Journal: bioRxiv

Article Title: Aberrant regulation of the Rap1 small GTPase in response to escalating, intermittent stress produces hippocampal synaptic and cognitive dysfunction

doi: 10.1101/2023.07.03.547282

Figure Lengend Snippet: Escalating, intermittent stress increases Rap1 levels in the P2 fraction of CA3 region homogenates. A and B. P2 fractions derived from CA3 homogenates of non-stressed mice and mice subjected to escalating, intermittent stress (tissue collected 24 hour after final stress exposure). Western blots were probed for the indicated small GTPases. Samples were derived from 3 independent stress experiments. C=non-stressed controls, S=stressed. C. Stress increased Rap1 in the P2 fraction, with no effect on the other small GTPases. *Bonferroni, p<0.05. n=15 non-stressed and 16 stressed mice. Direct comparison p values (Fisher’s LSD): Rap1 p=0.0079; Ras p=0.2799; Rac1 p=0.1957; Cdc42 p=0.1060. D and E. P1 fractions derived from CA3 homogenates of non-stressed mice and stressed mice. Blots were probed for the indicated small GTPases. Samples were derived from 3 independent stress experiments. C=non-stressed controls, S=stressed. F. Lack of effect of stress on the levels of any small GTPases in P1 fractions. n=15 non-stressed and 16 stressed mice. Direct comparison p values (Fisher’s LSD): Rap1 p=0.2576; Ras p=0.9073; Rac1 p=0.9904; Cdc42 p=0.1965. G and H. S2 fractions derived from CA3 homogenates of non-stressed mice and stressed mice. Blots were probed for the indicated small GTPases. Sample were derived from 3 independent stress experiments. C=non-stressed controls, S=stressed. I. Lack of effect of stress on the levels of any small GTPases in S2 fractions. n=15 non-stressed and 16 stressed mice. Direct comparison p values (Fisher’s LSD): Rap1 p=0.6271; Ras p=0.4169; Rac1 p=0.6289; Cdc42 p=0.4973. All summary data are the mean + SEM.

Article Snippet: The following primary antibodies were used: Actin (Fisher Scientific, Cat# PIMA515739; 1:5000 dilution), Rac1 (Cytokeleton, Cat# ARC03; 1:1000 dilution), Cdc42 (Cell Signaling, Cat# 2466: 1:1000 dilution), Rap1 (Cell Signaling, Cat# 2326; 1:1000 dilution), and Ras (detects K-Ras, H-Ras, and N-Ras; Cell Signaling, Cat# 3339; 1:1000 dilution).

Techniques: Derivative Assay, Western Blot, Comparison

Journal: bioRxiv

Article Title: Aberrant regulation of the Rap1 small GTPase in response to escalating, intermittent stress produces hippocampal synaptic and cognitive dysfunction

doi: 10.1101/2023.07.03.547282

Figure Lengend Snippet: Rap1 reduces the stability of select dendritic spine subtypes in CA3 pyramidal neurons. A. Schematic of experimental design. Mice were infused with HSV co-expressing Rap1 and GFP (HSV-Rap1-GFP) or HSV-GFP into the CA3. 4 days post-viral infusion, mice were transcardially perfused. B. Representative low magnification image showing the left hemisphere hippocampus. Surgical coordinates used result in the targeting of HSV-GFP to the CA3 and CA2 region of the dorsal hippocampus. Scale bar=500µm C. Representative images of CA3 region pyramidal neuron secondary dendrites of the apical tree expressing GFP or Rap1-GFP. Scale bar=10µm. D. Graph depicts the total dendritic spine density of CA3 pyramidal neurons expressing GFP and Rap1-GFP. *Two-tailed t-test, p<0.05. 3-5 neurons were imaged per mouse and spine values across all neurons within a single mouse were averaged. n=4 GFP and 4 Rap1-GFP mice. E. Graph depicts thin spine density in CA3 pyramidal neurons expressing GFP or Rap1-GFP. *Two-tailed t-test, p<0.05. n is same as in 5D. F. Graph depicts stubby spine density in CA3 pyramidal neurons expressing GFP and Rap1-GFP. No significant differences were detected. n is same as in 5D. G. Graph depicts mushroom spine density in CA3 pyramidal neurons expressing GFP and Rap1-GFP. No significant differences were detected. n is same as in 5D. H. No significant differences in the cumulative thin spine head diameter curve were identified between the GFP and Rap1-GFP conditions. n=782 spines in HSV-GFP mice and 693 spines in HSV-Rap1-GFP mice. I. No significant differences in the cumulative stubby spine head diameter curve were identified between the GFP and Rap1-GFP conditions. n=124 spines in HSV-GFP mice and 146 spines in HSV-Rap1-GFP mice. J. A modest, yet significant rightward shift in the cumulative mushroom spine head diameter curve was identified in stressed mice vs. non-stressed mice. *Mantel-Cox, p<0.05. n=140 spines in non-stressed mice and 162 spines in stressed mice. All summary data are the mean + SEM.

Article Snippet: The following primary antibodies were used: Actin (Fisher Scientific, Cat# PIMA515739; 1:5000 dilution), Rac1 (Cytokeleton, Cat# ARC03; 1:1000 dilution), Cdc42 (Cell Signaling, Cat# 2466: 1:1000 dilution), Rap1 (Cell Signaling, Cat# 2326; 1:1000 dilution), and Ras (detects K-Ras, H-Ras, and N-Ras; Cell Signaling, Cat# 3339; 1:1000 dilution).

Techniques: Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Aberrant regulation of the Rap1 small GTPase in response to escalating, intermittent stress produces hippocampal synaptic and cognitive dysfunction

doi: 10.1101/2023.07.03.547282

Figure Lengend Snippet: The spatial distribution of overexpressed Rap1 varies within different spine subtypes. A. Cultured hippocampal neuron SIM images of individual dendritic spines showing Rap1 nanodomains (Myc-tag immuno-labeled) in orange. Scale bar=1µm. B. Graph depicts the percent of filopodia and indicated spine subtypes that contain Myc-Rap1 nanodomains upon overexpression in mature cultured hippocampal neurons. C. Among thin spines containing overexpressed Rap1, the chart depicts the proportion located in each indicated spine region. D. Among mushroom spines containing overexpressed Rap1, the chart depicts the proportion located in each indicated spine region. E. Among the indicated protrusions exhibiting overexpressed Rap1, the graph depicts the nanodomain density of Myc-Rap1 throughout the entire filopodia or spine region (head and neck combined). ***Bonferroni, p<0.001. n=34 filopodia, 88 thin spines, 40 mushroom spines from 7 neurons. F. Among thin and mushroom spines exhibiting overexpressed Rap1 in the neck, graph depicts the nanodomain density of Myc-Rap1 in the neck. *Two-tailed t-test, p<0.05. n=61 thin spines and 21 mushroom spines from 7 neurons. G. Among thin and mushroom spines exhibiting overexpressed Rap1 in the head, graph depicts the nanodomain density of Myc-Rap1 in the head. ***Two-tailed t-test, p<0.0001. n=38 thin spines and 31 mushroom spines from 7 neurons. H. Representative line scans through a thin spine and a mushroom spine. Graph shows the intensity of the Rap1 signal as a function of distance across the head region and neck region for each spine subtype. I. Graph depicts the area under the curve of Rap1 intensity. Quantification is from a randomly selected population of thin and mushroom spines. One-way ANOVA with Dunnett’s multiple comparison correction, **p<0.01, ***p<0.001. n=90 thin neck, 14 mushroom neck, 90 thin head, 14 mushroom head; from 4 neurons. All summary data are the mean + SEM.

Article Snippet: The following primary antibodies were used: Actin (Fisher Scientific, Cat# PIMA515739; 1:5000 dilution), Rac1 (Cytokeleton, Cat# ARC03; 1:1000 dilution), Cdc42 (Cell Signaling, Cat# 2466: 1:1000 dilution), Rap1 (Cell Signaling, Cat# 2326; 1:1000 dilution), and Ras (detects K-Ras, H-Ras, and N-Ras; Cell Signaling, Cat# 3339; 1:1000 dilution).

Techniques: Cell Culture, Labeling, Over Expression, Two Tailed Test, Comparison

Journal: bioRxiv

Article Title: Aberrant regulation of the Rap1 small GTPase in response to escalating, intermittent stress produces hippocampal synaptic and cognitive dysfunction

doi: 10.1101/2023.07.03.547282

Figure Lengend Snippet: Overexpression of Rap1 in the CA3 region impairs recognition memory. A. Schematic of experimental design. Mice were infused with HSV-Rap1-GFP or HSV-GFP into the CA3. 4 days later mice underwent habituation in an open field. The next day mice were assessed for object-in-place recognition memory. During trial 2, mice should prefer to explore the objects that swapped locations compared to trial 1 (purple star and green triangle in the schematic). B. Graph depicts time GFP and Rap1-GFP mice spent exploring objects during trial 1 of the object-in-place task. No differences between groups were identified. n=8 GFP and 8 Rap-GFP mice. C. Graph depicts the trial 1 object-in-place recognition ratio for the GFP and Rap1-GFP mice. No differences between groups were identified at any time bins. n=8 GFP and 8 Rap-GFP mice. D. Graph depicts the trial 2 object-in-place recognition ratio for the GFP and Rap1-GFP mice. Differences were identified at all time bins except the 0-3 minute bin. *p<0.05, **p<0.01. n=8 GFP and 8 Rap-GFP mice. E. Graph depicts recognition ratio across all 7 minutes of trial 1 and trial 2 of the object-in-place task. **p<0.01. n=8 GFP and 8 Rap-GFP mice. F Schematic of experimental design for Y-maze testing. Mice were assessed for spontaneous alternation in the Y-maze 1 week prior to HSV infusion and 4 days post-HSV infusion. G. HSV-Rap1 did not affect spontaneous alternation performance post-HSV infusion relative to the pretest. n=3 GFP, 4 Rap1 mice. All summary data are the mean + SEM.

Article Snippet: The following primary antibodies were used: Actin (Fisher Scientific, Cat# PIMA515739; 1:5000 dilution), Rac1 (Cytokeleton, Cat# ARC03; 1:1000 dilution), Cdc42 (Cell Signaling, Cat# 2466: 1:1000 dilution), Rap1 (Cell Signaling, Cat# 2326; 1:1000 dilution), and Ras (detects K-Ras, H-Ras, and N-Ras; Cell Signaling, Cat# 3339; 1:1000 dilution).

Techniques: Over Expression

Journal: Molecular and Cellular Biology

Article Title: TIN2-Tethered TPP1 Recruits Human Telomerase to Telomeres In Vivo

doi: 10.1128/mcb.00240-10

Figure Lengend Snippet: FIG. 6. The TPP1 OB-fold is required to rescue telomerase recruitment to telomeres. An shRNA-resistant form of TPP1 is able to restore hTR localization to telomeres in TPP1-depleted cells. However, an shRNA-resistant form of TPP1 lacking the OB-fold cannot restore localization. (A) Parental and TPP1-depleted super-telomerase HeLa cells were subjected to FISH and IF to detect hTR (red), coilin (blue), and TRF2 (green). Merge panels show superimposition of hTR, coilin, and TRF2. Next, parental cells were cotransfected with shTPP1 and either TPP1* or TPP1OB*. Treated cells were subjected to FISH and IF to detect hTR (red), FLAG (blue), and RAP1 (telomere marker, green). Merge panels show superimposition of hTR, FLAG, and RAP1. (B) Plot of the average number of telomere-associated hTR foci per cell in the parental cells and each experimental group. Error bars indicate standard errors calculated with N equal to the number of samples quantitated.

Article Snippet: Next, cells were incubated with one of several combinations of the following primary antibodies at the indicated dilution for 1 h at room temperature: mouse anti-p80 coilin (1:5,000, ) (1), mouse anti-TRF2 (1:1,000; Imgenex Corp., San Diego, CA), rabbit anti-hTERT (1:400; Rockland, Gilbertsville, PA), mouse anti-FLAG (1:500; Sigma-Aldrich, St. Louis, MO), rabbit anti-RAP1(1: 2,000; Novus Biologicals, Littleton, CO), and rabbit anti-53BP1(1:500; Bethyl, Montgomery, TX).

Techniques: shRNA, Marker