Journal: Molecular and cellular neurosciences

Article Title: Class 4 Semaphorins and Plexin-B receptors regulate GABAergic and glutamatergic synapse development in the mammalian hippocampus

doi: 10.1016/j.mcn.2018.06.008

Figure Lengend Snippet: (A) Excitatory synapse density in 14 DIV hippocampal neurons for a given condition normalized to the synapse density of the negative control condition. Synapse density is defined as the overlap of PSD95/Synapsin I puncta on GFP-transfected neurons. Neurons were co-transfected with a GFP expressing vector and either an empty vector control, a shRNA targeting Sema4A, a myc-tagged Sema4A cDNA, or co-transfection of myc-Sema4A RNAi resistant cDNA with a shRNA targeting Sema4A. In this case, the negative control is neurons co-transfected with an empty vector and a GFP-expressing vector. The dotted line at 100% indicates the control condition normalized to itself. (A, right) Representative stretches of GFP-transfected dendrite immunostained for PSD95 (red) and Synapsin I (magenta). (B) Excitatory synapse density in 14 DIV hippocampal neurons in response to Sema4A-Fc and Sema4D-Fc treatment normalized to the synapse density of the negative control condition. Synapse density is defined by the overlap of PSD95/Synapsin I puncta on MAP2-positive dendrites. In this case, the negative control is neurons treated with Fc control. The dotted line at 100% indicates the control condition normalized to itself. (B, right) Representative stretches of MAP2-positive dendrite immunostained for PSD95 (red), Synapsin I (magenta), and MAP2 (green). (C) Excitatory synapse density in 14 DIV hippocampal neurons for a given condition normalized to the synapse density of the negative control condition. Synapse density is defined as the overlap of PSD95/Synapsin I puncta on GFP-transfected neurons. Neurons were co-transfected with a GFP expressing vector and either an empty vector control, a shRNA targeting Rem2, a shRNA targeting Plexin-B1, or a shRNA targeting Plexin-B2. In this case, the negative control is neurons co-transfected with an empty vector and a GFP-expressing vector. The dotted line at 100% indicates the control condition normalized to itself. (A, right) Representative stretches of GFP-transfected dendrite immunostained for PSD95 (red) and Synapsin I (magenta). (D) Excitatory synapse density in 14 DIV hippocampal neurons in response to Sema4A-Fc normalized to the synapse density of the negative control condition. Synapse density is defined by the overlap of PSD95/Synapsin I puncta on GFP-transfected neurons. Neurons were co-transfected with a GFP expressing vector and either an empty vector control, a shRNA targeting Plexin-B1, or a shRNA targeting Plexin-B2 and were treated with either Fc control or Sema4A-Fc. In this case, the negative control is neurons treated with Fc control and co-transfected with an empty vector and a GFP-expressing vector (gray bar, far left). The dotted line at 100% indicates the control condition normalized to itself. The error for the negative control condition is contained within the error bar for each experimental condition (see Materials and Methods). (D, right) Representative stretches of GFP transfected dendrite immunostained for PSD95 (red) and Synapsin I (magenta). For treatments: 2nM Fc control, Sema4A-Fc or Sema4D-Fc were added to 13 DIV hippocampal neurons overnight (12–16hrs). n ≥ 55 neurons per condition from three experiments; n = 44 neurons for Rem2 RNAi. *p < 0.05, Two-Way ANOVA with Tukey’s post hoc Test. Error bars are ± standard error of the average ratio. Scale bars, 5µm.

Article Snippet: The following antibodies were used: ms α GAD65 (1:1000; Millipore Cat# MAB351, RRID:AB_2263126), rb α GABA A R γ2 , (1:150; Millipore Cat# AB5559, RRID:AB_177523), rb α Synapsin I (1:1000; Millipore Cat# AB1543, RRID:AB_2200400), ms α PSD95 (1:500; UC Davis/NIH NeuroMab Facility Cat# 75-028, RRID:AB_2307331), ms α MAP2, (1:1000; Sigma-Aldrich Cat# M4403, RRID:AB_477193), ms α 488 (1:1000; Jackson ImmunoResearch Labs Cat# 115-545-003, RRID:AB_2338840), ms α Cy3 (1:500; Jackson ImmunoResearch Labs Cat# 715-165-150, RRID:AB_2340813), and rb α Cy5 (1:500; Thermo Fisher Scientific Cat# A10523, RRID:AB_2534032).

Techniques: Negative Control, Transfection, Expressing, Plasmid Preparation, Control, shRNA, Cotransfection

Journal: The Journal of Biological Chemistry

Article Title: The ubiquitin-proteasome system functionally links neuronal Tomosyn-1 to dendritic morphology

doi: 10.1074/jbc.M117.815514

Figure Lengend Snippet: Tomo-1 localizes within postsynaptic compartments and is sensitive to shRNA-mediated knockdown. A, ICC of Tomo-1 (green) in HEK293T cells following expression of mCherry (mCH) (red) with (i) empty vector, (ii) Tomo-2, (iii) Tomo-1, or (iv) Tomo-1 (secondary antibody only). Scale bar = 10 μm. B, anti–Tomo-1 Western blot analysis of lysates from nontransfected HEK293T cells versus cells transfected with Tomo-1 or Tomo-2. C, representative ICC image of hippocampal neuron displaying merged fluorescence of endogenous Tomo-1 (green), PSD95 (red), and nuclei (blue, DAPI). Scale bar = 10 μm. D, representative intensity line scans of Tomo-1 (green) and PSD95 (red) fluorescence of an individual straightened dendrite indicate coincident immunofluorescence (lower plot). Merged Tomo-1 + PSD95 fluorescence (lower micrograph). px, pixels. E, cytofluorogram of Tomo-1 and PSD95 intensities from the dashed box region in D (Pearson's overlap coefficient, r = 0.885, r2 = 0.783; Manders' correlation coefficients, M1 = 0.759, representing fraction of PSD95 overlapping Tomo-1, M2 = 0.889, representing fraction of Tomo-1 overlapping PSD95). A.U., arbitrary units.

Article Snippet: Affinity-purified Rb anti-Tomosyn-1 polyclonal antibody (catalogue no. 183103), affinity-purified Rb anti-Tomosyn-2 polyclonal antibody (catalogue no. 183203), and the Ms anti-PSD95 monoclonal antibody (catalogue no. 124011) were from Synaptic Systems (Gottingen, Germany).

Techniques: shRNA, Knockdown, Expressing, Plasmid Preparation, Western Blot, Transfection, Fluorescence, Immunofluorescence

Journal: The Journal of Biological Chemistry

Article Title: The ubiquitin-proteasome system functionally links neuronal Tomosyn-1 to dendritic morphology

doi: 10.1074/jbc.M117.815514

Figure Lengend Snippet: The E3 ligase HRD1 is present throughout neuronal processes and interacts with Tomo-1. A, representative ICC image showing merged immunoreactive fluorescence of endogenous HRD1 (green), PSD95 (red), and nuclei (DAPI, blue) in cultured neurons. Scale bar = 10 μm. Note presence of HRD1 in dendrites. B, representative fluorescence intensity line scans of HRD1 (green) and PSD95 (red) of an individual straightened dendrite indicate coincident immunofluorescence (lower plot). Merged Tomo-1+PSD95 fluorescence is also shown (lower micrograph). px, pixels. C, cytofluorogram analysis of fluorescence intensity relationship between HRD1 and PSD95 from dashed box region on dendrite highlighted in B (Pearson's overlap coefficient, r = 0.437, r2 = 0.191; Manders' correlation coefficients, M1 = 0.724, representing fraction of PSD95 overlapping HRD1; M2 = 0.517, fraction of HRD1 overlapping PSD95), indicates a lack of specific co-localization. A.U., arbitrary units. D, representative Tomo-1 and HRD1 interaction assessed via proximity ligation analysis demonstrates substantive numbers of fluorescent puncta in somatic regions and along neuronal processes (12 DIV). Scale bar = 10 μm. Inset expands outlined region. PLA testing for interaction between Tomo-1 and the synaptic protein Munc18 (top) resulted in low levels of fluorescent puncta similar to secondary antibody treatment alone (not shown).

Article Snippet: Affinity-purified Rb anti-Tomosyn-1 polyclonal antibody (catalogue no. 183103), affinity-purified Rb anti-Tomosyn-2 polyclonal antibody (catalogue no. 183203), and the Ms anti-PSD95 monoclonal antibody (catalogue no. 124011) were from Synaptic Systems (Gottingen, Germany).

Techniques: Fluorescence, Cell Culture, Immunofluorescence, Ligation