Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Synaptic Gα12/13 signaling establishes hippocampal PV inhibitory circuits

doi: 10.1073/pnas.2407828121

Figure Lengend Snippet: The Gα12/13 pathway is essential for inhibitory synaptic function in hippocampal neurons. ( A ) representative primary hippocampal neuron immunolabeled for Gα13 together with HA-tagged endogenous LPHN3 and the somatodendritic marker MAP2. ( B-D ), colocalization of Gα13 with LPHN1-3 in primary hippocampal neurons. Primary neurons from mouse lines with endogenously tagged LPHN1-3 ( , , ) were colabeled with Gα13, MAP2, and Myc-LPHN1 ( B ), LPHN2-mVenus ( C ), or HA-LPHN3 ( D ). ( E – H ) primary neurons colabeled for Gα13 and Syn1/2 ( E ), vGAT ( F ), SHANK2 ( G ), or Homer1 ( H ). MAP2 was used as a somatodendritic marker. ( I ) validation of Gα12/13 KD and rescue system. Primary hippocampal neurons were infected with lentiviruses encoding indicated conditions together with mClover3 as a reporter and immunolabeled for Gα13 and MAP2. ( J – L ) mIPSC recordings from primary hippocampal neurons infected with lentiviruses expressing either Gα12/13 shRNA scramble (Ctl) or Gα12/13 shRNA (KD), together with mClover3. ( J ) representative mIPSC traces. ( K ) cumulative probability plot of interevent intervals and summary graph of the mean mIPSC frequency [ Inset ]. ( L ) cumulative probability plot and summary graph [ Inset ] of mIPSC amplitude measurements. ( M – O ), similar to ( J – L ), except for mEPSC measurements. Numerical data are cumulative histograms or means ± SEM. Statistical significance was determined via Kolmogorov–Smirnov test for cumulative histograms or two-tailed t test for summary graphs using the number of neurons as “n” values (*** P < 0.001; * P < 0.05). SI Appendix , Fig. S4 for colocalization measurements, additional validation of shRNA constructs, and additional electrophysiological parameters.

Article Snippet: The following antibodies and reagents were used: HA rabbit (Cell Signaling Technologies, cat# 3724), GFP rabbit (Life Technologies, cat# A11122), Myc rabbit (Sigma, cat# C3956), Homer1 rabbit (Synaptic Systems, cat# 160 003), MAP2 mouse (Sigma, cat# M1406), MAP2 chicken (EnCor Biotechnology, cat# CPCA-MAP2), SHANK2 guinea pig (Synaptic Systems, cat# 162204), Syn1/2 rabbit (Synaptic Systems, cat# 106 002), VGAT guinea pig (Synaptic Systems, cat# 131004), vGLUT1 guinea pig (Millipore, cat# AB5905), Gephyrin mouse (Synaptic Systems cat# 147111), GNA13 mouse (Life Technologies, cat# 67188), β-actin mouse (Sigma, A1978), Parvalbumin rabbit (SWANT, cat # PV27a), Alexa Fluor 647 Phalloidin (Invitrogen cat# A22287), and corresponding fluorescently-conjugated goat secondary antibodies from Life Technologies.

Techniques: Immunolabeling, Marker, Biomarker Discovery, Infection, Expressing, shRNA, Two Tailed Test, Construct

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Synaptic Gα12/13 signaling establishes hippocampal PV inhibitory circuits

doi: 10.1073/pnas.2407828121

Figure Lengend Snippet: Gα12/13 selectively regulates inhibitory synaptic density. ( A and B ) representative images ( A ) and quantifications ( B ) of neurons colabeled for vGAT, Gephyrin, and MAP2 in the indicated experimental conditions. ( C and D ) similar to A and B , except for neurons colabeled with SHANK2, Homer1, and MAP2. ( E and F ) representative spine images ( E ) and summary graphs ( F ) depicting spine density from Gα12 scramble (Ctl) or Gα12 shRNA (KD) conditions. ( G and H ) similar to E and F , except for Gα13 scramble (Ctl) or Gα13 shRNA (KD) conditions. ( I and J ) similar to E and F , except for double Gα12/13 scramble (Ctl) or Gα12/13 shRNA (KD) conditions. ( K ) representative neurons sparsely transfected with Gα12/13 control shRNAs, KD shRNAs, or rescue constructs that coexpress mClover3. ( L ) Sholl analysis of sparsely transfected neurons. Numerical data are means ± SEM from 4 to 5 independent biological replicates. Statistical significance was determined via one-way ANOVA with post hoc Tukey tests (* P < 0.05), two-tailed t test, or two-way ANOVA in Fig. 6 L . SI Appendix , Fig. S8 for additional morphological parameters.

Article Snippet: The following antibodies and reagents were used: HA rabbit (Cell Signaling Technologies, cat# 3724), GFP rabbit (Life Technologies, cat# A11122), Myc rabbit (Sigma, cat# C3956), Homer1 rabbit (Synaptic Systems, cat# 160 003), MAP2 mouse (Sigma, cat# M1406), MAP2 chicken (EnCor Biotechnology, cat# CPCA-MAP2), SHANK2 guinea pig (Synaptic Systems, cat# 162204), Syn1/2 rabbit (Synaptic Systems, cat# 106 002), VGAT guinea pig (Synaptic Systems, cat# 131004), vGLUT1 guinea pig (Millipore, cat# AB5905), Gephyrin mouse (Synaptic Systems cat# 147111), GNA13 mouse (Life Technologies, cat# 67188), β-actin mouse (Sigma, A1978), Parvalbumin rabbit (SWANT, cat # PV27a), Alexa Fluor 647 Phalloidin (Invitrogen cat# A22287), and corresponding fluorescently-conjugated goat secondary antibodies from Life Technologies.

Techniques: shRNA, Transfection, Control, Construct, Two Tailed Test

Journal: bioRxiv

Article Title: Impaired neurogenesis and synaptogenesis in iPSC-derived Parkinson’s patient cortical neurons with D620N VPS35 mutation

doi: 10.1101/2024.08.07.606995

Figure Lengend Snippet: ( a , d , g ) Dendritic filament length (sum), number of branching points and average numbers of dendrites intersecting Sholl circles up to 200 μm distance per subgroup over time. ( b, e, h ) Group comparison between control and PD patient cell-lines. ( c ) Representative fluorescence images for human autaptic neurons for each subgroup at 4 WID. ci and cii are control neurons and ciii and civ are neurons induced from PD cell-lines. For immunohistochemical staining, MAP2 (green) was used as dendritic, Synapsin1/2 (red) as presynaptic and Shank2 (blue) as postsynaptic marker.

Article Snippet: The primary antibodies used were: MAP2 (chicken, 1:1000, Novus Biological), SHANK2 (guinea pig, 1:250, Synaptic Systems), Synapsin 1/2 (rabbit, 1:500, Synaptic Systems).

Techniques: Comparison, Control, Fluorescence, Immunohistochemical staining, Staining, Marker

Journal: bioRxiv

Article Title: Impaired neurogenesis and synaptogenesis in iPSC-derived Parkinson’s patient cortical neurons with D620N VPS35 mutation

doi: 10.1101/2024.08.07.606995

Figure Lengend Snippet: (a) Development of the number of synapses per subgroup over time. ( b ) Group comparison of controls vs PD patient cell-lines. ( c, d ) Representative fluorescence images for human autaptic neurons for each subgroup at 4 WID. Synapse number were counted by colocalizing Synapsin1/2 puncta (presynaptic marker, red) and Shank2 puncta (postsynaptic marker, blue) within proximity to dendrites (MAP2, green). Control cell ( c ) and PD patient cell ( d ) with magnification of synapse: i MAP2, Synapsin 1/2, Shank2, ii Synapsin 1/2, Shank2; iii Synapsin 1/2; iv Shank2.

Article Snippet: The primary antibodies used were: MAP2 (chicken, 1:1000, Novus Biological), SHANK2 (guinea pig, 1:250, Synaptic Systems), Synapsin 1/2 (rabbit, 1:500, Synaptic Systems).

Techniques: Comparison, Fluorescence, Marker, Control