Journal: Neurobiology of aging

Article Title: Chronic Gq activation of ventral hippocampal neurons and astrocytes differentially affects memory and behavior.

doi: 10.1016/j.neurobiolaging.2023.01.007

Figure Lengend Snippet: Fig. 1. Chronic activation of Gq pathways in CaMKII+ neurons and GFAP+ astrocytes in vCA1. (A) Viral strategy schematic for the neuronal groups. AAV5-CaMKII- hM3D(Gq)-mCherry or control vector AAV5-CaMKII-mCherry was bilaterally injected into the vCA1 region of wild type mice. (B) Representative images of RFP/NeuN + co-staining (red and green, respectively) and DAPI + cells (blue). (C) Viral strategy schematic for the astrocyte groups. AAV9-GFAP-hM3D(Gq)-mCherry or control vector AAV5-GFAP-mCherry was bilaterally injected into the vCA1 region of wild type mice. (D) Representative images of RFP/GFAP + co-staining (red and green, respectively) and DAPI + cells (blue). (E) Schematic representation of chronically activating neuron or astrocyte Gq receptors through administration of the water-soluble DREADD ligand de- schloroclozapine dihydrochloride (DCZ) for either 3, 6, or 9 months. Mice underwent a battery of behavioral tests at each end point (F-I) Histological assessment of NeuN + cells to determine whether our manipulations were killing vCA1 neurons in the CaMKII groups (F and G) and/or GFAP groups (H and J). NeuN counts were assessed with a two-way analysis of variance (ANOVA) with time point and group as factors. Error bars indicate SEM. p ≤0.05, ∗∗p ≤0.01, ∗∗∗p ≤0.001, ∗∗∗∗p ≤0.0 0 01, ns = not significant. Per group: n = 3 mice x 18 tiles (region of interest (ROI): vCA1) each were quantified for statistical analysis of NeuN counts. Scale bars indicate 50 micrometers.

Article Snippet: SOURCE IDENTIFIER Experimental models: Organisms/Strains Wild-type male C57Bl/6 Charles River Labs C57BL/6 Antibodies Rabbit polyclonal anti-Iba1/AIF1 SySy 234 003 (catalog #) Mouse monoclonal anti-GFAP NeuroMab 75–240 (catalog #) Guinea polyclonal anti-NeuN/Fox3 SySy 266–004 (catalog #) Rabbit polyclonal anti-cFos SySy 226–008 (catalog #) Rabbit polyclonal anti-RFP Rockland 600–401-379-RTU Goat anti rabbit Alexa Fluor 488 Secondary antibody Invitrogen A-11008 (catalog #) Goat anti-guinea pig Alexa Fluor 488 Secondary antibody Invitrogen A-11073 (catalog #) Goat anti-mouse Alexa Fluor 488 Secondary antibody Invitrogen A-11001 (catalog #) Goat anti-rabbit Alexa Fluor 555 Secondary antibody Invitrogen A27039 (catalog #) Chemicals Triton X-100 FisherScientific NC0478124 Paraformaldehyde (PFA) Sigma-Aldrich 158127–3KG Bovine serum albumin (BSA) Sigma-Aldrich B4287–25G Deschloroclozapine HelloBio HB8555 Deschloroclozapine dihydrochloride HelloBio HB9126 Vectashield Hard Set Mounting Medium with DAPI Vector Laboratories, Inc. H-1500-10 Software/Programs Fiji (ImageJ) https://fiji.sc/ FreezeFrame4 Coulbourn Instruments AnyMaze Stoelting Co. Prism GraphPad Software v9.4.1 Ilastik Berg et al. (2019) 3DMorph York et al. (2018) Viral constructs AAV9-CaMKII-hM3Dq-mCherry AddGene 50476–AAV9 AAV9-CaMKII-mCherry AddGene 114469–AAV9 AAV5-GFAP-hM3Dq-mCherry AddGene 50487–AAV5 AAV5-GFAP-hM3Dq-mCherry AddGene 58909–AAV5

Techniques: Activation Assay, Control, Plasmid Preparation, Injection, Staining, Battery

Journal: Translational Psychiatry

Article Title: Impairment in stimulus-response learning as a cognitive biomarker in a model of synucleinopathy

doi: 10.1038/s41398-025-03795-5

Figure Lengend Snippet: A Schematic of α-synuclein pre-formed fibril production. α-synuclein type 1 pre-formed fibrils (PFF-1) were generated from monomers expressed in E.coli at room temperature overnight, while α-synuclein type 2 pre-formed fibrils (PFF-2) were generated from monomers expressed in E.coli grown at 37 °C for four hours. Following this, both fibril precursors underwent monomer purification and identical fibrilization protocols and were characterized in vitro. B SDS-PAGE of PFF-1 and PFF-2 after sedimentation revealed similar populations of insoluble (pellet) vs soluble (supernatant) materials, both consisting of predominantly insoluble species. C Transmission Electron Microscopy (TEM) of unsonicated PFF-1 and PFF-2 displayed similar architecture and range of fibril sizes, both appearing as elongated fibrils of mixed length. D Far UV circular dichroism (UV-CD) measurements identified a significant β-sheet presence in fibrils compared monomers with subtle differences between PFF-1 and PFF-2s. Thioflavin T (ThT) binding assay revealed a stronger binding capacity of PFF-1 (E) relative to PFF-2 (F) after fibrilization and during a seeding assay with additional monomer.

Article Snippet: Human aSyn PFF-1 and PFF-2 are available from a commercial source (StressMarq Biosciences Cat# SPR-322 and SPR-317, respectively), which are expressed, purified, and fibrilized using methods previously described [ , ].

Techniques: Generated, Purification, In Vitro, SDS Page, Sedimentation, Transmission Assay, Electron Microscopy, Circular Dichroism, Binding Assay

Journal: Translational Psychiatry

Article Title: Impairment in stimulus-response learning as a cognitive biomarker in a model of synucleinopathy

doi: 10.1038/s41398-025-03795-5

Figure Lengend Snippet: A M83 mice underwent stereotaxic surgery for inoculation of aSyn PFF-1, aSyn PFF-2 or PBS control into the right dorsal striatum. At 8 weeks post injection, they subsequently underwent a motor test battery, including assessments of motor strength, motor coordination and gait. Following completion, they were assessed for the cognitive ability to acquire stimulus-response associations, with the ‘Visuomotor Conditional Learning’ at 9-12 weeks post injection. At 16 weeks post-injection, they repeated the motor test battery, and then were processed for pathology and biochemistry. B Brain homogenates from aSyn PFF-1 and PFF-2 inoculated mice both displayed resistance to proteinase K but exhibited variable banding patterns upon digestion, as assessed by Western Blot using an antibody for total aSyn. C, D Furthermore, the same brain homogenates were evaluated by dotblots using an antibody with high affinity for aggregated aSyn (chBIIB054), revealing that while both fibril types exhibited significant binding, aSyn PFF-2 brain homogenates presented with a stronger signal (Welch’s ANOVA test, W 2, 5.34 = 7.03, p < 0.05, in which Welch-corrected unpaired t-tests revealed to be driven by a significant difference between PBS control and PFF-2 (p < 0.05). Graphics made with Biorender.com.

Article Snippet: Human aSyn PFF-1 and PFF-2 are available from a commercial source (StressMarq Biosciences Cat# SPR-322 and SPR-317, respectively), which are expressed, purified, and fibrilized using methods previously described [ , ].

Techniques: Control, Injection, Battery, Western Blot, Binding Assay

Journal: Translational Psychiatry

Article Title: Impairment in stimulus-response learning as a cognitive biomarker in a model of synucleinopathy

doi: 10.1038/s41398-025-03795-5

Figure Lengend Snippet: A aSyn PFF-1, PFF-2 or PBS control was unilaterally injected into the right dorsal striatum. Brains for immunohistochemistry (B, C) and immunoblots (D-I) were collected at 16 weeks post injection. B Immunostained brain slices imaged at 63X on a Leica Stellaris 5 Confocal Microscope for aSyn phosphorylated at S129 (green= pS129; blue= Hoescht), scale bar 25 µm. C Although inoculation was localized within the dorsal striatum, pathology spread to many areas including orbitofrontal cortex, piriform cortex, medulla, and substantia nigra. 40X, Leica Stellaris 5 Confocal Microscope, scale bar 50 µm. D–I To quantify this pathology further, protein extract from the striatum of PFF- and PBS-inoculated mice were extracted and quantified with immunoblots. D–F The levels of pS129 and total human aSyn found in the detergent-soluble (RIPA) fractions from PFF-1 and PFF-2 samples were expressed as the percentage fold change relative to PBS samples. No significant differences were found between PFF-1 and PFF-2 when comparing pS129 (unpaired t-test, p > 0.05) or human aSyn (unpaired t-test, p > 0.05). G–I The levels of pS129 and total human aSyn found in the detergent-insoluble fractions (UREA solubilization of RIPA-insoluble pellets) were expressed in the same way as the detergent-soluble fractions (RIPA). No significant differences were found between PFF-1 and PFF-2 when comparing pS129 (unpaired t-test, p > 0.05) or human aSyn (unpaired t-test, p > 0.05). Graphics made with Biorender.com.

Article Snippet: Human aSyn PFF-1 and PFF-2 are available from a commercial source (StressMarq Biosciences Cat# SPR-322 and SPR-317, respectively), which are expressed, purified, and fibrilized using methods previously described [ , ].

Techniques: Control, Injection, Immunohistochemistry, Western Blot, Microscopy

Journal: Translational Psychiatry

Article Title: Impairment in stimulus-response learning as a cognitive biomarker in a model of synucleinopathy

doi: 10.1038/s41398-025-03795-5

Figure Lengend Snippet: A Schematic of the forelimb grip force test, used to measure muscle strength. Subjects grasped a smooth, triangular pull bar with both forelimbs and the force exerted in Newtons (N) was measured. B aSyn PFF-2 but not PFF-1 inoculated mice displayed a significant reduction in forelimb muscle strength at 16 weeks post injection (WPI) relative to 8 (2-way RM ANOVA, Group x Session: main effect of group (F 2,72 = 4.409, p < 0.05), main effect of session (F 1,72 = 13.51, p < 0.001), and significant interaction (F 2,72 = 6.677, p < 0.01), in which Šídák’s multiple comparisons revealed to be driven by a significant difference within between 8 and 16 weeks within the PFF-2 group (adj. p < 0.0001). C Schematic of the accelerating Rota-rod, used to measure motor coordination and motor learning. Subjects were placed on a stationary rod which began to accelerate linearly from 5-36 revolutions per minute over 5 min, and latency to fall (s) was calculated automatically. D No significant difference between groups was found at 8 WPI (2-way RM ANOVA, Group x Session: main effect of session (F 9,675 = 15.41, p < 0.0001), but no main effect of group (p > 0.05). A significant interaction was first observed, (F 18, 675 = 1.767, p < 0.05), but Šídák’s multiple comparisons revealed no significant differences between groups (adj. p > 0.05)). However, (E) aSyn PFF-2 mice exhibited a shorter latency to fall at 16 WPI relative to aSyn PFF-1 and PBS controls (2-way RM ANOVA, Group x Session: Main effect of group (F 2,71 = 7.436, p < 0.01), main effect of session (F 9,639 = 3.205, p < 0.001), but no interaction (p > 0.05). To examine this main effect further, Šídák’s multiple comparisons revealed a significant difference between aSyn PFF-2 and PBS controls (adj. p < 0.01) and aSyn PFF-2 and PFF-1 (adj. p < 0.01)). F Schematic of the catwalk XT Gait Analysis, used to measure gait and locomotion. Footprints were captured while subjects voluntarily traversed a glass-plated CatWalk runway, towards a dark goal box at the end. G A significant difference between 8 and 16 weeks was revealed in the stride length of aSyn PFF-2 inoculated mice (2-way RM ANOVA, Group x Session: no main effect of group (p > 0.05), but a main effect of session (F 1,24 = 4.493, p < 0.05), and significant interaction (F 2,24= 3.646 , p < 0.05), in which Šídák’s multiple comparisons revealed a significant difference between timepoints within the aSyn PFF-2 group (adj. p < 0.01). H A significant difference between 8 and 16 weeks was revealed in the swing speed of aSyn PFF-2 inoculated mice (2-way RM ANOVA, Group x Session: no main effect of group (p > 0.05), but a main effect of session (F 1,24= 6.537 , p < 0.05), and significant interaction (F 2,24= 4.300 , p < 0.05) in which Šídák’s multiple comparisons revealed a significant difference between timepoints within the aSyn PFF-2 group (adj. p < 0.01). I A significant difference between 8 and 16 weeks was revealed in the step cycle of aSyn PFF-2 inoculated mice (2-way RM ANOVA, Group x Session: no main effect of group (p > 0.05), but a main effect of session (F 1,24= 9.232 , p < 0.01) and significant interaction (F 2,24= 4.035 , p < 0.05) in which Šídák’s multiple comparisons revealed a significant difference between timepoints within the aSyn PFF-2 group (adj. p < 0.01). Data presented as Mean + SEM, group x session Two-way RM ANOVA, ** p < 0.01, **** p < 0.0001. Graphics made with Biorender.com.

Article Snippet: Human aSyn PFF-1 and PFF-2 are available from a commercial source (StressMarq Biosciences Cat# SPR-322 and SPR-317, respectively), which are expressed, purified, and fibrilized using methods previously described [ , ].

Techniques: Injection

Journal: Translational Psychiatry

Article Title: Impairment in stimulus-response learning as a cognitive biomarker in a model of synucleinopathy

doi: 10.1038/s41398-025-03795-5

Figure Lengend Snippet: A Schematic of Visuomotor Conditional Learning (VMCL) task. Cognitive assessments were conducted within touchscreen systems equipped with a touch-sensitive screen, a reward magazine attached to a reward pump for delivery of strawberry milkshake liquid reward and ABET cognition software ( above ). VMCL was employed to evaluate the acquisition of stimulus-response (S-R) contingencies, with subjects learning the conditional rule: “if visual stimulus A is presented, make motor response to the right-flanking window; if visual stimulus B is presented, make motor response to the flight-flanking window” ( below ). All subjects underwent VMCL testing for 20 sessions, 5-7 sessions per week. B M83 mice inoculated with aSyn PFF-1 and PFF-2 were significantly impaired at acquiring VMCL, as demonstrated by lower percent correct responses relative to PBS-inoculated mice (2-way RM ANOVA, Group x Session: main effect of group (F 2,60=10.53 , p < 0.001), main effect of session (F 2.686, 161.2=10.53 , p < 0.0001) and significant interaction (F 8,240=3.131 , p < 0.01), in which Šídák’s multiple comparisons revealed to be driven by a significant difference between PBS control and PFF-1 in sessions 2 (adj. p < 0.05), 4 (adj. p < 0.05) and 5 (adj. p < 0.05), and a significant difference between PBS control and PFF-2 in sessions 2 (adj. p < 0.01), 3 (adj. p < 0.001), 4 (adj. p < 0.01), and 5 (adj. p < 0.01)). C While all groups began at chance in Block 1 (1-way ANOVA, p > 0.05), (D) both aSyn-inoculated groups were significantly impaired relative to controls in Block 5 (1-way ANOVA, F 8.764 , p < 0.001, in which Šídák’s multiple comparisons revealed to be driven by a significant difference between PBS controls and PFF-1 (adj. p < 0.05), and between PBS controls PFF-2 (adj. p < 0.001)). E No significant difference was found in the percentage of missed trials across groups (RM Mixed-Effects Model, Group x Session: main effect of session (F 2.050,118.4= 20.55 , p < 0.0001), but no main effect of group or interaction (p > 0.05)), (F) but aSyn PFF-1 and PFF-2 mice exhibited a greater number of correction trials (2-way RM ANOVA, Group x Session: main effect of group (F 2,60= 6.008 , p < 0.01), main effect of session (F 3.240,194.4= 66.22 , p < 0.0001) but no interaction (p > 0.05)), and (G) an elevated perseveration index compared to controls (RM Mixed-Effects Model, Group x Session: main effect of group (F 2,60=4.648 , p < 0.05), main effect of session (F 3.191,171.5=24.48 , p < 0.0001), but no interaction (p > 0.05)). Furthermore, comparing task latencies, no significant difference was found in the latency to make correct choices (H) (2-way RM ANOVA: Group x Session: p > 0.05), but aSyn PFF-1 and PFF-2 mice took significantly longer to make incorrect choices (I) across VMCL acquisition compared to PBS controls (2-way RM ANOVA: Group x Session: main effect of group (F 2,60=5.384 , p < 0.01), main effect of session (F 3.202,192.1= 62.37 , p < 0.0001), and significant interaction (F 8,240=1.985 , p < 0.05), in which Šídák’s multiple comparisons revealed to be driven by a significant difference between PBS control and PFF-1 in sessions 2 (adj. p < 0.01), 3 (adj. p < 0.01) and 5 (adj. p < 0.05), and a significant difference between PBS control and PFF-2 in sessions 3 (adj. p < 0.05) and 5 (adj. p < 0.05)). No significant difference was found for the latency to collect rewards (J) (2-way RM ANOVA, Group x Session: main effect of session (F 2.275,135.9=17.67 , p < 0.0001), but no main effect of group or interaction (p > 0.05)). Data presented as Mean + SEM, * p < 0.05, ** p < 0.01, *** p < 0.001. Graphics made with Biorender.com.

Article Snippet: Human aSyn PFF-1 and PFF-2 are available from a commercial source (StressMarq Biosciences Cat# SPR-322 and SPR-317, respectively), which are expressed, purified, and fibrilized using methods previously described [ , ].

Techniques: Software, Control, Blocking Assay

Journal: iScience

Article Title: ILF3 prion-like domain regulates gene expression and fear memory under chronic stress

doi: 10.1016/j.isci.2023.106229

Figure Lengend Snippet:

Article Snippet: The primary antibodies used were the anti-ILF3 rabbit monoclonal antibody (Abcam), anti-TDP-43 rabbit polyclonal antibody (10782-2-AP, Proteintech, Rosemont, IL, USA), anti-hnRNPA1 rabbit monoclonal antibody (#8443, Cell Signaling Technology, Danvers, MA), and anti-c-Fos rabbit monoclonal antibody (#2250, Cell Signaling Technology).

Techniques: Recombinant, RNA HS Assay, CRISPR, Sequencing, Control, Software, Battery

Journal: Science Advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: ( A ) Co-IP analysis of the interaction between MYC-tagged LRRK2 and flag-tagged CDGI in cotransfected HEK 293T cells. Co-IP with anti-MYC was followed by anti-flag immunoblotting. ( B ) Co-IP analysis of the interaction between LRRK2 and CDGI in mouse brain lysates. Lysates prepared from LRRK2 WT and KO mouse whole brains were subjected to IP with anti-LRRK2 followed by anti-CDGI and anti-LRRK2 immunoblotting. ( C ) Co-IP analysis of the interaction between LRRK2 and CDGI in mouse brain lysates. Lysates prepared from CDGI WT and KO mouse whole brains were subjected to IP with anti-CDGI followed by anti-LRRK2 and anti-CDGI immunoblotting. ( D ) Co-IP analysis of the interaction between LRRK2 and CDGI in mouse striatal lysates. Lysates prepared from LRRK2 WT and KO mouse striatum were subjected to IP with anti-LRRK2 followed by anti-CDGI and anti-LRRK2 immunoblotting. ( E ) Co-IP analysis of the interaction between MYC-tagged LRRK2 and flag-tagged F1 (the GEF domain), F2 (the EF-hands domain), F3 (the DAG domain), or full-length (FL) CDGI in cotransfected HEK 293T cells. Co-IP with anti-MYC was followed by anti-flag or anti-MYC immunoblotting. A schematic representation of CDGI F1, F2, and F3 domains is shown. ( F ) Co-IP analysis of the interaction between V5-tagged CDGI and flag-tagged LRRK2 fragments in cotransfected HEK 293T cells. Co-IP with anti-V5 was followed by anti-flag or anti-V5 immunoblotting. A schematic representation of the different LRRK2 fragments is shown. ( G ) Co-IP analysis of the interaction between V5-tagged CDGI and MYC-tagged LRRK2-WT and TN mutant in cotransfected HEK 293T cells. Co-IP with anti-MYC was followed by anti-V5 immunoblotting. ( H ) Co-IP analysis of the interaction between flag-tagged CDGI-F1 domain and Myc-tagged LRRK2-WT and TN mutant in cotransfected HEK 293T cells. Co-IP with anti-MYC was followed by anti-flag immunoblotting. ( I ) Coimmunostaining of CDGI (green), LRRK2 (red), and DARPP32 (magenta) in mouse striatal sections. aa, amino acid.

Article Snippet: LRRK2 GSKI mice were purchased from Taconic (13940) ( ).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Mutagenesis

Journal: Science Advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: ( A ) GTP loading analysis of LRRK2 by an in vivo 32 P-orthophosphate labeling in HEK 293T cells overexpressing the indicated plasmids. The migration of GTP and GDP is indicated. The Ca 2+ ionophore A23187 and TPA were applied. ( B ) Quantification of LRRK2 GTP binding activity by the ratio GTP to (GTP + GDP). ( C ) GTP loading analysis of LRRK2 R1441C (RC) and G2019S (GS) with CDG-WT or GW by an in vivo 32 P-orthophosphate labeling in HEK 293T cells. TN (T1348N) was a negative control. ( D ) Quantification of LRRK2 GTP binding activity by the ratio GTP to (GTP + GDP). ( E ) The levels of LRRK2 bound to GTP were analyzed by pull-down assays with GTP-agarose from HEK 293T cells. ( F ) Quantification of LRRK2 bound to GTP-agarose beads. Data were normalized to LRRK2-WT alone in (B), (D), and (F). ( G ) GTP loading analysis of LRRK2 by an in vivo 32 P-orthophosphate labeling in WT striatal neurons compared to CDGI KO neurons transduced by LV-CDGI-WT or LV-CDGI-GW. ( H ) Quantification of LRRK2 GTP binding activity by the ratio GTP to (GTP + GDP). Data were normalized to WT neurons. ( I ) A diagram of the guanine nucleotide exchange assay is illustrated. Free BODIPY-FL-GDP is quenched with a low fluorescence in the solutions while showing increased fluorescence upon binding to GTPases. GEF catalyzes the exchange of preloaded BODIPY-FL-GDP for GTP causing a decrease in fluorescence. ( J ) Recombinant LRRK2 preloaded with BODIPY-FL-GDP was incubated with recombinant GST-CDGI proteins in the presence of excess cold GTP. Nucleotide exchange on LRRK2 was monitored by the fluorescence intensity change with different concentrations of CDGI-WT every 36 s for 15 min. Data are the means ± SEM, n = 3. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. n.s., not statistically significant.

Article Snippet: LRRK2 GSKI mice were purchased from Taconic (13940) ( ).

Techniques: In Vivo, Labeling, Migration, Binding Assay, Activity Assay, Negative Control, Fluorescence, Recombinant, Incubation

Journal: Science Advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: ( A to C ) Cellular fractionation assay of LRRK2 intensity on membrane and cytosol fractions in HEK 293T cells cotransfected with LRRK2 and CDGI-WT or GW. After 48 hours, cells were harvested and fractionated into cytosol and membrane fractions. Samples were immunoblotted with anti-MYC, V5, LAMP1, Rab10, and phosphor-Rab10. Data were normalized to LRRK2-MYC alone. ( D and E ) Liquid nitrogen freeze-thaw methods assessing LRRK2 membrane association. HEK 293T cells with stably expressed eGFP-LRRK2 were transfected with or without mCherry-CDGI. After 48 hours, cells were permeabilized by liquid nitrogen freeze-thaw to deplete cytosol and then fixed, immunostained with LAMP1, and visualized by confocal microscopy. Scale bar, 20 μM. Data were normalized to eGFP-LRRK2 stable cells transfected with mCherry. ( F and G ) Cellular fractionation assay of LRRK2 intensity on membrane and cytosol fractions in CDGI WT striatal neurons compared to CDGI KO neurons. CDGI WT and KO striatal neurons were treated with A23187 and TPA before being harvested and fractionated into cytosol and membrane fractions. Data were normalized to CDGI-WT neurons. Data are the means ± SEM, n = 3. One-way ANOVA followed by Tukey’s post hoc test was used for the data analysis of multiple comparisons, and Student’s t tests (unpaired, two-tailed) were used for the data analysis of two comparisons. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: LRRK2 GSKI mice were purchased from Taconic (13940) ( ).

Techniques: Cell Fractionation, Membrane, Stable Transfection, Transfection, Confocal Microscopy, Two Tailed Test

Journal: Science Advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: ( A ) Representative images of eye morphology of 1-week-old flies of the indicated genotypes by light microscopy. Drosophila LRRK2 ( dLRRK ) RNAi knockdown ( dLRRK RI ), human LRRK2 WT ( LRRK2 WT ), and LRRK2 mutant GS or RC ( LRRK2 GS or LRRK2 RC ) were coexpressed with human CDGI WT ( CDGI WT ) or CDGI-GW ( CDGI GW ) in fly eyes by a GMR-GAL4 driver ( GMR > CDGI/LRRK2 ). For each genotype, images were taken from at least 10 flies. Scale bar, 100 μm. ( B ) Representative images of eye morphology of 1-week-old flies of the indicated genotypes by SEM. Scale bar, 100 μm. ( C ) The fly eye size was quantified by ImageJ for each genotype. n = 10. Data are mean ± SEM, one-way ANOVA followed by Tukey’s post hoc test, * P < 0.05, ** P < 0.01, and **** P < 0.0001.

Article Snippet: LRRK2 GSKI mice were purchased from Taconic (13940) ( ).

Techniques: Light Microscopy, Knockdown, Mutagenesis

Journal: Science Advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: ( A ) Representative images of NeuN staining of mouse striatum. Ten-month-old WT, LRRK2 GSKI, RCKI, and KO mice were injected with AAV1-CDGI-WT or GW into the striatum. After 9 to 10 months of viral injection, the striatal neurons were immunolabeled by a NeuN antibody. Scale bar, 250 μm. ( B ) Quantification of NeuN-positive neurons in the striatum using an unbiased stereological method with stereo investigator software from five animals per group. ( C ) Representative images of DA neuron staining in mouse SNpc. After 9 to 10 months of viral injection, the DA neurons were immunolabeled by a TH antibody. Scale bar, 500 μm. ( D ) Quantification of TH-positive neurons in SNpc using an unbiased stereological method with stereo investigator software from five to seven animals per group. Data are mean ± SEM, two-way ANOVA followed by Tukey’s post hoc test, * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: LRRK2 GSKI mice were purchased from Taconic (13940) ( ).

Techniques: Staining, Injection, Immunolabeling, Software

Journal: Science Advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: Ten-month-old WT, LRRK2 GSKI, RCKI, and KO mice were injected with AAV1-CDGI-WT or GW into the striatum. After 9 to 10 months of viral injection, a battery of behavioral tests was performed. ( A ) Open-field test. The total time spent at the center was analyzed. ( B ) Open-field test. The total time spent at the corner was analyzed. ( C ) Rotarod test. The average retention time was analyzed. ( D ) Pole test to monitor behavioral abnormalities. The total time to descend to the bottom was recorded. Data are mean ± SEM, n = 5 to 9 per group, one-way ANOVA followed by Tukey’s post hoc test, * P < 0.05.

Article Snippet: LRRK2 GSKI mice were purchased from Taconic (13940) ( ).

Techniques: Injection, Battery

Journal: Science Advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: LRRK2 GTPase cycle is between inactive off GDP-bound state and active on GTP-bound state. CDGI binds to LRRK2 serving as a physiological GEF for LRRK2 to increase LRRK2 GTP binding activity and GDP release, and membrane association and in turn regulates LRRK2-induced striatal and DA neurodegeneration and behavioral deficits.

Article Snippet: LRRK2 GSKI mice were purchased from Taconic (13940) ( ).

Techniques: Binding Assay, Activity Assay, Membrane

Journal: iScience

Article Title: ILF3 prion-like domain regulates gene expression and fear memory under chronic stress

doi: 10.1016/j.isci.2023.106229

Figure Lengend Snippet:

Article Snippet: Ribosome-bound RNA was isolated using a Direct-zol RNA MicroPrep Kit (Zymo Research, Irvine, CA).

Techniques: Recombinant, RNA HS Assay, CRISPR, Sequencing, Control, Software, Battery

Journal: Scientific Reports

Article Title: The role of glycosyltransferase enzyme GCNT3 in colon and ovarian cancer prognosis and chemoresistance

doi: 10.1038/s41598-018-26468-4

Figure Lengend Snippet: GCNT3 overexpression reduces 5FU resistance in CRC cells. (Panel A) Protein expression levels of GCNT3 in non-infected colorectal cancer (CRC) cells, stable cell lines overexpressing GCNT3 and a battery of shGCNT3. Proteins were detected by western blot using specific antibodies against GCNT3, β-Actin and β-Tubulin, as a loading control. Full-length blots/gels are presented in Supplementary Fig. . (Panel B) mRNA expression levels of GCNT3 measured by RT-QPCR, in non-infected CRC cells, stable cell lines overexpressing GCNT3 and shGCNT3 number 7. Data represent mean ± SEM of three independent experiments. (Panel C) Representative immunofluorescence images of GCNT3 (green) and Tubulin (red) of NoORF, GCNT3, Scrambl and shGCNT3 7 cells. Nuclei were stained with DAPI (blue). Scale bars 50 µm. (Panel D) Comparison of 5-fluoracil (5FU) IC50 values (concentration needed for 50% of viability inhibition) between non-infected CRC cells. Cell viability assays were performed after 72 h treatment. Data represent mean ± SEM of at least two independent experiments each performed in triplicate. (Panel E) Induction of GCNT3 expression by 5FU in CRC cells. Tumour cells were treated with 30 µM 5FU, during 72 h, and their mRNA GCNT3 expression was measured by RT-QPCR and represented in comparison to controls (vehicle-treated cells). Results are expressed as the mean ± SEM. of three independent experiments, each performed in triplicate. Student’s t test was applied to assess statistically significant differences (*p < 0.05). (Panel F) Comparison of 5FU IC50 values between NoORF and GCNT3 cells. Cell viability assays were performed after 72 h treatment. Data represent mean ± SEM of at least three independent experiments each performed in triplicate. Asterisk indicates statistically different values in GCNT3 cells respect to the control (NoORF cells), (*p < 0.05).

Article Snippet: In clinical samples, GCNT3 gene expression was analyzed using the specific TaqMan probe (Hs00953355_m1, Life Technologies, Carlsbad CA, USA).

Techniques: Over Expression, Expressing, Infection, Stable Transfection, Battery, Western Blot, Control, Quantitative RT-PCR, Immunofluorescence, Staining, Comparison, Concentration Assay, Inhibition

Journal: Scientific Reports

Article Title: The role of glycosyltransferase enzyme GCNT3 in colon and ovarian cancer prognosis and chemoresistance

doi: 10.1038/s41598-018-26468-4

Figure Lengend Snippet: GCNT3 overexpression reduces proliferation, invasion and changes metabolic capacities of CRC cells. (Panel A) xCELLigence proliferation assay of NoORF, GCNT3, Scramble and shGCNT3 7 cells. The rate of proliferation is determined by analyzing the slope of the proliferation line between the 12 and 60 h interval. Complete growth curve of NoORF and GCNT3 SW620 cells from 0 to 72 h after seeding is represented. Results were expressed as 12 h normalized cell index value. Data are represented as mean ± SEM of four independent experiments each performed in triplicate. Student’s t test was applied to assess statistically significant differences (*p < 0.05). (Panel B) Boyden chamber transwell assay of GCNT3 SW620 cell invasion through Matrigel. After 96 h, SW620 cells were fixed and stained with crystal violet (bottom panels) and counted under an optical microscope. Pictures were taken using an Olympus CKX41 microscope (Olympus, Tokyo, Japan), with a 20X LCAch objective and registered using analysis getIT software (Olympus). Scale bars 100 µm. Data are represented as mean ±SEM of three independent experiments each performed in triplicate. Student’s t test was applied to assess statistically significant differences (**p < 0.01). (Panel C) Oxygen consumption rate (OCR) of NoORF and GCNT3 SW620 cells. Bioenergetics parameters were obtained by adding 2 µM Oligomycin to block ATP-linked OCR, 0.2 µM FCCP to uncouple mitochondria for maximal OCR and 0.5 µM Rotenone/Antimycin A (Rot/AA) to shut down mitochondrial respiration. Right panel reflects the quantification of basal respiration (oxygen consumption used to meet cellular ATP demand, calculated by subtracting non-mitochondrial OCR obtained upon Rot/AA addition) and spare respiratory capacity (capability to respond to an energetic demand, calculated as the difference between maximal and basal OCR). Left panel represents OCR measurements over time for cells stably expressing NoORF or GCNT3. We show representative experiments of 6 measures (n = 3).

Article Snippet: In clinical samples, GCNT3 gene expression was analyzed using the specific TaqMan probe (Hs00953355_m1, Life Technologies, Carlsbad CA, USA).

Techniques: Over Expression, Proliferation Assay, Boyden Chamber Transwell Assay, Staining, Microscopy, Software, Blocking Assay, Stable Transfection, Expressing

Journal: Scientific Reports

Article Title: The role of glycosyltransferase enzyme GCNT3 in colon and ovarian cancer prognosis and chemoresistance

doi: 10.1038/s41598-018-26468-4

Figure Lengend Snippet: Genomic analysis of GCNT3 overexpression. (Panel A) Description of candidate genes selected for validation. (Panel B) Experimental validation of GCNT3 microarray results by qRT-PCR. Bar graph showing the correlation of microarray data with qRT-PCR transcript levels in three CRC cellular models. The X axis shows the selected panel of validated genes and the Y-axis represent the relative fold change by microarray or qRT-PCR. Data represent mean ± SEM of three independent experiments each performed in triplicate. Student’s t test was applied to assess statistically significant differences (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: In clinical samples, GCNT3 gene expression was analyzed using the specific TaqMan probe (Hs00953355_m1, Life Technologies, Carlsbad CA, USA).

Techniques: Over Expression, Biomarker Discovery, Microarray, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: The role of glycosyltransferase enzyme GCNT3 in colon and ovarian cancer prognosis and chemoresistance

doi: 10.1038/s41598-018-26468-4

Figure Lengend Snippet: Proteomic analysis of GCNT3 overexpression. (Panel A) Immunoprecipitation of NoORF and GCNT3 SW620 cell extracts using V5 antibody. V5-GCNT3 tag protein was detected by Western blot (Inp: Input, IP: Immunoprecipitate, FT; Flow through). Experiments performed in duplicate. Full-length blots/gels are presented in Supplementary Fig. . (Panel B) Protein-protein interaction network of GCNT3 using STRING. Statistically significant interactors of proteomic study were included in the analysis (p-value > 0.0005). Only top-10 statistically significant proteins included in nodes are named, as well as, PAM and CANX.

Article Snippet: In clinical samples, GCNT3 gene expression was analyzed using the specific TaqMan probe (Hs00953355_m1, Life Technologies, Carlsbad CA, USA).

Techniques: Over Expression, Immunoprecipitation, Western Blot

Journal: Scientific Reports

Article Title: The role of glycosyltransferase enzyme GCNT3 in colon and ovarian cancer prognosis and chemoresistance

doi: 10.1038/s41598-018-26468-4

Figure Lengend Snippet: Biological processes enriched by GCNT3 overexpression in CRC cells. REViGO Scatterplot of GO categories enriched in GCNT3 genomic (Panel A) and proteomic (Panel B) analysis. GO enrichment analysis for statistically significant transcripts and proteins. The remaining terms after the redundancy reduction were plotted in a two dimensional space. Bubble sizes indicates the p-value (log10 p-value). Semantic space is based on the semantic similarity, which is the degree of relatedness between two entities by measuring the similarity of their annotation meanings. The list of enriched GO terms is subjected to redundancy reduction, based on the “most informative common ancestor” approach in REVIGO and is represented by cluster representatives in a scatterplot. The x- and y-axes of the scatterplot represent the distance between the cluster representatives.

Article Snippet: In clinical samples, GCNT3 gene expression was analyzed using the specific TaqMan probe (Hs00953355_m1, Life Technologies, Carlsbad CA, USA).

Techniques: Over Expression

Journal: Scientific Reports

Article Title: The role of glycosyltransferase enzyme GCNT3 in colon and ovarian cancer prognosis and chemoresistance

doi: 10.1038/s41598-018-26468-4

Figure Lengend Snippet: Clinical relevance of GCNT3 expression in epithelial ovarian cancer (EOC). (Panel A) Association between GCNT3 expression and time to treatment failure (TTF) in EOC. Kaplan–Meier plots for GCNT3 expression in 56 EOC patients. (Panel B) Association of GCNT3 gene expression profiles with response. The expression levels of GCNT3 in non-responders and responders groups are shown. The median value of GCNT3 expression is indicated by the horizontal bar on the graph (Man-Whitney U-test for P values). (Panel C) ROC analysis of the GCNT3 signature in EOC patients. The AUC was 0.667. (Panel D) Forest plot showing the meta-analysis of hazard ratio (HR) and 95% confidence interval (CI) estimates for TTF for the prognostic significance of GCNT3 expression in EOC patients from six different studies. (Panel E) Protein and mRNA expression levels of GCNT3 in non-infected EOC cells and Caov3 stable cell lines overexpressing GCNT3. Proteins were detected by western blot using specific antibodies against GCNT3 and β-Actin. mRNA expression levels of GCNT3 and VEGFA were measured by RT-QPCR. Data represent mean ± SEM of three independent experiments. Student’s t test was applied to assess statistically significant differences (***p < 0.001). Full-length blots/gels are presented in Supplementary Fig. . (Panel F) Boyden chamber transwell assay of GCNT3 Caov3 invasion through Matrigel. After 96 h, Caov3 cells were fixed and stained with crystal violet (bottom panels) and counted under an optical microscope. Pictures were taken using an Olympus CKX41 microscope (Olympus, Tokyo, Japan), with a 20X LCAch objective and registered using analysis getIT software (Olympus). Scale bars 100 µm. On the left, xCELLigence proliferation assay of NoORF and GCNT3 Caov3 cells. The rate of proliferation is determined by analyzing the slope of the proliferation line between the 12 and 60 h interval. Data are represented as mean ± SEM of three independent experiments each performed in triplicate. Student’s t test was applied to assess statistically significant differences (**p < 0.01).

Article Snippet: In clinical samples, GCNT3 gene expression was analyzed using the specific TaqMan probe (Hs00953355_m1, Life Technologies, Carlsbad CA, USA).

Techniques: Expressing, Gene Expression, Infection, Stable Transfection, Western Blot, Quantitative RT-PCR, Boyden Chamber Transwell Assay, Staining, Microscopy, Software, Proliferation Assay