Journal: bioRxiv

Article Title: Leishmania exploits the macrophage endoplasmic reticulum-shaping protein CLIMP-63 to modulate mitochondrial biogenesis and bioenergetics

doi: 10.64898/2026.03.19.712868

Figure Lengend Snippet: BMM (treated with siRNA to either CLIMP-63 or RTN4 or treated with control siRNA, Ctr) were infected with L. donovani or L. amazonensis metacyclic promastigotes and at various time points post-phagocytosis parasite replication and PV size were assessed. (A) Quantification of L. donovani parasite burden in CLIMP-63 depleted BMM at 6, 24, 48, and 72 h post-infection. (B) Quantification of L. amazonensis parasite burden in CLIMP-63 depleted BMM at 6, 24, 48, and 72 h post-infection. (C) Quantification of PV size in CLIMP-63-depleted BMM infected with L. amazonensis at 48 and 72 h post-phagocytosis. (D) Quantification of L. donovani parasite burden in RTN4-depleted BMM at 6, 24, 48, and 72 h post-infection. (E) Quantification of L. amazonensis parasite burden in RTN4-depleted BMM at 6, 24, 48, and 72 h post-infection. (F) Quantification of PV size in RTN4-depleted BMM infected with L. amazonensis at 48 and 72 h post-phagocytosis. Data in (A, B, D, E) are presented as the means ± SEM of values of one representative experiment of three independent experiments. Data in (C, F) are presented as a violin plot with means ± standard deviations (SD) of values from three independent experiments for a total of 450 PVs. Statistics were calculated using one-way analyses of variance (ANOVA) with Sidak’s multiple comparison test with * P ≤ 0.05, *** P ≤ 0.001 and **** P ≤ 0.0001 significance. Blots showing the efficacy the siRNA-mediated CLIMP-63 and RTN4 knockdowns are shown in S3 Fig and S4 Fig, respectively.

Article Snippet: The mouse anti-CLIMP-63 monoclonal antibody (sc-393544) was from Santa Cruz Biotechnology, the rabbit anti-RTN4 polyclonal antibody (ab186735) and the rat anti-BrdU (ab6326) were from Abcam, the rabbit polyclonal antibody RTN4 (10950-1-AP) was from Proteintech, the mouse anti-phosphoglycan (Galβ1,4Manα1-PO4) CA7AE monoclonal antibody ( ) was from Cedarlane, the rabbit anti-β-actin polyclonal antibody was from Cell Signalling, the rabbit anti-Tom20 polyclonal antibody (EPR15581-54) was from Abcam, and the rat anti-LAMP-1 monoclonal antibody 1D4B developed by J.T.

Techniques: Control, Infection, Comparison

Journal: bioRxiv

Article Title: Fingolimod acutely facilitates the activation of TRKB

doi: 10.64898/2026.02.27.707919

Figure Lengend Snippet: TRKB signaling in vitro , related to . (A) Quantification of TRKB mRNA levels by qPCR in N2A cells non-transfected and transfected with TRKB.wt plasmid. (B) 30min BDNF treatment (10ng/ml) increase TRKB homodimerization by PCA in N2A cells. Unpaired t-test, t=8,351, df=30, p<0.0001, n=16. (C) BDNF (10ng/ml) increased TRKB phosphorylation at tyrosine 816 in cortical neurons in ELISA. Unpaired t-test, t=3,433, df=14, p=0.004, n=8. (D) BDNF (10ng/ml) increased interaction in cortical neurons between TRKB and its signaling partner PLCgamma1 in ELISA. Unpaired t-test, t=2,646, df=13, p=0.02, n=7/8. (E) BDNF (10ng/ml) promotes interaction between TRKB and activated Src family kinase in cortical neurons measured by ELISA. Unpaired t-test, t=2,512, df=12, p=0.027, n=6/8. Data is expressed as mean+SEM.

Article Snippet: The following day, after 3x 10 min PBS washes, biotinylated BDNF (0, 2, 4, 6, 8, 10 ng/ml, Alomone Labs, #B-250-B) was added with or without FNG (0, 0.1, 1, 10 μM) for 1h at room temperature.

Techniques: In Vitro, Transfection, Plasmid Preparation, Phospho-proteomics, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Fingolimod acutely facilitates the activation of TRKB

doi: 10.64898/2026.02.27.707919

Figure Lengend Snippet: FNG induces TRKB signaling in vitro . (A) Acute administration of FNG (0.1, 1, 10 μM, 30 min) on N2A cells induces TRKB homodimerization (n=8/group) in PCA. Statistical analysis performed by One-way ANOVA and Tukey’s post hoc test, F (3, 76) = 13,05 p(0.1 μM)=0.0013, p(1 μM)=0.007, p(10 μM)=0.0011. (B) Acute administration of FNG and the effect on TRKB phosphorylation at tyrosine 816 measured by ELISA in primary cultured cortical cells from rat embryos (n=12/group). One-way ANOVA and Šidák’s post hoc test, F (3, 44) = 18,11, p<0.0001. (C) Acute administration of 10 μM FNG on TRKB:PLCg1 interaction measured by ELISA (n=5/group). Unpaired t-test, t=3,734, df=8, p=0.0053. (D) Detection of plasma membrane localized TRKB following acute FNG treatment (n=18/group). Kruskal-Wallis and Dunn’s post hoc test, KW= 17,81, p=0.0011. (E) Effect of the BDNF-blocking peptide TRKB_FC on the effects of acute FNG treatment in TRKB-PLCg1 interaction measured by ELISA (n=18/group). Two-way ANOVA and Tukey’s post hoc test, F (1, 100) = 15,93 p=0.0001 for the treatment effect. Data is expressed as mean+SEM.

Article Snippet: The following day, after 3x 10 min PBS washes, biotinylated BDNF (0, 2, 4, 6, 8, 10 ng/ml, Alomone Labs, #B-250-B) was added with or without FNG (0, 0.1, 1, 10 μM) for 1h at room temperature.

Techniques: In Vitro, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Cell Culture, Clinical Proteomics, Membrane, Blocking Assay

Journal: bioRxiv

Article Title: Fingolimod acutely facilitates the activation of TRKB

doi: 10.64898/2026.02.27.707919

Figure Lengend Snippet: TRKB activation by FNG is an early event in FNG action and requires BDNF. (A) Acute administration of FNG and the activation of Src family kinase measured by ELISA on primary rat cortical neurons (n=5/group). One way ANOVA and Fisher’s LSD, F (3, 16) = 40,13, p(1 μM, 10 μM)<0.0001. (B) Effect of acute FNG treatments on TKRB dimerization in N2A cells in presence of kinase inhibitors PP1 and k252a (n=12/group). One way ANOVA and Tuckey’s post hoc test, F (5, 66) = 19,72, p<0.0001. (C) Ligand binding assay for TRKB and biotinylated BDNF in presence of increasing dose of FNG (n= 6/group). (D) Acute administration of FNG does not induce dimerization of the TRKB mutant Y433F (n=12/group). Two-way ANOVA and Šidák’s post hoc test, F (2, 55) = 6,216, p(Y433F +/- )=0.0032, p(Y433F)=0.0002. Data is expressed as mean+SEM. (E) Effect of increasing concentration of FNG on the binding of biotinylated fluoxetine on TRKB (n=6/group).

Article Snippet: The following day, after 3x 10 min PBS washes, biotinylated BDNF (0, 2, 4, 6, 8, 10 ng/ml, Alomone Labs, #B-250-B) was added with or without FNG (0, 0.1, 1, 10 μM) for 1h at room temperature.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Ligand Binding Assay, Mutagenesis, Concentration Assay, Binding Assay

Journal: bioRxiv

Article Title: Fingolimod acutely facilitates the activation of TRKB

doi: 10.64898/2026.02.27.707919

Figure Lengend Snippet: The effect of FNG administration on contextual fear conditioning. (A) Combined effects of pravastatin (10mg/kg for 14 days in drinking water) and fingolimod (1mg/kg, 2h prior) on the ability of female mice to discriminate between context A and context B, expressed as discrimination index. Pravastatin treatment impairs the discrimination ability of the two contexts, Three-way ANOVA F(1, 35) = 30,66, p<0.0001. (B) 10 min foot shock conditioning session for BDNF.wt and BDNF.het mice. FNG treatment (1 mg/kg, 2h prior) did not affect the freezing behaviour. Two way ANOVA, F(1, 21) = 9,068, p=0,9925, while BDNF.hets generally freeze more compared to control, Two way ANOVA F(1, 21)=12.78, p=0.0018. (C) FNG treatment (1 mg/kg, 2h prior, blue bars) had no effect on BDNF.wt animals exposed for 5 min to a novel environment but could rescue the generalized freezing of the BDNF.het animals. F(1, 21) = 6,279, p(interaction)=0,0205, p(controls)=0.0001, p(BDNF.het)=0.004283 by two-way ANOVA and uncorrected Fisher’s LSD multiple comparisons (D) FNG treatment did not have any effect on the freezing behavior of BDNF.wt and BDNF.het mice placed back in the conditioning environment without foot shocks. Two way ANOVA, F (1, 21) = 0,05193, p=0,8219. Similarly as the conditioning, BDNF.het freeze more compared to wild-type littermates F(1, 21)=8.636, p= p=0.0078. In a cohort of animals pre-treated with pravastatin (10mg/kg for 14 days in drinking water) (Panels E,F,G) FNG (1mg/kg, 2h prior) did not affect the conditioning. BDNF.het mice freeze significantly more compared to control animals (E), Two way ANOVA F(1,16)=11.8, p=0.0034. (F) Similarly as for the condition, BDNF.het mice freeze more than the wt littermates, Two way ANOVA, F(1,16)=24.49; p=0.0001. FNG treatment (1mg/kg, 2h prior) could not rescue the freezing behaviour in BDNF.het mice and had no significant effects on BDNF.wt animals. (G) Animals placed back in the conditioning environment without foot shock did not show any behavioral effect after FNG treatment (1mg/kg, 2h prior). As in the previous conditions, BDNF.het mice generally freeze more than the wt, Two way ANOVA, F(1,16)=9.374, p=0.0075. Data expressed as mean+SEM, n=5/7 per group.

Article Snippet: The following day, after 3x 10 min PBS washes, biotinylated BDNF (0, 2, 4, 6, 8, 10 ng/ml, Alomone Labs, #B-250-B) was added with or without FNG (0, 0.1, 1, 10 μM) for 1h at room temperature.

Techniques: Control

Journal: Brain Sciences

Article Title: Neonatal Handling Positively Modulates Anxiety, Sensorimotor Gating, Working Memory, and Cortico-Hippocampal Neuroplastic Adaptations in Two Genetically Selected Rat Strains Differing in Emotional and Cognitive Traits

doi: 10.3390/brainsci15080776

Figure Lengend Snippet: Western blot analysis of the BDNF ( A , B ), trkB ( C , D ), and the PSA-NCAM ( E , F ), in the prefrontal (prelimbic/infralimbic) cortex of the RHA and the RLA rats, either untreated controls (CTRL) or treated with neonatal handling (NH). The values represent the densitometric analysis of the BDNF/GAPDH ( B ), trkB/GAPDH ( D ), and the PSA-NCAM/GAPDH ( F ) band grey optical density (O.D.) ratios. The bars denote the mean ± S.E.M. of 9–10 rats, in each experimental group. ***: p < 0.001 (post hoc Duncan’s multiple range test).

Article Snippet: Molecular weight (mw) standards (Precision Plus Protein Western C Standards, Cat# 161–0376, Bio-Rad, Hercules, CA, USA) and recombinant human BDNF protein (rhBDNF) (Cat# B-257, Alomone Labs, Jerusalem, Israel) were run in parallel.

Techniques: Western Blot

Journal: Brain Sciences

Article Title: Neonatal Handling Positively Modulates Anxiety, Sensorimotor Gating, Working Memory, and Cortico-Hippocampal Neuroplastic Adaptations in Two Genetically Selected Rat Strains Differing in Emotional and Cognitive Traits

doi: 10.3390/brainsci15080776

Figure Lengend Snippet: Western blot analysis of the BDNF ( A , B ), trkB ( C , D ), and the PSA-NCAM ( E , F ), in the anterior cingulate cortex of the RHA and the RLA rats, either untreated controls (CTRL) or treated with neonatal handling (NH). The values represent the densitometric analysis of the BDNF/GAPDH ( B ), trkB/GAPDH ( D ), and the PSA-NCAM/GAPDH ( F ) band grey optical density (O.D.) ratios. The bars denote the mean ± S.E.M. of 9–10 rats, in each experimental group. **: p < 0.01 (post hoc Duncan’s multiple range test).

Article Snippet: Molecular weight (mw) standards (Precision Plus Protein Western C Standards, Cat# 161–0376, Bio-Rad, Hercules, CA, USA) and recombinant human BDNF protein (rhBDNF) (Cat# B-257, Alomone Labs, Jerusalem, Israel) were run in parallel.

Techniques: Western Blot

Journal: Brain Sciences

Article Title: Neonatal Handling Positively Modulates Anxiety, Sensorimotor Gating, Working Memory, and Cortico-Hippocampal Neuroplastic Adaptations in Two Genetically Selected Rat Strains Differing in Emotional and Cognitive Traits

doi: 10.3390/brainsci15080776

Figure Lengend Snippet: Western blot analysis of the BDNF ( A , B ), trkB ( C , D ), and the PSA-NCAM ( E , F ), in the ventral hippocampus of the RHA and the RLA rats, either untreated controls (CTRL) or treated with neonatal handling (NH). The values represent the densitometric analysis of the BDNF/GAPDH ( B ), trkB/GAPDH ( D ), and the PSA-NCAM/GAPDH ( F ) band grey optical density (O.D.) ratios. The bars denote the mean ± S.E.M. of 9–10 rats, in each experimental group. *: p < 0.05; **: p < 0.02 (post hoc Duncan’s multiple range test).

Article Snippet: Molecular weight (mw) standards (Precision Plus Protein Western C Standards, Cat# 161–0376, Bio-Rad, Hercules, CA, USA) and recombinant human BDNF protein (rhBDNF) (Cat# B-257, Alomone Labs, Jerusalem, Israel) were run in parallel.

Techniques: Western Blot

Journal: Brain Sciences

Article Title: Neonatal Handling Positively Modulates Anxiety, Sensorimotor Gating, Working Memory, and Cortico-Hippocampal Neuroplastic Adaptations in Two Genetically Selected Rat Strains Differing in Emotional and Cognitive Traits

doi: 10.3390/brainsci15080776

Figure Lengend Snippet: Western blot analysis of the BDNF ( A , B ), trkB ( C , D ), and the PSA-NCAM ( E , F ), in the dorsal hippocampus of the RHA and the RLA rats, either untreated controls (CTRL) or treated with neonatal handling (NH). The values represent the densitometric analysis of the BDNF/GAPDH ( B ), trkB/GAPDH ( D ), and the PSA-NCAM/GAPDH ( F ) band grey optical density (O.D.) ratios. The bars denote the mean ± S.E.M. of 9–10 rats, in each experimental group.

Article Snippet: Molecular weight (mw) standards (Precision Plus Protein Western C Standards, Cat# 161–0376, Bio-Rad, Hercules, CA, USA) and recombinant human BDNF protein (rhBDNF) (Cat# B-257, Alomone Labs, Jerusalem, Israel) were run in parallel.

Techniques: Western Blot