIRF images of U87MG cells reveal a detachment of the cell membrane from the culture dish surface after Sema3A is added to the culture medium. Scale bar, 10 μm. I , Impedance recording of live cells over time shows that U87MG cells overexpressing miR126-5p are unresponsive to Sema3A added to the culture medium because their impedance continuously increases, whereas the impedance of U87MG cells overexpressing miR142 decreases after treatment .

    Techniques Used: Expressing, Over Expression

    Overexpression of miR126-5p in primary SOD1 G93A myocytes blocks motor axon degeneration and preserves NMJ activity in a compartmental coculture. A , B , Western blot analysis of transfected myocyte extract overexpressing miR126-5p or miR142 and their CM validates miR126-5p as a regulator of Sema3A specifically in muscles. ERK was used as a loading control (Student's t test, n = 3, * p = 0.0499 and * p = 0.05, respectively). C , Schematic view of the experimental procedure in D , E . HB9::GFP SC explants and primary myocytes of SOD1 G93A mice were cocultured in a MFC. The growth of the HB9::GFP axons was assessed both by time-lapse imaging of the microgroove compartment and by imaging axons that traversed the distal compartment. D , Representative time-lapse images and quantification of HB9::GFP axon growth when cocultured with SOD1 miR126 myocytes (top) or SOD1 miR142 myocytes (bottom). SOD1 miR126 myocytes in the distal compartment enhanced the axonal traversal of the distal compartment compared with the SOD1 miR142 myocytes. The data are shown as the mean rate of axons that traversed the distal compartment out of the total number of axons in each field ± SEM (Student's t test, n = 3, * p = 0.04216). E , Representative time-lapse images and quantification of HB9::GFP axon growth when cocultured with PR 50 miR126 myocytes (top) or PR 50 miR142 myocytes (bottom). PR 50 miR126 myocytes in the distal compartment enhanced the axonal traversal of the distal compartment compared with PR 50 miR142 myocytes. The data are shown as the mean rate of axons that traversed the distal compartment out of the total number of axons in each field ± SEM (Student's t test, n = 3, *** p = 0.0039). F , Representative myocyte contraction plot showing the bursting contractile behavior of innervated myocytes in vitro . G , Quantification of the percentage of innervated myocytes that contract in a bursting pattern shows a diminished rate of bursting behavior in SOD1 G93A myocytes compared with LM controls. SOD1 miR126 myocytes show an increase in the rate of bursting myocytes back to the LM levels. The data are shown as the mean percentage of bursting myocytes ± SEM (Student's t test, n = 3, * p = 0.0291, * p = 0.0156, ** p = 0.005656). *** p < 0.001.
    Figure Legend Snippet: Overexpression of miR126-5p in primary SOD1 G93A myocytes blocks motor axon degeneration and preserves NMJ activity in a compartmental coculture. A , B , Western blot analysis of transfected myocyte extract overexpressing miR126-5p or miR142 and their CM validates miR126-5p as a regulator of Sema3A specifically in muscles. ERK was used as a loading control (Student's t test, n = 3, * p = 0.0499 and * p = 0.05, respectively). C , Schematic view of the experimental procedure in D , E . HB9::GFP SC explants and primary myocytes of SOD1 G93A mice were cocultured in a MFC. The growth of the HB9::GFP axons was assessed both by time-lapse imaging of the microgroove compartment and by imaging axons that traversed the distal compartment. D , Representative time-lapse images and quantification of HB9::GFP axon growth when cocultured with SOD1 miR126 myocytes (top) or SOD1 miR142 myocytes (bottom). SOD1 miR126 myocytes in the distal compartment enhanced the axonal traversal of the distal compartment compared with the SOD1 miR142 myocytes. The data are shown as the mean rate of axons that traversed the distal compartment out of the total number of axons in each field ± SEM (Student's t test, n = 3, * p = 0.04216). E , Representative time-lapse images and quantification of HB9::GFP axon growth when cocultured with PR 50 miR126 myocytes (top) or PR 50 miR142 myocytes (bottom). PR 50 miR126 myocytes in the distal compartment enhanced the axonal traversal of the distal compartment compared with PR 50 miR142 myocytes. The data are shown as the mean rate of axons that traversed the distal compartment out of the total number of axons in each field ± SEM (Student's t test, n = 3, *** p = 0.0039). F , Representative myocyte contraction plot showing the bursting contractile behavior of innervated myocytes in vitro . G , Quantification of the percentage of innervated myocytes that contract in a bursting pattern shows a diminished rate of bursting behavior in SOD1 G93A myocytes compared with LM controls. SOD1 miR126 myocytes show an increase in the rate of bursting myocytes back to the LM levels. The data are shown as the mean percentage of bursting myocytes ± SEM (Student's t test, n = 3, * p = 0.0291, * p = 0.0156, ** p = 0.005656). *** p < 0.001.

    Techniques Used: Over Expression, Activity Assay, Western Blot, Transfection, Imaging, In Vitro

    Alterations in Semaphorin3A regulation by miR126-5p trigger MN degeneration in ALS. miR126-5p is a negative regulator of Sema3 signaling in skeletal muscles. Downregulation of miR126-5p in ALS disease drives the overexpression and secretion of Sema3A and potentially other NMJ-destabilizing factors in skeletal muscles. The downregulation in miR126-5p in diseased MNs drives the overexpression of NRP1 specifically in axons. The excess binding and activation of the NRP1 receptor by its overexpressed ligand Sema3A as a result of miR126-5p alteration promote NMJ disruption and axon degeneration in a spatially confined process.
    Figure Legend Snippet: Alterations in Semaphorin3A regulation by miR126-5p trigger MN degeneration in ALS. miR126-5p is a negative regulator of Sema3 signaling in skeletal muscles. Downregulation of miR126-5p in ALS disease drives the overexpression and secretion of Sema3A and potentially other NMJ-destabilizing factors in skeletal muscles. The downregulation in miR126-5p in diseased MNs drives the overexpression of NRP1 specifically in axons. The excess binding and activation of the NRP1 receptor by its overexpressed ligand Sema3A as a result of miR126-5p alteration promote NMJ disruption and axon degeneration in a spatially confined process.

    Techniques Used: Over Expression, Binding Assay, Activation Assay

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    Structured Review

    Alomone Labs anti sema3a
    Anti Sema3a, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti sema3a/product/Alomone Labs
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti sema3a - by Bioz Stars, 2023-02
    90/100 stars

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    Alomone Labs anti sema3a
    Presymptomatic elevation in the levels of <t>Sema3A</t> and NRP1 in ALS models. A , B , Western blot analysis of P30 and P60 GC muscle extracts revealed that the levels of Sema3A are elevated in presymptomatic SOD1 G93A muscles compared with their corresponding LM control wherein at earlier stages we found no significant difference . Tubulin was used as a loading control. P30 (Student's t test, n = 3, * p = 0.042). P60 (Student's t test, n = 4, * p = 0.038). C , qPCR analysis of presymptomatic P60 and P30 GC muscle extracts also shows an elevation in the mRNA levels of Sema3A in SOD1 G93A (Student's t test, SOD1 G93A , n = 5, LM, n = 4, * p = 0.049). D , Immunostaining of primary myocytes after 7 d in culture shows elevated levels of Sema3A in primary myocytes of SOD1 G93A . White represents Sema3A. Blue represents nuclear DAPI staining. Scale bars, 5 μm. E , Image analysis reveals an increase in Sema3A intensity in SOD1 G93A primary myocytes (Student's t test, n = 3, ∼80 cells, *** p = 0.00001). F , Western blot analysis of primary myocyte-CM revealed a higher level of Sema3A in the CM of SOD1 G93A . Cultures were lysed after CM collection, and equal loading volumes of lysates were immunoblotted for ERK to validate CM, which was produced from a similar mass of myocytes (Student's t test, n = 3, * p = 0.018). G , H , Immunostaining of fixed whole P60 GC muscles shows distinct Sema3A expression in the NMJs of SOD1 G93A mice. White represents Sema3A. Red represents TMR-BTX labeling of acetylcholine receptors on postsynapse. Blue represents presynaptic NFH in neurons. The percentage of muscle fibers expressing Sema3A in their NMJs in P60 SOD1 G93A mice is higher (∼100 NMJ per 1 biological repeat; Student's t test, SOD1 G93A , n = 4; WT, n = 3; * p = 0.011). Scale bars, 10 μm. We also examined Sema3A expression in later stages of the disease . I , Western blot analysis of GC muscle extracts from P60 mice revealed elevated NRP1 levels in the muscles of SOD1 G93A . Tubulin was used as a loading control (Student's t test, n = 3, * p = 0.048). J , Western blot analysis of SN extract from P60 mice shows an elevation in the levels of NRP1 in the SNs of SOD1 G93A mice ( n = 3). K , Western blot analysis of primary MN lysates after 3 d in culture reveals an elevation in the NRP1 levels in SOD1 G93A MNs, which are not regulated by Sema3A binding . ERK was used as a loading control (Student's t test, n = 3, * p = 0.031). L–N , Immunostaining of primary MNs after 3 d in culture shows an elevation in the levels of NRP1 in both axons (inset, ∼4.1-fold) and somata (∼1.9-fold) of SOD1 G93A MNs. White represents NRP1. Blue represents NFH. Somata (Student's t test, n = 3, ∼40 cells, *** p = 0.00021); axons (Student's t test, n = 3, ∼40 cells, * p = 0.012). Scale bars, 10 μm. O , P , Immunostaining of fixed whole P60 GC muscles shows distinct NRP1 expression in the NMJs of SOD1 G93A mice. White represents NRP1. Red represents BTX. Blue represents NFH. The percentage of muscle fibers expressing NRP1 in their NMJs in P60 SOD1 G93A mice is higher (Student's t test, SOD1 G93A , n = 4; WT, n = 3; * p = 0.042). Scale bars, 5 μm. We further examined NRP1 expression in later stages of the disease . Elevations in Sema3A and its coreceptor were found also in human sALS samples . A–C , E , F , I–K , M , N , Data represent the mean fold difference over the LM control ± SEM.
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    Presymptomatic elevation in the levels of Sema3A and NRP1 in ALS models. A , B , Western blot analysis of P30 and P60 GC muscle extracts revealed that the levels of Sema3A are elevated in presymptomatic SOD1 G93A muscles compared with their corresponding LM control wherein at earlier stages we found no significant difference . Tubulin was used as a loading control. P30 (Student's t test, n = 3, * p = 0.042). P60 (Student's t test, n = 4, * p = 0.038). C , qPCR analysis of presymptomatic P60 and P30 GC muscle extracts also shows an elevation in the mRNA levels of Sema3A in SOD1 G93A (Student's t test, SOD1 G93A , n = 5, LM, n = 4, * p = 0.049). D , Immunostaining of primary myocytes after 7 d in culture shows elevated levels of Sema3A in primary myocytes of SOD1 G93A . White represents Sema3A. Blue represents nuclear DAPI staining. Scale bars, 5 μm. E , Image analysis reveals an increase in Sema3A intensity in SOD1 G93A primary myocytes (Student's t test, n = 3, ∼80 cells, *** p = 0.00001). F , Western blot analysis of primary myocyte-CM revealed a higher level of Sema3A in the CM of SOD1 G93A . Cultures were lysed after CM collection, and equal loading volumes of lysates were immunoblotted for ERK to validate CM, which was produced from a similar mass of myocytes (Student's t test, n = 3, * p = 0.018). G , H , Immunostaining of fixed whole P60 GC muscles shows distinct Sema3A expression in the NMJs of SOD1 G93A mice. White represents Sema3A. Red represents TMR-BTX labeling of acetylcholine receptors on postsynapse. Blue represents presynaptic NFH in neurons. The percentage of muscle fibers expressing Sema3A in their NMJs in P60 SOD1 G93A mice is higher (∼100 NMJ per 1 biological repeat; Student's t test, SOD1 G93A , n = 4; WT, n = 3; * p = 0.011). Scale bars, 10 μm. We also examined Sema3A expression in later stages of the disease . I , Western blot analysis of GC muscle extracts from P60 mice revealed elevated NRP1 levels in the muscles of SOD1 G93A . Tubulin was used as a loading control (Student's t test, n = 3, * p = 0.048). J , Western blot analysis of SN extract from P60 mice shows an elevation in the levels of NRP1 in the SNs of SOD1 G93A mice ( n = 3). K , Western blot analysis of primary MN lysates after 3 d in culture reveals an elevation in the NRP1 levels in SOD1 G93A MNs, which are not regulated by Sema3A binding . ERK was used as a loading control (Student's t test, n = 3, * p = 0.031). L–N , Immunostaining of primary MNs after 3 d in culture shows an elevation in the levels of NRP1 in both axons (inset, ∼4.1-fold) and somata (∼1.9-fold) of SOD1 G93A MNs. White represents NRP1. Blue represents NFH. Somata (Student's t test, n = 3, ∼40 cells, *** p = 0.00021); axons (Student's t test, n = 3, ∼40 cells, * p = 0.012). Scale bars, 10 μm. O , P , Immunostaining of fixed whole P60 GC muscles shows distinct NRP1 expression in the NMJs of SOD1 G93A mice. White represents NRP1. Red represents BTX. Blue represents NFH. The percentage of muscle fibers expressing NRP1 in their NMJs in P60 SOD1 G93A mice is higher (Student's t test, SOD1 G93A , n = 4; WT, n = 3; * p = 0.042). Scale bars, 5 μm. We further examined NRP1 expression in later stages of the disease . Elevations in Sema3A and its coreceptor were found also in human sALS samples . A–C , E , F , I–K , M , N , Data represent the mean fold difference over the LM control ± SEM.

    Journal: The Journal of Neuroscience

    Article Title: miR126-5p Downregulation Facilitates Axon Degeneration and NMJ Disruption via a Non–Cell-Autonomous Mechanism in ALS

    doi: 10.1523/JNEUROSCI.3037-17.2018

    Figure Lengend Snippet: Presymptomatic elevation in the levels of Sema3A and NRP1 in ALS models. A , B , Western blot analysis of P30 and P60 GC muscle extracts revealed that the levels of Sema3A are elevated in presymptomatic SOD1 G93A muscles compared with their corresponding LM control wherein at earlier stages we found no significant difference . Tubulin was used as a loading control. P30 (Student's t test, n = 3, * p = 0.042). P60 (Student's t test, n = 4, * p = 0.038). C , qPCR analysis of presymptomatic P60 and P30 GC muscle extracts also shows an elevation in the mRNA levels of Sema3A in SOD1 G93A (Student's t test, SOD1 G93A , n = 5, LM, n = 4, * p = 0.049). D , Immunostaining of primary myocytes after 7 d in culture shows elevated levels of Sema3A in primary myocytes of SOD1 G93A . White represents Sema3A. Blue represents nuclear DAPI staining. Scale bars, 5 μm. E , Image analysis reveals an increase in Sema3A intensity in SOD1 G93A primary myocytes (Student's t test, n = 3, ∼80 cells, *** p = 0.00001). F , Western blot analysis of primary myocyte-CM revealed a higher level of Sema3A in the CM of SOD1 G93A . Cultures were lysed after CM collection, and equal loading volumes of lysates were immunoblotted for ERK to validate CM, which was produced from a similar mass of myocytes (Student's t test, n = 3, * p = 0.018). G , H , Immunostaining of fixed whole P60 GC muscles shows distinct Sema3A expression in the NMJs of SOD1 G93A mice. White represents Sema3A. Red represents TMR-BTX labeling of acetylcholine receptors on postsynapse. Blue represents presynaptic NFH in neurons. The percentage of muscle fibers expressing Sema3A in their NMJs in P60 SOD1 G93A mice is higher (∼100 NMJ per 1 biological repeat; Student's t test, SOD1 G93A , n = 4; WT, n = 3; * p = 0.011). Scale bars, 10 μm. We also examined Sema3A expression in later stages of the disease . I , Western blot analysis of GC muscle extracts from P60 mice revealed elevated NRP1 levels in the muscles of SOD1 G93A . Tubulin was used as a loading control (Student's t test, n = 3, * p = 0.048). J , Western blot analysis of SN extract from P60 mice shows an elevation in the levels of NRP1 in the SNs of SOD1 G93A mice ( n = 3). K , Western blot analysis of primary MN lysates after 3 d in culture reveals an elevation in the NRP1 levels in SOD1 G93A MNs, which are not regulated by Sema3A binding . ERK was used as a loading control (Student's t test, n = 3, * p = 0.031). L–N , Immunostaining of primary MNs after 3 d in culture shows an elevation in the levels of NRP1 in both axons (inset, ∼4.1-fold) and somata (∼1.9-fold) of SOD1 G93A MNs. White represents NRP1. Blue represents NFH. Somata (Student's t test, n = 3, ∼40 cells, *** p = 0.00021); axons (Student's t test, n = 3, ∼40 cells, * p = 0.012). Scale bars, 10 μm. O , P , Immunostaining of fixed whole P60 GC muscles shows distinct NRP1 expression in the NMJs of SOD1 G93A mice. White represents NRP1. Red represents BTX. Blue represents NFH. The percentage of muscle fibers expressing NRP1 in their NMJs in P60 SOD1 G93A mice is higher (Student's t test, SOD1 G93A , n = 4; WT, n = 3; * p = 0.042). Scale bars, 5 μm. We further examined NRP1 expression in later stages of the disease . Elevations in Sema3A and its coreceptor were found also in human sALS samples . A–C , E , F , I–K , M , N , Data represent the mean fold difference over the LM control ± SEM.

    Article Snippet: Antibodies were used at the following concentrations: anti-Neurofilament Heavy Chain 1:500 (NFH, Abcam, ab72996; 1:1000), synaptophysin (Millipore, MAB5258; 1:300), synaptotagmin (Alomone Labs, ant-003; 1:300), anti-NRP1 (1:100), anti-Sema3A (1:100), anti-NRP2 (1:100), and anti-Sema3B (1:100).

    Techniques: Western Blot, Immunostaining, Staining, Produced, Expressing, Labeling, Binding Assay

    Sema3A as well as primary myocytes expressing diverse ALS-causing mutations impair the growth of wild-type HB9::GFP motor axons and enhance their retraction and degeneration. A , Experimental procedure illustration and representative time-lapse images of HB9::GFP motor axons in the distal compartment of an MFC with no muscles after applying Sema3A to the distal compartment. After 6 h, axons in the distal compartment of chambers that were treated with Sema3A undergo degeneration, whereas axons in the control chamber or axons cotreated with NRP1 antibody and Sema3A continue growing. Scale bar, 20 μm. B , Quantification of the rate of degraded axons in the distal compartment revealed a higher percentage of degradation in chambers that were exposed to Sema3A compared with either control or coapplication of Sema3A and NRP1 antibody (∼60 axons for Sema3A treatment, ∼70 axons for Control; Student's t test; n = 4; mean ± SEM, *** p = 0.00022). C , Schematic view of the experimental procedure in D–F . HB9::GFP SC explants and primary myocytes of SOD1 G93A , TDP43 A315T , C9orf72-PR 50 , C9orf72-GR 50 , or LM, GFP, and SOD1 wt as controls were cocultured in an MFC , and the growth of HB9::GFP axons was assessed by time-lapse imaging of the microgroove compartment. D , Representative time-lapse images of the HB9::GFP axon growth when cocultured with (left to right) LM, SOD1 G93A , and SOD1 G93A + NRP1 antibody. The presence of SOD1 G93A myocytes in the distal compartment triggers the retraction and degeneration of HB9::GFP motor axons growing in the groove compartment and prevents their traversing. When NRP1 antibody is applied to the distal compartment, together with SOD1 G93A -expressing myocytes, axons are less prone to degenerate. Scale bar, 5 μm. E , Quantification of the rate of axons traversing the distal compartment in B shows the mean percentage of axons that traversed the distal compartment out of the total axons in each field ( n = 3; NRP1 antibody experiment, n = 4; Student's t test, * p = 0.025, * p = 0.0433). F , Quantification of the rate of axons traversing the distal compartment shows the mean percentage of axons that traversed the distal compartment out of the total number of axons in each field in coculture with TDP43 A315T , C9orf72-PR 50 , C9orf72-GR 50 myocytes, or GFP as a control. The traversing rate of HB9::GFP motor axons into the distal compartment in each of the cocultures with muscle-expressing ALS mutations is significantly reduced ( n = 3; Student's t test, GR 50 , * p = 0.0137; PR 50 , * p = 0.0374; TDP, * p = 0.0304). G , Representative images of fixed and immunostained SOD1 G93A motor axons in the distal compartment of an MFC after applying LM or SOD1 G93A myocyte CM to the distal compartment. After 48 h, axons in the distal compartment of chambers that were treated with SOD1 G93A CM underwent degeneration, whereas axons that were treated with LM CM remained intact. WT MN axons remained intact after application of either CM . When NRP1 antibody is applied to the distal compartment, together with SOD1 G93A CM, axons are less prone to degenerate, suggesting the involvement of multiple factors . Green represents acetylated tubulin. Scale bar, 20 μm. H , Quantification of the rate of degenerated SOD1 G93A axons in the distal compartment treated with control CM, SOD1 G93A CM, or SOD1 G93A CM that was coincubated with anti-NRP1 antibody (Student's t test; n = 3; *** p = 5 × 10 −7 , * p = 0.018). ** p < 0.01.

    Journal: The Journal of Neuroscience

    Article Title: miR126-5p Downregulation Facilitates Axon Degeneration and NMJ Disruption via a Non–Cell-Autonomous Mechanism in ALS

    doi: 10.1523/JNEUROSCI.3037-17.2018

    Figure Lengend Snippet: Sema3A as well as primary myocytes expressing diverse ALS-causing mutations impair the growth of wild-type HB9::GFP motor axons and enhance their retraction and degeneration. A , Experimental procedure illustration and representative time-lapse images of HB9::GFP motor axons in the distal compartment of an MFC with no muscles after applying Sema3A to the distal compartment. After 6 h, axons in the distal compartment of chambers that were treated with Sema3A undergo degeneration, whereas axons in the control chamber or axons cotreated with NRP1 antibody and Sema3A continue growing. Scale bar, 20 μm. B , Quantification of the rate of degraded axons in the distal compartment revealed a higher percentage of degradation in chambers that were exposed to Sema3A compared with either control or coapplication of Sema3A and NRP1 antibody (∼60 axons for Sema3A treatment, ∼70 axons for Control; Student's t test; n = 4; mean ± SEM, *** p = 0.00022). C , Schematic view of the experimental procedure in D–F . HB9::GFP SC explants and primary myocytes of SOD1 G93A , TDP43 A315T , C9orf72-PR 50 , C9orf72-GR 50 , or LM, GFP, and SOD1 wt as controls were cocultured in an MFC , and the growth of HB9::GFP axons was assessed by time-lapse imaging of the microgroove compartment. D , Representative time-lapse images of the HB9::GFP axon growth when cocultured with (left to right) LM, SOD1 G93A , and SOD1 G93A + NRP1 antibody. The presence of SOD1 G93A myocytes in the distal compartment triggers the retraction and degeneration of HB9::GFP motor axons growing in the groove compartment and prevents their traversing. When NRP1 antibody is applied to the distal compartment, together with SOD1 G93A -expressing myocytes, axons are less prone to degenerate. Scale bar, 5 μm. E , Quantification of the rate of axons traversing the distal compartment in B shows the mean percentage of axons that traversed the distal compartment out of the total axons in each field ( n = 3; NRP1 antibody experiment, n = 4; Student's t test, * p = 0.025, * p = 0.0433). F , Quantification of the rate of axons traversing the distal compartment shows the mean percentage of axons that traversed the distal compartment out of the total number of axons in each field in coculture with TDP43 A315T , C9orf72-PR 50 , C9orf72-GR 50 myocytes, or GFP as a control. The traversing rate of HB9::GFP motor axons into the distal compartment in each of the cocultures with muscle-expressing ALS mutations is significantly reduced ( n = 3; Student's t test, GR 50 , * p = 0.0137; PR 50 , * p = 0.0374; TDP, * p = 0.0304). G , Representative images of fixed and immunostained SOD1 G93A motor axons in the distal compartment of an MFC after applying LM or SOD1 G93A myocyte CM to the distal compartment. After 48 h, axons in the distal compartment of chambers that were treated with SOD1 G93A CM underwent degeneration, whereas axons that were treated with LM CM remained intact. WT MN axons remained intact after application of either CM . When NRP1 antibody is applied to the distal compartment, together with SOD1 G93A CM, axons are less prone to degenerate, suggesting the involvement of multiple factors . Green represents acetylated tubulin. Scale bar, 20 μm. H , Quantification of the rate of degenerated SOD1 G93A axons in the distal compartment treated with control CM, SOD1 G93A CM, or SOD1 G93A CM that was coincubated with anti-NRP1 antibody (Student's t test; n = 3; *** p = 5 × 10 −7 , * p = 0.018). ** p < 0.01.

    Article Snippet: Antibodies were used at the following concentrations: anti-Neurofilament Heavy Chain 1:500 (NFH, Abcam, ab72996; 1:1000), synaptophysin (Millipore, MAB5258; 1:300), synaptotagmin (Alomone Labs, ant-003; 1:300), anti-NRP1 (1:100), anti-Sema3A (1:100), anti-NRP2 (1:100), and anti-Sema3B (1:100).

    Techniques: Expressing, Imaging

    miR126-5p is depleted in SOD1 G93A muscles and regulates Sema3 and NRP expression. A , NanoString chip screen heat map of significantly altered miRs in P60 muscles of SOD1 G93A compared with LM mice (extended table under ). Red and green represent a high or low abundance of miRs, respectively. * p < 0.05 (Student's t test; n = 3). B , miR126-5p was the most significantly downregulated miRNA in SOD1 G93A muscles (Student's t test; n = 3, ** p = 0.003). C , qPCR analysis of P60 GC muscle extracts further validates the decrease in miR 126-5p in SOD1 G93A ( n = 3). D–G , qPCR analysis of Sema3A, NRP1, Sema3B, and NRP2 transcript levels in HeLa cells overexpressing either miR126-5p or miR142 demonstrates a reduction in their expression levels specifically under miR126-5p overexpression (Student's t test; n = 3, * p = 0.0438, * p = 0.034, * p = 0.031, and * p = 0.0434, respectively). H , Representative TIRF images of U87MG cells reveal a detachment of the cell membrane from the culture dish surface after Sema3A is added to the culture medium. Scale bar, 10 μm. I , Impedance recording of live cells over time shows that U87MG cells overexpressing miR126-5p are unresponsive to Sema3A added to the culture medium because their impedance continuously increases, whereas the impedance of U87MG cells overexpressing miR142 decreases after treatment .

    Journal: The Journal of Neuroscience

    Article Title: miR126-5p Downregulation Facilitates Axon Degeneration and NMJ Disruption via a Non–Cell-Autonomous Mechanism in ALS

    doi: 10.1523/JNEUROSCI.3037-17.2018

    Figure Lengend Snippet: miR126-5p is depleted in SOD1 G93A muscles and regulates Sema3 and NRP expression. A , NanoString chip screen heat map of significantly altered miRs in P60 muscles of SOD1 G93A compared with LM mice (extended table under ). Red and green represent a high or low abundance of miRs, respectively. * p < 0.05 (Student's t test; n = 3). B , miR126-5p was the most significantly downregulated miRNA in SOD1 G93A muscles (Student's t test; n = 3, ** p = 0.003). C , qPCR analysis of P60 GC muscle extracts further validates the decrease in miR 126-5p in SOD1 G93A ( n = 3). D–G , qPCR analysis of Sema3A, NRP1, Sema3B, and NRP2 transcript levels in HeLa cells overexpressing either miR126-5p or miR142 demonstrates a reduction in their expression levels specifically under miR126-5p overexpression (Student's t test; n = 3, * p = 0.0438, * p = 0.034, * p = 0.031, and * p = 0.0434, respectively). H , Representative TIRF images of U87MG cells reveal a detachment of the cell membrane from the culture dish surface after Sema3A is added to the culture medium. Scale bar, 10 μm. I , Impedance recording of live cells over time shows that U87MG cells overexpressing miR126-5p are unresponsive to Sema3A added to the culture medium because their impedance continuously increases, whereas the impedance of U87MG cells overexpressing miR142 decreases after treatment .

    Article Snippet: Antibodies were used at the following concentrations: anti-Neurofilament Heavy Chain 1:500 (NFH, Abcam, ab72996; 1:1000), synaptophysin (Millipore, MAB5258; 1:300), synaptotagmin (Alomone Labs, ant-003; 1:300), anti-NRP1 (1:100), anti-Sema3A (1:100), anti-NRP2 (1:100), and anti-Sema3B (1:100).

    Techniques: Expressing, Over Expression

    Overexpression of miR126-5p in primary SOD1 G93A myocytes blocks motor axon degeneration and preserves NMJ activity in a compartmental coculture. A , B , Western blot analysis of transfected myocyte extract overexpressing miR126-5p or miR142 and their CM validates miR126-5p as a regulator of Sema3A specifically in muscles. ERK was used as a loading control (Student's t test, n = 3, * p = 0.0499 and * p = 0.05, respectively). C , Schematic view of the experimental procedure in D , E . HB9::GFP SC explants and primary myocytes of SOD1 G93A mice were cocultured in a MFC. The growth of the HB9::GFP axons was assessed both by time-lapse imaging of the microgroove compartment and by imaging axons that traversed the distal compartment. D , Representative time-lapse images and quantification of HB9::GFP axon growth when cocultured with SOD1 miR126 myocytes (top) or SOD1 miR142 myocytes (bottom). SOD1 miR126 myocytes in the distal compartment enhanced the axonal traversal of the distal compartment compared with the SOD1 miR142 myocytes. The data are shown as the mean rate of axons that traversed the distal compartment out of the total number of axons in each field ± SEM (Student's t test, n = 3, * p = 0.04216). E , Representative time-lapse images and quantification of HB9::GFP axon growth when cocultured with PR 50 miR126 myocytes (top) or PR 50 miR142 myocytes (bottom). PR 50 miR126 myocytes in the distal compartment enhanced the axonal traversal of the distal compartment compared with PR 50 miR142 myocytes. The data are shown as the mean rate of axons that traversed the distal compartment out of the total number of axons in each field ± SEM (Student's t test, n = 3, *** p = 0.0039). F , Representative myocyte contraction plot showing the bursting contractile behavior of innervated myocytes in vitro . G , Quantification of the percentage of innervated myocytes that contract in a bursting pattern shows a diminished rate of bursting behavior in SOD1 G93A myocytes compared with LM controls. SOD1 miR126 myocytes show an increase in the rate of bursting myocytes back to the LM levels. The data are shown as the mean percentage of bursting myocytes ± SEM (Student's t test, n = 3, * p = 0.0291, * p = 0.0156, ** p = 0.005656). *** p < 0.001.

    Journal: The Journal of Neuroscience

    Article Title: miR126-5p Downregulation Facilitates Axon Degeneration and NMJ Disruption via a Non–Cell-Autonomous Mechanism in ALS

    doi: 10.1523/JNEUROSCI.3037-17.2018

    Figure Lengend Snippet: Overexpression of miR126-5p in primary SOD1 G93A myocytes blocks motor axon degeneration and preserves NMJ activity in a compartmental coculture. A , B , Western blot analysis of transfected myocyte extract overexpressing miR126-5p or miR142 and their CM validates miR126-5p as a regulator of Sema3A specifically in muscles. ERK was used as a loading control (Student's t test, n = 3, * p = 0.0499 and * p = 0.05, respectively). C , Schematic view of the experimental procedure in D , E . HB9::GFP SC explants and primary myocytes of SOD1 G93A mice were cocultured in a MFC. The growth of the HB9::GFP axons was assessed both by time-lapse imaging of the microgroove compartment and by imaging axons that traversed the distal compartment. D , Representative time-lapse images and quantification of HB9::GFP axon growth when cocultured with SOD1 miR126 myocytes (top) or SOD1 miR142 myocytes (bottom). SOD1 miR126 myocytes in the distal compartment enhanced the axonal traversal of the distal compartment compared with the SOD1 miR142 myocytes. The data are shown as the mean rate of axons that traversed the distal compartment out of the total number of axons in each field ± SEM (Student's t test, n = 3, * p = 0.04216). E , Representative time-lapse images and quantification of HB9::GFP axon growth when cocultured with PR 50 miR126 myocytes (top) or PR 50 miR142 myocytes (bottom). PR 50 miR126 myocytes in the distal compartment enhanced the axonal traversal of the distal compartment compared with PR 50 miR142 myocytes. The data are shown as the mean rate of axons that traversed the distal compartment out of the total number of axons in each field ± SEM (Student's t test, n = 3, *** p = 0.0039). F , Representative myocyte contraction plot showing the bursting contractile behavior of innervated myocytes in vitro . G , Quantification of the percentage of innervated myocytes that contract in a bursting pattern shows a diminished rate of bursting behavior in SOD1 G93A myocytes compared with LM controls. SOD1 miR126 myocytes show an increase in the rate of bursting myocytes back to the LM levels. The data are shown as the mean percentage of bursting myocytes ± SEM (Student's t test, n = 3, * p = 0.0291, * p = 0.0156, ** p = 0.005656). *** p < 0.001.

    Article Snippet: Antibodies were used at the following concentrations: anti-Neurofilament Heavy Chain 1:500 (NFH, Abcam, ab72996; 1:1000), synaptophysin (Millipore, MAB5258; 1:300), synaptotagmin (Alomone Labs, ant-003; 1:300), anti-NRP1 (1:100), anti-Sema3A (1:100), anti-NRP2 (1:100), and anti-Sema3B (1:100).

    Techniques: Over Expression, Activity Assay, Western Blot, Transfection, Imaging, In Vitro

    Alterations in Semaphorin3A regulation by miR126-5p trigger MN degeneration in ALS. miR126-5p is a negative regulator of Sema3 signaling in skeletal muscles. Downregulation of miR126-5p in ALS disease drives the overexpression and secretion of Sema3A and potentially other NMJ-destabilizing factors in skeletal muscles. The downregulation in miR126-5p in diseased MNs drives the overexpression of NRP1 specifically in axons. The excess binding and activation of the NRP1 receptor by its overexpressed ligand Sema3A as a result of miR126-5p alteration promote NMJ disruption and axon degeneration in a spatially confined process.

    Journal: The Journal of Neuroscience

    Article Title: miR126-5p Downregulation Facilitates Axon Degeneration and NMJ Disruption via a Non–Cell-Autonomous Mechanism in ALS

    doi: 10.1523/JNEUROSCI.3037-17.2018

    Figure Lengend Snippet: Alterations in Semaphorin3A regulation by miR126-5p trigger MN degeneration in ALS. miR126-5p is a negative regulator of Sema3 signaling in skeletal muscles. Downregulation of miR126-5p in ALS disease drives the overexpression and secretion of Sema3A and potentially other NMJ-destabilizing factors in skeletal muscles. The downregulation in miR126-5p in diseased MNs drives the overexpression of NRP1 specifically in axons. The excess binding and activation of the NRP1 receptor by its overexpressed ligand Sema3A as a result of miR126-5p alteration promote NMJ disruption and axon degeneration in a spatially confined process.

    Article Snippet: Antibodies were used at the following concentrations: anti-Neurofilament Heavy Chain 1:500 (NFH, Abcam, ab72996; 1:1000), synaptophysin (Millipore, MAB5258; 1:300), synaptotagmin (Alomone Labs, ant-003; 1:300), anti-NRP1 (1:100), anti-Sema3A (1:100), anti-NRP2 (1:100), and anti-Sema3B (1:100).

    Techniques: Over Expression, Binding Assay, Activation Assay