Journal: Life Science Alliance

Article Title: SorCS1 inhibits amyloid-β binding to neurexin and rescues amyloid-β–induced synaptic pathology

doi: 10.26508/lsa.202201681

Figure Lengend Snippet: (A) Representative images showing the results of cell surface–binding assay testing for interaction between SorCS1-Fc and known synaptic organizers. SorCS1-Fc (1 µM) was added to COS-7 cells expressing the indicated construct. Note that SorCS1-Fc binds to COS-7 cells expressing HA-NRX1βS4(−), but not to those expressing any of the other organizers. For the N-terminal extracellular HA-tagged constructs, surface HA was immunostained to verify the expression of the construct on the COS-7 cell surface. Scale bars: 30 µm. (B) Representative images showing the binding of SorCS1-Fc (1 µM) to COS-7 cells expressing the indicated isoform of extracellularly HA-tagged NRX constructs. S4(+) and S4(−) indicate with and without an insert at splicing site 4, respectively, and ΔHRD indicates lack of the N-terminal histidine-rich domain (HRD) of β-NRX. HA fluorescent signals correspond to surface HA. Scale bar: 30 µm. (C) Quantification of bound SorCS1-Fc for each NRX construct. n = 30 cells for each construct from three independent experiments, one-way ANOVA, P < 0.0001. *** P < 0.001 compared with HA-CD4 and § P < 0.001 between NRXβ and NRXβΔHRD by Tukey’s multiple comparisons test. Data are presented as mean ± SEM. (D) Pull-down assays of purified recombinant His-tagged SorCS1 ectodomain protein with Fc, NRX1βS4(−)-Fc, or NRX1βS4(−)ΔHRD-Fc proteins indicate that the SorCS1 ectodomain and the NRX1β ectodomain form a complex when the NRX1β HRD is present.

Article Snippet: The pre-immobilized beads were then incubated with 50 nM recombinant mouse SorCS1 ectodomain tagged with a C-terminal 6-His tag (SorCS1-His, Cat# 4395-SR-050; R&D systems) in ECS for 2 h at 4°C.

Techniques: Binding Assay, Expressing, Construct, Purification, Recombinant

Journal: Life Science Alliance

Article Title: SorCS1 inhibits amyloid-β binding to neurexin and rescues amyloid-β–induced synaptic pathology

doi: 10.26508/lsa.202201681

Figure Lengend Snippet: Immunoblotting with anti-His tag antibody (left) and with anti-Fc antibody (right) on the same blot. The boxed regions were cropped to prepare the representative images shown in . Note that in lane 6, the anti-His antibody not only detected the SorCS1-His band at around 150 kD, but also an extra-band at around 60 kD, which corresponds to the size of NRX1β-Fc (lane 6 in the right hand panel). On the other hand, the anti-His antibody did not detect any band between 50–75 kD in lane 7, which was loaded with NRX1βΔHRD-Fc. These results suggest that the anti-His antibody can recognize the HRD of NRX1β and confirm that the Fc protein loaded in the lane 7 is lacking the HRD. Unexpectedly, the migration of NRX1βΔHRD-Fc on SDS–PAGE was slower than that of NRX1β-Fc, presumably because of the phenomenon called as “gel shifting.”

Article Snippet: The pre-immobilized beads were then incubated with 50 nM recombinant mouse SorCS1 ectodomain tagged with a C-terminal 6-His tag (SorCS1-His, Cat# 4395-SR-050; R&D systems) in ECS for 2 h at 4°C.

Techniques: Western Blot, Migration, SDS Page

Journal: Life Science Alliance

Article Title: SorCS1 inhibits amyloid-β binding to neurexin and rescues amyloid-β–induced synaptic pathology

doi: 10.26508/lsa.202201681

Figure Lengend Snippet: (A) Representative images of triple-labelling for bound biotin-AβOs, bound SorCS1-Fc, and surface HA of COS-7 cells expressing extracellularly HA-tagged NRX1βS4(−). Recombinant biotin-AβOs (100 nM, monomer equivalent) with/without SorCS1-Fc (1,500 nM) were extracellularly applied to COS-7 cells expressing HA-NRX1βS4(−). (B) Quantification of biotin-AβOs bound to COS-7 cells expressing HA-NRX1βS4(−) in the presence of various concentrations of SorCS1-Fc (0–1,500 nM). The half maximal inhibitory concentration (IC 50 ) is 92.8 nM. (C) Representative images of triple-labelling for bound SorCS1-Fc, bound biotin-AβOs, and surface HA of COS-7 cells expressing HA-NRX1βS4(−). SorCS1-Fc (250 nM) with/without biotin-AβOs (2,000 nM, monomer equivalent) was applied to COS-7 cells expressing HA-NRX1βS4(−). (D) Quantification of SorCS1-Fc bound to COS-7 cells expressing HA-NRX1βS4(−) in the presence of various concentrations of biotin-AβOs (0–2,000 nM, monomer equivalent). (E) Representative images of triple-labelling for bound biotin-AβOs, surface HA, and total myc (surface and intracellular myc both) of COS-7 cells co-expressing HA-NRX1βS4(−) with either intracellularly myc-tagged-SorCS1b (SorCS1b-myc) or SorCS1b-myc lacking the VPS10 domain (SorCS1bΔVPS10-myc). Biotin-AβOs (250 nM, monomer equivalent) were applied to COS-7 cells co-expressing HA-NRX1βS4(−) with SorCS1b-myc or SorCS1bΔVPS10-myc. COS-7 cells expressing HA-CD4 were used as a negative control. (F) Quantification of biotin-AβOs bound to COS-7 cells co-expressing HA-NRX1βS4(−) with SorCS1b-myc or SorCS1bΔVPS10-myc. (G) Representative images of triple-labelling for bound NLGN1-Fc, surface HA, and total myc of COS-7 cells co-expressing HA-NRX1βS4(−) with SorCS1b-myc or SorCS1bΔVPS10-myc. NLGN1-Fc (20 nM) was applied to COS-7 cells expressing the indicated constructs. (H) Quantification of NLGN1-Fc bound to COS-7 cells co-expressing HA-NRX1βS4(−) with SorCS1b-myc or SorCS1bΔVPS10-myc. (I) Representative images obtained in protein-clustering assays using neuroligin 1 (NLGN1)-Fc–coated beads or Fc-coated beads (a negative control) in cultures of hippocampal neurons (DIV21) co-transfected with either SorCS1b-IRES-GFP and HA-NRX1βS4(−), SorCS1b-IRES-GFP and HA-NRX1βS4(−)ΔHRD, or SorCS1bΔVPS10-IRES-GFP and HA-NRX1βS4(−). (J, K) Quantification of the average intensity of HA-NRX1β or HA-NRX1βΔHRD (J) and SorCS1b or SorCS1bΔVPS10 (K) around beads coated with NLGN1-Fc or Fc. n = 30 cells for each condition from three independent experiments for (B, D, F, H) and n > 100 beads for each condition from three independent experiments for (J, K), one-way ANOVA, P < 0.0001. *** P < 0.001, * P < 0.05, N.S., not significant by Dunnett’s test compared with the 0 nM control condition for (B, D) and Tukey’s multiple comparisons test for (F, H, J, K). Data are presented as mean ± SEM. Scale bar: 30 µm (A, C, E, G) and 5 µm (I).

Article Snippet: The pre-immobilized beads were then incubated with 50 nM recombinant mouse SorCS1 ectodomain tagged with a C-terminal 6-His tag (SorCS1-His, Cat# 4395-SR-050; R&D systems) in ECS for 2 h at 4°C.

Techniques: Expressing, Recombinant, Concentration Assay, Negative Control, Construct, Transfection, Control

Journal: Life Science Alliance

Article Title: SorCS1 inhibits amyloid-β binding to neurexin and rescues amyloid-β–induced synaptic pathology

doi: 10.26508/lsa.202201681

Figure Lengend Snippet: (A) Representative images showing immunoreactivity for total myc (surface and intracellular myc both) and surface SorCS1 in COS-7 cells transfected with extracellularly myc-tagged CD4 (myc-CD4, top panels) or intracellularly myc-tagged SorCS1b (SorCS1b-myc, bottom panels). Immunostaining for surface SorCS1 and total myc was performed before and after cell permeabilization, respectively. The SorCS1 antibody that we used recognizes an epitope in the N-terminal extracellular region of SorCS1, allowing us to label SorCS1 expressed on the cell surface. Surface SorCS1 signal was detected only in COS cells transfected with SorCS1b-myc, but not myc-CD4. (B) Representative images showing surface myc and surface SorCS1 in the indicated transfection conditions. Immunostaining for surface myc and surface SorCS1 was performed without cell permeabilization. Without permeabilization, the intracellular myc tag of SorCS1b-myc was not detected (left bottom), indicating that our protocol successfully prevents antibody internalization and recognition of intracellular epitopes. (A, B) Thus, the SorCS1 immunoreactivity detected without cell permeabilization (bottom-right in [B] and bottom-right in [A]) indeed comes from surface SorCS1b rather than intracellular SorCS1b. (C) A representative image showing immunoreactivity for total myc and surface SorCS1 in COS-7 cells transfected with intracellularly myc-tagged SorCS1b lacking the VPS10 domain (SorCS1bΔVPS10-myc). Surface SorCS1 signal was detected in COS cells transfected with SorCS1bΔVPS10-myc, indicating that SorCS1bΔVPS10-myc can be targeted to the cell membrane. Thus, the SorCS1 antibody that we used recognizes an epitope in the SorCS1 extracellular region outside the VPS10 domain. Scale bars: 30 µm.

Article Snippet: The pre-immobilized beads were then incubated with 50 nM recombinant mouse SorCS1 ectodomain tagged with a C-terminal 6-His tag (SorCS1-His, Cat# 4395-SR-050; R&D systems) in ECS for 2 h at 4°C.

Techniques: Transfection, Immunostaining, Membrane

Journal: Life Science Alliance

Article Title: SorCS1 inhibits amyloid-β binding to neurexin and rescues amyloid-β–induced synaptic pathology

doi: 10.26508/lsa.202201681

Figure Lengend Snippet: (A, B) Representative images of cultured hippocampal neurons (DIV21) transfected with the IRES-GFP or SorCS1b-IRES-GFP expression vectors followed by immunostaining of surface SorCS1 and MAP2 before and after cell permeabilization, respectively. The GFP and MAP2 signals were used to distinguish axons (GFP-positive but MAP-negative neurites, arrowheads in (A)) from dendrites (GFP- and MAP2-positive neurites). Immunoreactivity for surface SorCS1 was detected in both axons and dendrites of neurons transfected with SorCS1b-IRES-GFP, but not in those transfected with IRES-GFP. (C) Representative images showing the axons of cultured hippocampal neurons (DIV21) co-transfected with SorCS1b-IRES-GFP and HA-NRX1βS4(−) (left), SorCS1b-IRES-GFP and HA-NRX1βS4(−)ΔHRD (middle), or SorCS1bΔVPS10-IRES-GFP and HA-NRX1βS4(−) (right) and immunostained for surface SorCS1 and surface HA before permeabilization and MAP2 after permeabilization. SorCS1b is nicely colocalized with HA-NRX1β (left), but not HA-NRX1βΔHRD (middle), especially at the contact sites between GFP-expressing axons and dendrites (MAP2-positive neurites) (arrows). (D) Quantification of colocalization between the indicated proteins using Pearson’s correlation coefficients. n = 30 cells for each condition from three independent experiments, one-way ANOVA, P < 0.0001, and *** P < 0.001 by Tukey’s multiple comparisons test. Scale bar: 30 µm (A) and 10 µm (B, C).

Article Snippet: The pre-immobilized beads were then incubated with 50 nM recombinant mouse SorCS1 ectodomain tagged with a C-terminal 6-His tag (SorCS1-His, Cat# 4395-SR-050; R&D systems) in ECS for 2 h at 4°C.

Techniques: Cell Culture, Transfection, Expressing, Immunostaining

Journal: Life Science Alliance

Article Title: SorCS1 inhibits amyloid-β binding to neurexin and rescues amyloid-β–induced synaptic pathology

doi: 10.26508/lsa.202201681

Figure Lengend Snippet: Representative low-magnification images showing VGLUT1 accumulation induced by NLGN1-Fc–coated beads at contacting axons. NLGN1-coated breads were exposed to cultured hippocampal neurons transfected with IRES-GFP, SorCS1b-IRES-GFP, or SorCS1bΔVPS10-IRES-GFP with AβO treatment (500 nM monomer equivalent, 24 h) or with vehicle treatment. The neurons were immunostained for VGLUT1, an excitatory presynaptic maker, and MAP2, a dendrite marker. For quantification, the beads contacting axons (GFP-positive and MAP2-negative neurites) (arrowheads) were selected. At contacting axons of IRES-GFP–transfected neurons, NLGN1-Fc–coated beads significantly induced VGLUT1 accumulation, and AβO treatment seemed to suppress the VGLUT1 accumulation. In contrast, it seems that AβO treatment failed to suppress NLGN1-induced VGLUT1 accumulation at contacting axons transfected with SorCS1-IRES-GFP but not those transfected with SorCS1bΔVPS10-IRES-GFP. These qualitative results are consistent with the quantitative results shown in . Scale bar: 15 µm.

Article Snippet: The pre-immobilized beads were then incubated with 50 nM recombinant mouse SorCS1 ectodomain tagged with a C-terminal 6-His tag (SorCS1-His, Cat# 4395-SR-050; R&D systems) in ECS for 2 h at 4°C.

Techniques: Cell Culture, Transfection, Marker

Journal: Life Science Alliance

Article Title: SorCS1 inhibits amyloid-β binding to neurexin and rescues amyloid-β–induced synaptic pathology

doi: 10.26508/lsa.202201681

Figure Lengend Snippet: (A) Representative images of cultured hippocampal neurons co-transfected with SorCS1b-IRES-GFP and HA-NRX1βS4(−) and exposed to AβOs (500 nM monomer equivalent) or vehicle treatment for 24 h. The neurons were immunostained for surface SorCS1 and surface HA before cell permeabilization and for MAP2 after permeabilization. Colocalization between SorCS1b and NRX1βS4(−) on the axon surface was maintained even after the AβO treatment, which was the same treatment condition as used in the other neuron-based AβO experiments in this study. (B) Quantification of colocalization between SorCS1 and HA-NRX1βS4(−) with or without AβOs. The AβO treatment did not affect the colocalization. n = 31 cells for vehicle and 22 cells for AβO-treated neurons from one independent experiment, t test, N.S., not significant. Data are presented as mean ± SEM. Scale bar: 30 µm.

Article Snippet: The pre-immobilized beads were then incubated with 50 nM recombinant mouse SorCS1 ectodomain tagged with a C-terminal 6-His tag (SorCS1-His, Cat# 4395-SR-050; R&D systems) in ECS for 2 h at 4°C.

Techniques: Cell Culture, Transfection

Journal: Life Science Alliance

Article Title: SorCS1 inhibits amyloid-β binding to neurexin and rescues amyloid-β–induced synaptic pathology

doi: 10.26508/lsa.202201681

Figure Lengend Snippet: (A) Representative images of cultured hippocampal neurons (DIV21) transfected with the SorCS1bΔVPS10-IRES-GFP expression vector followed by immunostaining of surface SorCS1 and MAP2 before and after cell permeabilization, respectively. The GFP and MAP2 signals were used to distinguish axons (GFP-positive but MAP2-negative neurites, arrowheads) from dendrites (GFP- and MAP2-positive neurites). Immunoreactivity for surface SorCS1 was detected in both axons and dendrites of neurons transfected with SorCS1b ΔVPS10-IRES-GFP. (B) Quantification of the average intensity of surface SorCS1 on axons of neurons transfected with SorCS1bΔVPS10-IRES-GFP and SorCS1b-IRES-GFP. Deletion of the VPS10 domain does not affect the surface expression level of SorCS1b. n = 30 cells for SorCS1bΔVPS10-IRES-GFP and 32 cells for SorCS1b-IRES-GFP from three independent experiments, t test, N.S., not significant. Data are presented as mean ± SEM. Scale bar: 30 µm.

Article Snippet: The pre-immobilized beads were then incubated with 50 nM recombinant mouse SorCS1 ectodomain tagged with a C-terminal 6-His tag (SorCS1-His, Cat# 4395-SR-050; R&D systems) in ECS for 2 h at 4°C.

Techniques: Cell Culture, Transfection, Expressing, Plasmid Preparation, Immunostaining

Journal: Psychopharmacology

Article Title: Perseveration in a spatial-discrimination serial reversal learning task is differentially affected by MAO-A and MAO-B inhibition and associated with reduced anxiety and peripheral serotonin levels

doi: 10.1007/s00213-017-4569-x

Figure Lengend Snippet: Effects of selective MAO inhibition on monoamine levels in a OFC, b DRN, c BLA, and d dorsomedial striatum (pmol/mg tissue). In c and d , dopamine levels are shown on the left y -axis while NA and 5-HT levels are shown on the right y -axis. Data are mean values ± SEM. Significance is denoted as follows: * p < 0.05, ** p < 0.005 versus vehicle, + p < 0.05 versus lazabemide. e Coronal sections showing regions of interest for ex vivo neurochemical analysis of monoamines following vehicle, moclobemide, and lazabemide administration. dmPFC dorsomedial PFC, OFC orbitofrontal cortex, dmS dorsomedial striatum, NAcb nucleus accumbens, BLA basolateral amygdala, CA1 hippocampal CA1 region, LH lateral hypothalamus, DRN dorsal raphé nuclei. Adapted from Paxinos and Watson

Article Snippet: Moclobemide and lazabemide hydrochloride were purchased from Tocris (UK) and dissolved in 15% hydroxypropyl-beta-cyclodextrin and 0.9% saline (“vehicle”).

Techniques: Inhibition, Ex Vivo

Journal: Psychopharmacology

Article Title: Perseveration in a spatial-discrimination serial reversal learning task is differentially affected by MAO-A and MAO-B inhibition and associated with reduced anxiety and peripheral serotonin levels

doi: 10.1007/s00213-017-4569-x

Figure Lengend Snippet: Final group sizes for animals that successfully completed the task under drug conditions

Article Snippet: Moclobemide and lazabemide hydrochloride were purchased from Tocris (UK) and dissolved in 15% hydroxypropyl-beta-cyclodextrin and 0.9% saline (“vehicle”).

Techniques:

Journal: Psychopharmacology

Article Title: Perseveration in a spatial-discrimination serial reversal learning task is differentially affected by MAO-A and MAO-B inhibition and associated with reduced anxiety and peripheral serotonin levels

doi: 10.1007/s00213-017-4569-x

Figure Lengend Snippet: Effects of moclobemide ( n = 18) and lazabemide ( n = 21) on total trials to achieve criterion ( a , b ) and the proportion of perseverative errors ( c , d ). Mean values ± SEM for a single post drug administration session are shown. Significance is denoted as follows: * p < 0.05, ** p < 0.01, *** p < 0.001 versus vehicle

Article Snippet: Moclobemide and lazabemide hydrochloride were purchased from Tocris (UK) and dissolved in 15% hydroxypropyl-beta-cyclodextrin and 0.9% saline (“vehicle”).

Techniques:

Journal: Psychopharmacology

Article Title: Perseveration in a spatial-discrimination serial reversal learning task is differentially affected by MAO-A and MAO-B inhibition and associated with reduced anxiety and peripheral serotonin levels

doi: 10.1007/s00213-017-4569-x

Figure Lengend Snippet: Effects of moclobemide ( n = 16) and lazabemide ( n = 20) on response latencies (s) following an incorrect ( a , b ) and correct ( c , d ) response. Data for two animals was not saved due to a technical failure with the equipment. * p < 0.05, ** p < 0.01, *** p < 0.001 versus vehicle

Article Snippet: Moclobemide and lazabemide hydrochloride were purchased from Tocris (UK) and dissolved in 15% hydroxypropyl-beta-cyclodextrin and 0.9% saline (“vehicle”).

Techniques:

Journal: Psychopharmacology

Article Title: Perseveration in a spatial-discrimination serial reversal learning task is differentially affected by MAO-A and MAO-B inhibition and associated with reduced anxiety and peripheral serotonin levels

doi: 10.1007/s00213-017-4569-x

Figure Lengend Snippet: Lose-shift probabilities for high ( n = 5) and low ( n = 5) perseveration rats that received moclobemide (16 mg/kg, 3 mg/kg), combination of lazabemide and moclobemide, and vehicle

Article Snippet: Moclobemide and lazabemide hydrochloride were purchased from Tocris (UK) and dissolved in 15% hydroxypropyl-beta-cyclodextrin and 0.9% saline (“vehicle”).

Techniques:

Journal: Psychopharmacology

Article Title: Perseveration in a spatial-discrimination serial reversal learning task is differentially affected by MAO-A and MAO-B inhibition and associated with reduced anxiety and peripheral serotonin levels

doi: 10.1007/s00213-017-4569-x

Figure Lengend Snippet: Levels of DA and 5-HT in regions of interest following vehicle (Veh, n = 5), lazabemide (L10, n = 6), and moclobemide (M3, n = 4; M16, n = 4) administration

Article Snippet: Moclobemide and lazabemide hydrochloride were purchased from Tocris (UK) and dissolved in 15% hydroxypropyl-beta-cyclodextrin and 0.9% saline (“vehicle”).

Techniques: