Journal: Cancers

Article Title: Roles of Annexin A1 Expression in Small Cell Lung Cancer.

doi: 10.3390/cancers17091407

Figure Lengend Snippet: Figure 1. Predictive characterization of the SCLC TME according to ANXA1 expression. ESTIMATE algorithm revealed significantly higher immune (p < 0.001) (A) and stroma scores (p < 0.001) (B) in ANXA1-high tumors and indicated higher estimate (p < 0.001) (C) and lower purity scores (p < 0.001) (D). EPIC algorithm shows the distribution of the main TME cell types in SCLC tumors derived from bulk RNA-seq data (E). ANXA1-high tumors were shown to harbor significantly more B-cells (p = 0.027), CAFs (p < 0.001), TAMs (p < 0.001), and NK cells (p < 0.001) compared to ANXA1- low tumors (F). Data are displayed on violin charts, where the median (solid line) and upper and lower quartiles (dashed lines) are indicated. Wilcoxon’s rank sum test was performed for pairwise comparisons, where no data showed a normal distribution according to the Shapiro–Wilk test. ns: non-significant, *: p < 0.05, ***: p < 0.001.

Article Snippet: For double IF staining, primary antibodies for CD45 (#40763, abcam), CD68 (#303565, abcam), CD3 (#5690, abcam), CD19 (#90176, Cell Signaling), vimentin (#MA5-11883, Invitrogen), and ANXA1 (#32934, Cell Signaling) and fluorescent antibodies, namely Alexa IgG anti-rabbit A488 and IgG anti-mouse A594, were used as secondaries (Invitrogen).

Techniques: Expressing, Derivative Assay, RNA Sequencing

Journal: Cancers

Article Title: Roles of Annexin A1 Expression in Small Cell Lung Cancer.

doi: 10.3390/cancers17091407

Figure Lengend Snippet: Figure 2. Differential expression and pathway analysis in the context of ANXA1 expression. Dif- ferentially expressed genes (DEGs) in ANXA1-high and ANXA1-low tumor subsets were analyzed using the HTG EdgeSeq oncological biomarker panel. n = 230 genes showed significantly increased expression in ANXA1-high tumors, while n = 16 genes were significantly overexpressed in ANXA1- low tumors (adjusted p-value < 0.05; LogFC > 2) (A). Pathway analysis using the Reactome database shows significant enrichment ratios (FDR ≤0.05) in blue for n = 3 pathways in ANXA1-high and n = 2 pathways in ANXA1-low tumors (B). Using ImmunDB’s Immunome panel, n = 117 genes were significant in ANXA1-high tumors and none in ANXA1-low tumors (C). Pathway analysis per- formed as described previously shows the 10 most enriched pathways (FDR ≤0.05) in ANXA1-high tumors. The affinity propagation method was used to reduce the redundancy of the gene sets in the enrichment (D).

Article Snippet: For double IF staining, primary antibodies for CD45 (#40763, abcam), CD68 (#303565, abcam), CD3 (#5690, abcam), CD19 (#90176, Cell Signaling), vimentin (#MA5-11883, Invitrogen), and ANXA1 (#32934, Cell Signaling) and fluorescent antibodies, namely Alexa IgG anti-rabbit A488 and IgG anti-mouse A594, were used as secondaries (Invitrogen).

Techniques: Quantitative Proteomics, Expressing, Biomarker Discovery

Journal: Cancers

Article Title: Roles of Annexin A1 Expression in Small Cell Lung Cancer.

doi: 10.3390/cancers17091407

Figure Lengend Snippet: Figure 3. Clinical characteristics of ANXA1 expression. Clinical parameters and overall survival (OS) were evaluated in the SCLC cohort according to ANXA1 expression. Spearman correlation and pairwise comparisons showed no significant associations between ANXA1 expression and diagnosis age (A), TMB (B), tumor stage (C), or gender (D). Kaplan–Meier [25] analysis indicated significantly decreased OS in ANXA1-high patients (HR: 1.809, 95% CI: 0.994 to 3.291, p = 0.0339, (E). Log-rank test was used to establish significance in KM analysis. Wilcoxon rank sum test was performed for pairwise comparison according to gender. Spearman’s correlation analysis was performed to assess coefficients (rs) and p-values (p) when comparing ANXA1 expression with diagnosis age, TMB, and tumor stage.

Article Snippet: For double IF staining, primary antibodies for CD45 (#40763, abcam), CD68 (#303565, abcam), CD3 (#5690, abcam), CD19 (#90176, Cell Signaling), vimentin (#MA5-11883, Invitrogen), and ANXA1 (#32934, Cell Signaling) and fluorescent antibodies, namely Alexa IgG anti-rabbit A488 and IgG anti-mouse A594, were used as secondaries (Invitrogen).

Techniques: Expressing, Biomarker Discovery, Comparison

Journal: Cancers

Article Title: Roles of Annexin A1 Expression in Small Cell Lung Cancer.

doi: 10.3390/cancers17091407

Figure Lengend Snippet: Figure 4. Tissue expression of ANXA1 protein. ANXA1 protein expression was evaluated using IHC and double IF staining on resected tumors from 9 early-stage SCLC patients. ANXA1 expression was observed in the tumor cells of 2 patients, with scattered stromal cell positivity (A,B). Two additional patients showed ANXA1 expression in the stroma and in non-tumoral cells within tumor nests (C,D). Double IF staining revealed ANXA1 co-expression with CD45 on immune cells (E, asterisks), whereas cancer cells of tumor nests showed positivity only for ANXA1. ANXA1+ tumor cells (F, asterisks) do not colocalize with vimentin+ CAFs in the stroma (F), whereas ANXA1 shows expression on multiple CD68+ TAMs both in the stroma (G) and in tumor nests (G, asterisks in inset). In contrast, some CD68+ TAMs show no co-expression of ANXA1 (G, arrows in inset).

Article Snippet: For double IF staining, primary antibodies for CD45 (#40763, abcam), CD68 (#303565, abcam), CD3 (#5690, abcam), CD19 (#90176, Cell Signaling), vimentin (#MA5-11883, Invitrogen), and ANXA1 (#32934, Cell Signaling) and fluorescent antibodies, namely Alexa IgG anti-rabbit A488 and IgG anti-mouse A594, were used as secondaries (Invitrogen).

Techniques: Expressing, Staining

Journal: Cancers

Article Title: Roles of Annexin A1 Expression in Small Cell Lung Cancer.

doi: 10.3390/cancers17091407

Figure Lengend Snippet: Figure 5. Experimental protocol and annexin A1 silencing. Groups and experimental protocol (A). Western blot evaluation of ANXA1 relative protein levels of native, shCTRL, and shANXA1 cells (B). One-way ANOVA, Tukey’s post hoc test, n = 6–8; *** p < 0.001. Data are expressed as mean ± SEM.

Article Snippet: For double IF staining, primary antibodies for CD45 (#40763, abcam), CD68 (#303565, abcam), CD3 (#5690, abcam), CD19 (#90176, Cell Signaling), vimentin (#MA5-11883, Invitrogen), and ANXA1 (#32934, Cell Signaling) and fluorescent antibodies, namely Alexa IgG anti-rabbit A488 and IgG anti-mouse A594, were used as secondaries (Invitrogen).

Techniques: Western Blot

Journal: bioRxiv

Article Title: Convergent evolution of a fungal effector enabling phagosome membrane penetration

doi: 10.1101/2025.03.06.641871

Figure Lengend Snippet: ( A – D ) Recruitment of TSG101 and CHMP3 to phagosomes in hMDMs. ( A ) Detection of TSG101 and GAL3, or ( B ) detection of TSG101 and CHMP3 on phagosomes containing A. fumigatus WT conidia in hMDMs. Regions indicated by white or yellow dashed-line frames are enlarged on the right or bottom, respectively. Channel intensity plots show the fluorescence signal across the yellow lines. ( C and D ) Phagosomes positive for ( C ) TSG101 and ( D ) CHMP3 were quantified. ( E – H ) Recruitment of ESCRT components to phagosomes in A549 cells. (E) Immunostaining of A549 cells incubated with A. fumigatus WT conidia, highlighting the indicated ESCRT markers. Yellow arrows mark phagosomes positive for both tested markers. DIC, differential interference contrast. ( F – H) Phagosomes positive for ( F ) CHMP3, ( G ) TSG101, and ( H ) ALG2 were quantified. A549 cells or p11-KO cells were incubated with conidia of WT or Δ hscA strains for 4 hours. Intracellular Ca 2+ was subsequently chelated by adding 25 μM BAPTA-AM to the medium, followed by an additional 4 hours of incubation at 37°C. (I) Chelation of Ca 2+ reduces the recruitment of p11 to phagosomes. ( J – L ) Recruitment of ANXA2 and ANXA1 to phagosomes. (J) A549 cells were incubated with A. fumigatus WT conidia and immunostained with antibodies against p11, ANXA2, and ANXA1. Yellow arrows indicate phagosomes positive for both tested markers, while white arrows denote a phagosome positive for ANXA2 but negative for p11. Phagosomes positive for ( K ) ANXA2 and ( L ) ANXA1 were quantified. ( M ) HscA, p11, and Ca 2+ -dependent recruitment of GAL3 to phagosomes. Statistics: Error bars represent the mean ± SD; p -values were determined using unpaired two-tailed t test (C and D) or one-way ANOVA, followed by Tukey’s multiple comparisons test. The number of individual experiments is indicated below each bar.

Article Snippet: To stain phagosomal markers, cells were incubated with primary antibodies overnight at 4°C, followed by incubation with secondary goat anti-mouse IgG Alexa Fluor 488 (Cat# A-11029, Thermo Fisher Scientific) or goat anti-rabbit IgG DyLight 633 (Cat# 35562, Thermo Fisher Scientific) at room temperature for 1 h. The primary antibodies or probes used were rabbit anti-ALG2 (1:100; Cat# 12303-1-AP, Proteintech), rabbit anti-ANXA2 (1:100; Cat# 8235, Cell Signaling Technology [CST]), rabbit anti-ANXA1 (1:200; Cat# 32934, CST), rabbit anti-CD9 (1:100; Cat# ab236630, Abcam), rabbit anti-CHMP3 (1:100; Cat# 15472-1-AP, Proteintech), rabbit anti-LAMP1 (1:200; Cat# 9091, CST), mouse anti-GAL3 (1:100; Cat# 60207-1-Ig, Proteintech), mouse anti-GFP (1:200; Cat# sc-9996, Santa Cruz), rabbit anti-HA (1:500; Cat# 3724, CST), mouse anti-Myc (1:100; Cat# 2276, CST), mouse anti-p11 (1:500; Cat# 610071, BD), rabbit anti-RAB7 (1:100; Cat# 9367, CST), mouse anti-TFEB (1:100, Cat# 91767, CST), mouse anti-TSG101 (1:200; Cat# sc-7964, Santa Cruz), and Alexa Fluor TM 633 Streptavidin (1 µg/mL; Cat# S21375, Thermo Fisher Scientific).

Techniques: Fluorescence, Immunostaining, Incubation, Two Tailed Test

Journal: CNS Neuroscience & Therapeutics

Article Title: Annexin A1 Mitigates Blood–Brain Barrier Disruption in a Sepsis‐Associated Encephalopathy Model by Enhancing the Expression of Occludin and Zonula Occludens‐1 ( ZO ‐1)

doi: 10.1111/cns.70173

Figure Lengend Snippet: Reduced expression of Annexin A1 (ANXA1) was observed in the brain cortex of septic mice. The septic mice model was established using cecal ligation and puncture (CLP). (A) ANXA1 mRNA levels in the cortex; (B) ANXA1 protein levels (*** p < 0.005 vs. WT group).

Article Snippet: The membrane was blocked with 5% non‐fat milk for 2 h and then incubated overnight at 4°C with primary antibodies against ANXA1 (Cat# 30797, 1:1000; CST, USA), occludin (Cat# 91131, 1:500; CST), ZO‐1 (Cat# 13663, 1:1000; CST), VEGF‐R2 (Cat# 2479, 1:1000; CST), and β‐actin (Cat# 4970, 1:5000; CST).

Techniques: Expressing, Ligation

Journal: CNS Neuroscience & Therapeutics

Article Title: Annexin A1 Mitigates Blood–Brain Barrier Disruption in a Sepsis‐Associated Encephalopathy Model by Enhancing the Expression of Occludin and Zonula Occludens‐1 ( ZO ‐1)

doi: 10.1111/cns.70173

Figure Lengend Snippet: Annexin A1 (ANXA1) deficiency reduced the 7‐day survival rate and exacerbated CLP‐induced neurobehavioral changes in septic mice. Mice were divided into four groups: WT, CLP, ANXA1 (−/−), and ANXA1 (−/−) + CLP. (A) The survival rate was analyzed at day 0, 1, 2, 3, 4, 5, 6, and 7 post‐operation; (B) cognitive function at days 3, 5, and 7 after the operation was assessed using the Y‐maze test (*** p < 0.005 vs. WT group; ### p < 0.005 vs. ANXA1 (−/−) group; && p < 0.01 vs. CLP group).

Article Snippet: The membrane was blocked with 5% non‐fat milk for 2 h and then incubated overnight at 4°C with primary antibodies against ANXA1 (Cat# 30797, 1:1000; CST, USA), occludin (Cat# 91131, 1:500; CST), ZO‐1 (Cat# 13663, 1:1000; CST), VEGF‐R2 (Cat# 2479, 1:1000; CST), and β‐actin (Cat# 4970, 1:5000; CST).

Techniques:

Journal: CNS Neuroscience & Therapeutics

Article Title: Annexin A1 Mitigates Blood–Brain Barrier Disruption in a Sepsis‐Associated Encephalopathy Model by Enhancing the Expression of Occludin and Zonula Occludens‐1 ( ZO ‐1)

doi: 10.1111/cns.70173

Figure Lengend Snippet: ANXA1 deficiency exacerbated the expression of inflammatory cytokines IL‐6, IL‐8, HMGB1, and TNF‐α in the brain cortex of septic mice. Mice were divided into four groups: WT, CLP, ANXA1 (−/−), and ANXA1 (−/−) + CLP. (A) MRNA levels of IL‐6, IL‐8, HMGB1, and TNF‐α in the cortex; (B) protein levels of IL‐6, IL‐8, HMGB1, and TNF‐α in the cortex (*** p < 0.005 vs. WT group; ### p < 0.005 vs. ANXA1 (−/−) group; && p < 0.01 vs. CLP group).

Article Snippet: The membrane was blocked with 5% non‐fat milk for 2 h and then incubated overnight at 4°C with primary antibodies against ANXA1 (Cat# 30797, 1:1000; CST, USA), occludin (Cat# 91131, 1:500; CST), ZO‐1 (Cat# 13663, 1:1000; CST), VEGF‐R2 (Cat# 2479, 1:1000; CST), and β‐actin (Cat# 4970, 1:5000; CST).

Techniques: Expressing

Journal: CNS Neuroscience & Therapeutics

Article Title: Annexin A1 Mitigates Blood–Brain Barrier Disruption in a Sepsis‐Associated Encephalopathy Model by Enhancing the Expression of Occludin and Zonula Occludens‐1 ( ZO ‐1)

doi: 10.1111/cns.70173

Figure Lengend Snippet: ANXA1 deficiency aggravated the expression of biomarkers of vascular endothelial dysfunction in the brain cortex of septic mice. Mice were divided into four groups: WT, CLP, ANXA1 (−/−), and ANXA1 (−/−) + CLP. (A) MRNA levels of VCAM‐1, ICAM‐1, and MCP‐1 in the cortex; (B) protein levels of VCAM‐1, ICAM‐1, and MCP‐1 in the cortex (*** p < 0.005 vs. WT group; ### p < 0.005 vs. ANXA1 (−/−) group; && p < 0.01 vs. CLP group).

Article Snippet: The membrane was blocked with 5% non‐fat milk for 2 h and then incubated overnight at 4°C with primary antibodies against ANXA1 (Cat# 30797, 1:1000; CST, USA), occludin (Cat# 91131, 1:500; CST), ZO‐1 (Cat# 13663, 1:1000; CST), VEGF‐R2 (Cat# 2479, 1:1000; CST), and β‐actin (Cat# 4970, 1:5000; CST).

Techniques: Expressing

Journal: CNS Neuroscience & Therapeutics

Article Title: Annexin A1 Mitigates Blood–Brain Barrier Disruption in a Sepsis‐Associated Encephalopathy Model by Enhancing the Expression of Occludin and Zonula Occludens‐1 ( ZO ‐1)

doi: 10.1111/cns.70173

Figure Lengend Snippet: ANXA1 deficiency exacerbated the dysfunction of BBB integrity and reduction of occludin and ZO‐1 in the brain cortex of septic mice. Mice were divided into four groups: WT, CLP, ANXA1 (−/−), and ANXA1 (−/−) + CLP. (A) BBB permeability was measured using EBD staining; (B) mRNA levels of occludin and ZO‐1; (C) protein levels of occludin and ZO‐1 as measured by immunostaining. Scale bar, 100 μm (*** p < 0.005 vs. WT group; ### p < 0.005 vs. ANXA1 (−/−) group; && p < 0.01 vs. CLP group).

Article Snippet: The membrane was blocked with 5% non‐fat milk for 2 h and then incubated overnight at 4°C with primary antibodies against ANXA1 (Cat# 30797, 1:1000; CST, USA), occludin (Cat# 91131, 1:500; CST), ZO‐1 (Cat# 13663, 1:1000; CST), VEGF‐R2 (Cat# 2479, 1:1000; CST), and β‐actin (Cat# 4970, 1:5000; CST).

Techniques: Permeability, Staining, Immunostaining

Journal: CNS Neuroscience & Therapeutics

Article Title: Annexin A1 Mitigates Blood–Brain Barrier Disruption in a Sepsis‐Associated Encephalopathy Model by Enhancing the Expression of Occludin and Zonula Occludens‐1 ( ZO ‐1)

doi: 10.1111/cns.70173

Figure Lengend Snippet: ANXA1 deficiency aggravated the increase in the expression of VEGF‐A and VEGF‐R2 in the brain cortex of septic mice. Mice were divided into four groups: WT, CLP, ANXA1 (−/−), and ANXA1 (−/−) + CLP. (A) mRNA levels of VEGF‐A and VEGF‐R2; (B) protein levels of VEGF‐A and VEGF‐R2. Scale bar, 100 μm (*** p < 0.005 vs. WT group; ### p < 0.005 vs. ANXA1 (−/−) group; && p < 0.01 vs. CLP group).

Article Snippet: The membrane was blocked with 5% non‐fat milk for 2 h and then incubated overnight at 4°C with primary antibodies against ANXA1 (Cat# 30797, 1:1000; CST, USA), occludin (Cat# 91131, 1:500; CST), ZO‐1 (Cat# 13663, 1:1000; CST), VEGF‐R2 (Cat# 2479, 1:1000; CST), and β‐actin (Cat# 4970, 1:5000; CST).

Techniques: Expressing

Journal: CNS Neuroscience & Therapeutics

Article Title: Annexin A1 Mitigates Blood–Brain Barrier Disruption in a Sepsis‐Associated Encephalopathy Model by Enhancing the Expression of Occludin and Zonula Occludens‐1 ( ZO ‐1)

doi: 10.1111/cns.70173

Figure Lengend Snippet: The peptide Annexin A1 (Ac2‐26) prevented LPS‐induced increased brain endothelial monolayer permeability in bEnd.3 brain microvascular endothelial cells (BVECs). Cells were stimulated with LPS (1 μg/mL) with or without ANXA1 (Ac2‐26) (0.25, 0.5 μM) for 48 h. (A) Endothelial permeability was assessed by FITC‐dextran permeation; (B) protein expression of occludin and ZO‐1 (*** p < 0.005 vs. control group; ## , ### p < 0.01, 0.005 vs. LPS group).

Article Snippet: The membrane was blocked with 5% non‐fat milk for 2 h and then incubated overnight at 4°C with primary antibodies against ANXA1 (Cat# 30797, 1:1000; CST, USA), occludin (Cat# 91131, 1:500; CST), ZO‐1 (Cat# 13663, 1:1000; CST), VEGF‐R2 (Cat# 2479, 1:1000; CST), and β‐actin (Cat# 4970, 1:5000; CST).

Techniques: Permeability, Expressing, Control

Journal: CNS Neuroscience & Therapeutics

Article Title: Annexin A1 Mitigates Blood–Brain Barrier Disruption in a Sepsis‐Associated Encephalopathy Model by Enhancing the Expression of Occludin and Zonula Occludens‐1 ( ZO ‐1)

doi: 10.1111/cns.70173

Figure Lengend Snippet: Annexin A1 (Ac2‐26) reduced the expression of VEGF‐A and VEGF‐R2 in bEnd.3 BVECs. Cells were stimulated with LPS (1 μg/mL) with or without ANXA1 (Ac2‐26) (0.25, 0.5 μM) for 48 h. (A) mRNA levels of VEGF‐A and VEGF‐R2; (B) protein levels of VEGF‐A; (C) protein levels of VEGF‐R2 (*** p < 0.005 vs. control group; ## , ### p < 0.01, 0.005 vs. LPS + group).

Article Snippet: The membrane was blocked with 5% non‐fat milk for 2 h and then incubated overnight at 4°C with primary antibodies against ANXA1 (Cat# 30797, 1:1000; CST, USA), occludin (Cat# 91131, 1:500; CST), ZO‐1 (Cat# 13663, 1:1000; CST), VEGF‐R2 (Cat# 2479, 1:1000; CST), and β‐actin (Cat# 4970, 1:5000; CST).

Techniques: Expressing, Control

Journal: CNS Neuroscience & Therapeutics

Article Title: Annexin A1 Mitigates Blood–Brain Barrier Disruption in a Sepsis‐Associated Encephalopathy Model by Enhancing the Expression of Occludin and Zonula Occludens‐1 ( ZO ‐1)

doi: 10.1111/cns.70173

Figure Lengend Snippet: VEGF‐A abolished the protective effects of ANXA1 (Ac2‐26) against LPS‐induced increased brain endothelial monolayer permeability in bEnd.3 BVECs. Cells were stimulated with LPS (1 μg/mL) with or without ANXA1 (Ac2‐26) (0.5 μM) or VEGF‐A (10 ng/mL). (A) Brain endothelial permeability was assessed by FITC‐dextran permeation; (B) protein expression of occludin and ZO‐1 (*** p < 0.005 vs. control group; ## p < 0.01 vs. LPS group; && p < 0.01 vs. LPS + ANXA1 group).

Article Snippet: The membrane was blocked with 5% non‐fat milk for 2 h and then incubated overnight at 4°C with primary antibodies against ANXA1 (Cat# 30797, 1:1000; CST, USA), occludin (Cat# 91131, 1:500; CST), ZO‐1 (Cat# 13663, 1:1000; CST), VEGF‐R2 (Cat# 2479, 1:1000; CST), and β‐actin (Cat# 4970, 1:5000; CST).

Techniques: Permeability, Expressing, Control

Journal: bioRxiv

Article Title: Anxa1+ dopamine neuron vulnerability defines prodromal Parkinson’s disease bradykinesia and procedural motor learning impairment

doi: 10.1101/2024.12.22.629963

Figure Lengend Snippet: (A) Schematic representation of FACS followed by bulk RNA-seq analysis of DANs from 10– 12-week-old MP-early (n = 5) and cMfn2KO-early (n = 5) mice, 3 weeks post-tamoxifen injection. (B) Graph displaying significantly downregulated DAN subtype markers (MP-early: blue; cMfn2KO-early: magenta; FDR < 0.05). (C) ISH (left) and IF (right) demonstrating co-expression of Anxa1, Aldh1a1, and Th in the SNc. Low- and high-magnification scale bars are 200 μm and 30 μm, respectively. (D) 3D reconstruction showing different DAN subpopulations visualized using anti-Anxa1, anti-Aldh1a1, and anti-Th antibodies in the SNc. (E) Coronal sections illustrating the spatial distribution of these subpopulations in the SNc. (F) ISH depicting Anxa1, Aldh1a1, and Th expression in the SNc of control (Ctrl) and MP-early mice. (G) Line graph showing the dorsoventral distribution of the three SNc DAN subpopulations in Ctrl (n = 5) and MP-early (n = 5) mice. (H) Boxplot illustrating the proportions of these subpopulations in Ctrl and MP-early mice (two-way ANOVA, Genotype × Subtype: F(2,24) = 49.66, p < 0.0001; *p = 0.0452, ***p < 0.0001, Sidak’s post-hoc test). (I) Illustration showing ANXA1 mRNA quantification in iPSC-DANs from PD patients carrying the SNCA-trp mutation. (J) Upper: Boxplots showing the expression of various SNc DAN subtype markers in iPSC- DANs. Lower: Boxplots comparing ALDH1A1 (left) and ANXA1 (right) mRNA levels between healthy controls and SNCA-trp iPSC-DANs. (K) Schematic representation of the low-concentration 6-OHDA lesion model. (L) ISH showing Anxa1, Aldh1a1, and Th expression in the SNc of intact and lesioned hemispheres. (M) Boxplot comparing the proportions of the three SNc DAN subpopulations in the intact versus lesioned hemispheres (n = 4; RM two-way ANOVA, Side × Subtype: F(2,9) = 36.33, p < 0.0001; *p < 0.05, ***p = 0.0002, Sidak’s post-hoc test). (N) Schematic illustration of cAtg7KO mice and IF images showing LB-like p62+ inclusions (arrows) in SNc DANs. (O) IF images showing the expression of Anxa1, Aldh1a1, and Th in the SNc of Ctrl and cAtg7KO mice. (P) Boxplot showing the proportions of the three SNc DAN subpopulations in Ctrl (n = 5) and cAtg7KO (n = 5) mice (two-way ANOVA, Genotype × Subtype: F(2,24) = 8.470, p = 0.0016; **p = 0.0082, Sidak’s post-hoc test). Boxplots display all data points, with the 25th and 75th percentiles (boxes), median (center), and maxima (whiskers). Abbreviations: PD: Parkinson’s Disease, SNc: Substantia Nigra pars compacta, DANs: Dopaminergic Neurons, FACS: Fluorescence-Activated Cell Sorting, RNA-seq: RNA Sequencing, Tmxf: tamoxifen, MP: MitoPark, cMfn2KO: Conditional Mitofusin 2 Knockout, FDR: False Discovery Rate, FC: fold change, Br: bregma, A: anterior, D: dorsal, L: lateral, ISH: In Situ Hybridization, IF: Immunofluorescence, ml: medial lemniscus, Ctrl: Control, iPSC- DANs: Induced Pluripotent Stem Cell-Derived Dopaminergic Neurons, CT: threshold cycle, SNCA-trp: SNCA-triplication, 6-OHDA: 6-Hydroxydopamine, mfb: medial forebrain bundle, cAtg7KO: Conditional Atg7 Knockout, LB: Lewy Bodies. See also Figure S1 .

Article Snippet: Following this blocking step, the sections were incubated overnight at 4°C with primary antibodies: rabbit anti-Anxa1 (Cat# 71-3400, Thermo-Fisher), goat anti-Aldh1a1 (Cat# AF5869, R&D Systems), rabbit anti-MOR (Cat# ab17934, Abcam), chicken anti-TH (Cat# ab76442, Abcam), mouse anti-TH (Cat# AB152, Sigma-Aldrich), rabbit anti-GFP (Cat# A6455, Invitrogen), chicken anti-GFP (Cat# A10262, Invitrogen), mouse anti-p62 (Cat# 610833, BD Biosciences) and rabbit anti-RFP (Cat# 600-401-379-RTU, Rockland), all diluted in 2.5% donkey serum and 0.3% Triton-X in PBS.

Techniques: RNA Sequencing Assay, Injection, Expressing, Control, Mutagenesis, Concentration Assay, Fluorescence, FACS, Knock-Out, In Situ Hybridization, Immunofluorescence, Derivative Assay

Journal: bioRxiv

Article Title: Anxa1+ dopamine neuron vulnerability defines prodromal Parkinson’s disease bradykinesia and procedural motor learning impairment

doi: 10.1101/2024.12.22.629963

Figure Lengend Snippet: (A) Schematic illustration representing the labeling of Anxa1+ fibers in Anxa1-Flp mice. (B) IF images showing the overlap between eYFP labeling and Anxa1-positive dopaminergic neurons (Anxa1+ DANs). Low-magnification scale bar: 200 μm; high-magnification scale bars: 30 μm. (C) Fluorescent images of eYFP+ labeled fibers and somata at various anteroposterior coronal levels within the SNc and SNr. (D) 3D reconstruction of eYFP+ labeled fibers localized in the SNr. (E) Schematic coronal sections illustrating the distribution of eYFP+ labeled fibers in the SNr. (F) Fluorescent images displaying eYFP+ labeled axons at distinct anteroposterior coronal levels within the CP and ACB. (G) 3D reconstruction of eYFP+ labeled fibers within the CP and ACB. (H) Schematic coronal sections showing the distribution of eYFP+ labeled fibers within the CP and ACB. (I) IF images illustrating eYFP+ labeled fibers in dendron bouquets within the SNr (left; scale bar: 30 μm) and in dorsolateral CP patches and subcallosal streak (right; scale bar: 500 μm). (J) Schematic diagram depicting bilateral Anxa1+ fiber labeling in combination with low-concentration 6-OHDA treatment. (K) IF images showing eYFP labeling alongside Th immunoreactivity in the SNc of the intact (left) and lesioned (right) sides (scale bars: 200 μm). (L) IF images depicting eYFP labeling alongside Th immunoreactivity in the CP of the intact (left) and lesioned (right) sides (scale bars: 500 μm). (M) Schematic coronal sections showing the distribution of eYFP+ labeled fibers within the CP and ACB of intact and lesioned side. (N) Heatmap showing the fluorescent intensity of eYFP in the intact and lesioned sides, with the difference in intensity displayed (n = 5 mice). Abbreviations: eYFP: enhanced Yellow Fluorescent Protein, DANs: Dopaminergic Neurons, SNc: Substantia Nigra pars compacta, SNr: Substantia Nigra pars reticulata, CP: Caudoputamen, A: anterior, D: dorsal, L: lateral, dlCP: dorso-lateral CP, ACB: Accumbens, Br: bregma, 6-OHDA: 6- Hydroxydopamine, mfb: medial forebrain bundle, OD: optic density, Diff.: difference. See also Figure S2 .

Article Snippet: Following this blocking step, the sections were incubated overnight at 4°C with primary antibodies: rabbit anti-Anxa1 (Cat# 71-3400, Thermo-Fisher), goat anti-Aldh1a1 (Cat# AF5869, R&D Systems), rabbit anti-MOR (Cat# ab17934, Abcam), chicken anti-TH (Cat# ab76442, Abcam), mouse anti-TH (Cat# AB152, Sigma-Aldrich), rabbit anti-GFP (Cat# A6455, Invitrogen), chicken anti-GFP (Cat# A10262, Invitrogen), mouse anti-p62 (Cat# 610833, BD Biosciences) and rabbit anti-RFP (Cat# 600-401-379-RTU, Rockland), all diluted in 2.5% donkey serum and 0.3% Triton-X in PBS.

Techniques: Labeling, Concentration Assay

Journal: bioRxiv

Article Title: Anxa1+ dopamine neuron vulnerability defines prodromal Parkinson’s disease bradykinesia and procedural motor learning impairment

doi: 10.1101/2024.12.22.629963

Figure Lengend Snippet: (A) Illustration (top) and fluorescent images (bottom) depicting the various groups of mice utilized for FP recordings in the CP (scale bars: 500 μm). (B) Schematic representation of coronal sections illustrating the recording locations within the CP for each experimental group. (C) Illustration (top) and image (bottom) demonstrating the experimental setup for FP recordings during reward delivery. (D) Z-scores of FP signals aligned to the water drop touch across all recordings from the Anxa1 GCaMP-dCP (mice = 7, n = 10 recordings) and DAT GCaMP-dCP (mice = 3, n = 4 recordings) groups. Line graphs represent average AUC across three time windows relative to the water drop touch for Anxa1 GCaMP-dCP (RM one-way ANOVA, F(1.725, 15.52) = 13.26, p = 0.0006; second vs. third: ***p = 0.0003, Sidak’s post-hoc test) and DAT GCaMP-dCP (RM one-way ANOVA, F(1.093, 3.278) = 10.39, p = 0.0421; first vs. second: p = 0.0228, Sidak’s post-hoc test). (E) Z-scores of FP signals aligned to the water drop touch for recordings from the dLight-dCP (mice = 6, n = 13 recordings) and dLight-cCP (mice = 6, n = 11 recordings) groups. Line graphs illustrate average AUC across three time windows relative to water drop touch for dLight-dCP (RM one-way ANOVA, F(1.918, 23.01) = 9.658, p = 0.001; second vs. third: *p = 0.0037, Sidak’s post-hoc test) and dLight-cCP (RM one-way ANOVA, F(1.829, 18.29) = 10.58, p = 0.0011; first vs. second: *p = 0.002, first vs. third: p = 0.0228, Sidak’s post-hoc test). (F-G) Comparison of AUC differences for the second- and third-time windows relative to the first (baseline) across all recorded groups of mice (one-sample two-tailed t-test; second DAT GCaMP- dCP: p = 0.0308, second dLight-cCP: *p = 0.0028, third dLight-cCP: p = 0.0148, Bonferroni correction applied). (H) Left: Illustration (top) and fluorescent images (bottom) depicting ChRmine-oScarlet expression in the SNc of DAT-ChRmine and Anxa1-ChRmine mice (scale bar: 300 μm). Right: Illustration of the experimental setup and timeline for the self-stimulation protocol. (I) Boxplot comparing active pokes during the first two days of the self-stimulation protocol across groups (Ctrl-Tdtomato: n = 6, Anxa1-ChRmine: n = 10, DAT-ChRmine: n = 9; Kruskal-Wallis test, p = 0.0007; Ctrl-Tdtomato vs. DAT-ChRmine: *p = 0.004, Anxa1-ChRmine vs. DAT-ChRmine: *p = 0.0029, Dunn’s post-hoc test). (J) Boxplot comparing preference for the stimulation-associated poke during the first two days of the self-stimulation protocol across groups (Ctrl-Tdtomato: n = 6, Anxa1-ChRmine: n = 10, DAT- ChRmine: n = 9; Kruskal-Wallis test, p = 0.0003; Ctrl-Tdtomato vs. DAT-ChRmine: p = 0.0131, Anxa1-ChRmine vs. DAT-ChRmine: **p = 0.0003, Dunn’s post-hoc test). (K) Line graphs depicting the number of left and right pokes across pre- and post-reversal experimental days for Ctrl-Tdtomato, Anxa1-ChRmine, and DAT-ChRmine mice (RM two-way ANOVA, Side × Days: F(4, 32) = 15.70, p < 0.0001; Right vs. Left: **p < 0.001, Sidak’s post-hoc test). (L) Line graphs showing preference for the stimulation-associated port across pre- and post-reversal experimental days for Ctrl-Tdtomato, Anxa1-ChRmine, and DAT-ChRmine groups (Friedman test, p = 0.0004; day −1 vs. day 1: *p = 0.008 and day 1 vs. day 4: ##p = 0.0011, Dunn’s post-hoc test). (M) Illustration (top) and fluorescent images (middle) depicting TeTx-GFP expression in the SNc and CP of Anxa1-TeTx mice (scale bars: 300 μm and 500 μm). Bottom: Illustration demonstrating the experimental setup and timeline for the fixed ratio (FR) and progressive ratio (PR) protocol. (N) Line graphs presenting the number of pokes during the FR and PR protocols across experimental days for the two groups. (O) Boxplots comparing total pokes in the FR5 (left) and PR (right) stages of the protocol between the two groups. Data in line graphs are expressed as mean ± SEM. Boxplots depict all data points, the 25th and 75th percentiles (box), the median (center), and the maxima (whiskers). Abbreviations: DANs: Dopaminergic neurons, FP: Fiber photometry, dCP: dorsal Caudoputamen, cCP: central Caudoputamen, DAT: Dopamine Transporter, AUC: Area Under the Curve, SNc: Substantia Nigra pars compacta, Ctrl: Control, Opto: optogenetic stimulation, TeTx: Tetanus Toxin, eGFP: enhanced Green Fluorescent Protein, Ac: active, In: inactive, FR: Fixed Ratio, PR: Progressive Ratio. See also Supplementary Video1 .

Article Snippet: Following this blocking step, the sections were incubated overnight at 4°C with primary antibodies: rabbit anti-Anxa1 (Cat# 71-3400, Thermo-Fisher), goat anti-Aldh1a1 (Cat# AF5869, R&D Systems), rabbit anti-MOR (Cat# ab17934, Abcam), chicken anti-TH (Cat# ab76442, Abcam), mouse anti-TH (Cat# AB152, Sigma-Aldrich), rabbit anti-GFP (Cat# A6455, Invitrogen), chicken anti-GFP (Cat# A10262, Invitrogen), mouse anti-p62 (Cat# 610833, BD Biosciences) and rabbit anti-RFP (Cat# 600-401-379-RTU, Rockland), all diluted in 2.5% donkey serum and 0.3% Triton-X in PBS.

Techniques: Comparison, Two Tailed Test, Expressing, Control

Journal: bioRxiv

Article Title: Anxa1+ dopamine neuron vulnerability defines prodromal Parkinson’s disease bradykinesia and procedural motor learning impairment

doi: 10.1101/2024.12.22.629963

Figure Lengend Snippet: (A) Illustration showing the experimental setup of the open field arena. Mouse behavior was recorded from below for 20 minutes while neuronal activity was measured using FP. (B) Boxplots comparing the correlation between the FP signal and linear speed (left boxplot) with the correlation between the shuffled signal and linear speed (right boxplot) for Anxa1 GCaMP- dCP (mice = 9, n = 9 recordings, **p = 0.0039, two-sided paired t-test), DAT GCaMP-dCP (mice = 5, n = 7 recordings), dLight-dCP (mice = 6, n = 8 recordings, **p = 0.0078, two-sided paired t- test), and dLight-cCP (mice = 6, n = 7 recordings, *p = 0.016, two-sided paired t-test). (C) Left: Image showing the pose estimation of six body parts using DLC. Right: Dendrogram depicting the hierarchical clustering of 37 behavioral motifs using VAME. Motifs are color-coded according to the simplified action categories: Stationary/Sniffing, Turn, Rear, Groom, Walk, and Sniff/Walk. (D) Graph showing the linear speed distribution for each behavioral motif. (E) Heatmaps displaying the mean z-scored FP signals (GCaMP and dLight) aligned to the onset of each motif. Motifs are ranked based on speed, as in d. (F-G) Graphs showing the correlation of FP signal AUC (time window: 0 to 1 s) between Anxa1-GCaMP-dCP and dLight-dCP ( F , R = 0.70, p < 0.01) or dLight-cCP ( G , R = −0.52, p < 0.01). (H) Graph showing the correlation between FP signal AUC (time window: 0 to 1 s) for Anxa1-GCaMP-dCP and mean motif linear speed (R = 0.83, p < 0.01). Boxplots show all data points, with the 25th and 75th percentiles (box), the median (center), and the maxima (whiskers). Abbreviations: OFT: open filed test, DANs: Dopaminergic neurons, FP: Fiber photometry, DAT: Dopamine transporter, dCP: dorsal Caudoputamen, cCP: central Caudoputamen, DLC: DeepLabCut, VAME: Variational Animal Motion Embedding, AUC: Area under the curve, R: Pearson correlation coefficient. See also Figure S3 and Supplementary Video2 .

Article Snippet: Following this blocking step, the sections were incubated overnight at 4°C with primary antibodies: rabbit anti-Anxa1 (Cat# 71-3400, Thermo-Fisher), goat anti-Aldh1a1 (Cat# AF5869, R&D Systems), rabbit anti-MOR (Cat# ab17934, Abcam), chicken anti-TH (Cat# ab76442, Abcam), mouse anti-TH (Cat# AB152, Sigma-Aldrich), rabbit anti-GFP (Cat# A6455, Invitrogen), chicken anti-GFP (Cat# A10262, Invitrogen), mouse anti-p62 (Cat# 610833, BD Biosciences) and rabbit anti-RFP (Cat# 600-401-379-RTU, Rockland), all diluted in 2.5% donkey serum and 0.3% Triton-X in PBS.

Techniques: Activity Assay

Journal: bioRxiv

Article Title: Anxa1+ dopamine neuron vulnerability defines prodromal Parkinson’s disease bradykinesia and procedural motor learning impairment

doi: 10.1101/2024.12.22.629963

Figure Lengend Snippet: (A) Illustration depicting the distinct groups of mice, and the experimental setup utilized for the open field test. (B) Representative path-tracking traces of the various experimental groups during the open field test. (C–E) Boxplots presenting the mean linear speed (C) , mean angular speed (D) , and immobility time (E) for each control group (Ctrl-Tdtomato: n = 18, Ctrl-Asc Acid: n = 9, Ctrl-early: n = 14, Ctrl-middle: n = 15) and experimental group (Anxa1-TeTx: n = 14, 6OHDA-low: n = 11, MP-early: n = 13, MP-middle: n = 14). Statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001 (two-sided unpaired t-test). (F) Graphs showing the acceleration distribution density for each group of mice. (G) Left: Illustration categorizing mouse behavior into five action motifs using VAME: Stationary/Sniffing, Turn, Rear, Groom, and Walk & Sniff/Walk. Right: PCA of motif usage, reporting the PC1 and PC2 for each group of mice. (H) Loading plot displaying PC1 and PC2 values for the different behavioral motifs, color-coded according to the action categories defined in (G). (I) Graphs depicting linear regression analyses between the mean motif speed and the eigenvalues of PC1 (top; R = −0.762, p < 0.0001) or PC2 (bottom; R = −0.1494, p = 0.3772). (J) Graphs illustrating the motif usage (Δtime) across different groups (data expressed as mean ± SEM). Motifs are arranged from bottom to top according to increasing speed. Colored symbols represent statistically significant altered motifs (Anxa1-TeTx: yellow, 6-OHDA-low: pink, MP-early: light blue, MP-middle: dark blue; multiple two-sided unpaired t-tests, adjusted p < 0.05, Holm-Sidak method). (K) Illustration showing the probability of one “walk” motif occurring after the other. (L) Statemap depiction of “walk” motifs (as nodes) and transition probabilities (as weighted edges) from control mice. (M) Statemap depiction of “walk” motifs (as nodes) and transition probabilities (as weighted edges) from Anxa1-TeTx mice (n = 3 mice; red: significant upregulated transition, yellow: significant downregulated transition; Fisher’s Exact Test, adjusted p < 0.05, Benjamini-Hochberg correction). The colored nodes represent the motifs with statistically significant altered usage in Anxa1-TeTx mice according to (J) (red: significant upregulated motif, yellow: significant downregulated transition motif). Boxplots display all data points, with the 25th and 75th percentiles (box), the median (center), and the maxima (whiskers). Abbreviations: DANs: Dopaminergic neurons, PD: Parkinson’s disease, OFT: open field test, Ctrl: Control, Asc: ascorbic, TeTx: Tetanus Toxin, 6-OHDA: 6-hydroxydopamine, MP: MitoPark, DLC: DeepLabCut, PCA: Principal component analysis, PC: Principal component, R: Pearson correlation coefficient. See also Figure S4 and S5.

Article Snippet: Following this blocking step, the sections were incubated overnight at 4°C with primary antibodies: rabbit anti-Anxa1 (Cat# 71-3400, Thermo-Fisher), goat anti-Aldh1a1 (Cat# AF5869, R&D Systems), rabbit anti-MOR (Cat# ab17934, Abcam), chicken anti-TH (Cat# ab76442, Abcam), mouse anti-TH (Cat# AB152, Sigma-Aldrich), rabbit anti-GFP (Cat# A6455, Invitrogen), chicken anti-GFP (Cat# A10262, Invitrogen), mouse anti-p62 (Cat# 610833, BD Biosciences) and rabbit anti-RFP (Cat# 600-401-379-RTU, Rockland), all diluted in 2.5% donkey serum and 0.3% Triton-X in PBS.

Techniques: Control

Journal: bioRxiv

Article Title: Anxa1+ dopamine neuron vulnerability defines prodromal Parkinson’s disease bradykinesia and procedural motor learning impairment

doi: 10.1101/2024.12.22.629963

Figure Lengend Snippet: (A) Illustration and image depicting the experimental setup for the 2-choice arrow maze task. (B) Representative heatmaps showing the position density of Ctrl-Tdtomato (left) and Anxa1-TeTx (right) mice on the first day (top) and fourth day (bottom) in the arrow maze task. (C) Boxplots displaying mean linear speed (left) and immobility time (right) for Ctrl-Tdtomato (n = 18) and Anxa1-TeTx (n = 10) mice on the first and fourth days of the arrow maze task. (RM two-way ANOVA for mean linear speed, Group effect: F(1,26) = 6.198, p = 0.0195; Ctrl-Tdtomato vs. Anxa1-TeTx: *p = 0.0473, Sidak’s post-hoc test). (D) Left: Boxplot illustrating the difference between rewarded and error transitions in Ctrl-Tdtomato (n = 18) and Anxa1-TeTx (n = 10) mice on the first and fourth days of the arrow maze task (RM two-way ANOVA, Group × Days: F(1,26) = 6.563, p = 0.0166; Day 1 vs. Day 2: ###p = 0.0009, Ctrl-Tdtomato vs. Anxa1-TeTx: **p < 0.0001, Sidak’s post-hoc test). Right: Boxplot illustrating locomotor efficacy for Ctrl-Tdtomato (n = 18) and Anxa1-TeTx (n = 10) mice on the first and fourth days of the arrow maze task (RM two-way ANOVA, Group × Days: F(1,26) = 6.065, p = 0.0207; Day 1 vs. Day 2: ###p < 0.0001, Ctrl-Tdtomato vs. Anxa1-TeTx: *p = 0.0027 and **p < 0.0001, Sidak’s post-hoc test). (E) Illustration representing the groups of mice (left) and the experimental design (right) for the forelimb-reaching task. (F) Example sequence of reaching efforts illustrating the three phases of movement: start, grasp, and retract, demonstrated in on-target (top) and off-target (bottom) reaching efforts. (G) Images depicting the method utilized to assess the performance of mice in the reaching task. (H) Heatmap showing the density of reaching efforts around the target for all groups of mice on the first (top) and fourth (bottom) days of the task. (I) Graph presenting the reach fraction distribution across 0.1 cm distance bins from the target for Ctrl mice (n = 8), Anxa1-TeTx mice (n = 4), and Anxa1-Arch mice (n = 6) on the first (top) and fourth (bottom) days of the task. (J) Boxplot summarizing the target accuracy fraction (defined as < 0.1 cm distance) for all groups of mice (RM two-way ANOVA, Group × Days: F(2,14) = 5.301, p = 0.0181; Day 1 vs. Day 2: #p = 0.0123, Ctrl vs. Anxa1-TeTx: **p = 0.0063, Ctrl vs. Anxa1-Arch: *p = 0.0268, Sidak’s post-hoc test). Boxplots display all data points, the 25th and 75th percentiles (box), the median (center), and the maxima (whiskers). Abbreviations: C: Central arm, L: Left arm, R: Right arm, DANs: Dopaminergic neurons, Ctrl: Control, TeTx: Tetanus toxin. Rew: reward, Er: error. See also Figure S6 and Supplementary Video3.

Article Snippet: Following this blocking step, the sections were incubated overnight at 4°C with primary antibodies: rabbit anti-Anxa1 (Cat# 71-3400, Thermo-Fisher), goat anti-Aldh1a1 (Cat# AF5869, R&D Systems), rabbit anti-MOR (Cat# ab17934, Abcam), chicken anti-TH (Cat# ab76442, Abcam), mouse anti-TH (Cat# AB152, Sigma-Aldrich), rabbit anti-GFP (Cat# A6455, Invitrogen), chicken anti-GFP (Cat# A10262, Invitrogen), mouse anti-p62 (Cat# 610833, BD Biosciences) and rabbit anti-RFP (Cat# 600-401-379-RTU, Rockland), all diluted in 2.5% donkey serum and 0.3% Triton-X in PBS.

Techniques: Sequencing, Control