Journal: bioRxiv

Article Title: Pathogenic KIF1A variants differentially disrupt axonal trafficking and impede synaptic development

doi: 10.64898/2026.01.14.699478

Figure Lengend Snippet: (A) Representative immunoblot of endogenous synaptophysin (Syp) and synaptobrevin-2 (Syb2) protein in wild-type and KIF1A mutant iNeurons at DIV21. No significant change is observed for total neuronal protein levels for either synaptic cargo ( Figure S1B ). (B-E) Examination of subcellular Syp and Syb2 content in the iNeuron soma (A): Representative immunocytochemistry images (B) of somatodendritic compartment (MAP2, white) and synaptic cargos (SYB2, green; SYB2, magenta). Quantification of mean soma intensity normalized to wild-type for SYB2 (D) and SYP (E). Plots display mean ± standard deviation of experimental replicates, n = 30 neurons from 3 independent experiments, reported p -values determined using linear mixed effect model. (F-H) Examination of subcellular Syp and Syb2 content at heterologous presynaptic compartments formed in response to neuroligin-1-expressing HEK cells (F): Representative immunocytochemistry images (G) of heterologous presynapses formed on NL1+ HEK cells (SYB2, green; SYP, magenta; TUBB3 axonal marker, blue) in wild-type and KIF1A mutant iNeurons. Quantification of mean soma intensity normalized to wild-type for SYB2 (D) and SYP (E). Plots display mean ± standard deviation of experimental replicates, n = 30 HEK cells interacting with numerous axons from 3 independent experiments, reported p -values determined using linear mixed effect model.

Article Snippet: The following primary antibodies were used: NF-H (mouse anti-NF-H; BioLegend, 801601; 1:1000), Synapsin I/II (guinea pig anti-Synapsin I/II; Synaptic Systems, 106-004; 1:1000), PSD-95 (rabbit anti-PSD-95; Synaptic Systems, 124-008; 1:500), MAP2 (mouse anti-MAP2; EMD Millipore, MAB3418; 1:200), Synaptophysin (mouse anti-Synaptophysin; Sigma-Aldrich, S5768; 1:200), and Synaptobrevin-2 (rabbit anti-Synaptobrevin-2; Cell Signaling, 13508; 1:250).

Techniques: Western Blot, Mutagenesis, Immunocytochemistry, Standard Deviation, Expressing, Marker

Journal: bioRxiv

Article Title: PQBP1-dependent alternative RNA splicing underlies high calorie diet-induced cognitive impairment

doi: 10.1101/2025.06.02.657545

Figure Lengend Snippet: a) Upper panels show density, length, diameter and volume of dendritic spines in the mouse splenial cortex. The density was changed while the other parameters were not significantly affected by HFD. Lower panels show VAMP2-positive dendritic spines of mice co-injected by AAV2-VAMP2-mCherry and AAV1-EGFP directed by synapsin-1 promoter.saralab) Dynamics of dendritic spines during 24 hours of observation by two-photon microscopy. Eliminated spines during 24 hours are indicated with red arrowheads. Elimination and stability was changed while formation was not affected by HFD.saralac) Western blot of cerebral cortex with a post-synapse marker PSD95. Three blots were independently performed with a mouse. Right panels show quantitative analysis of signal intensities of the PSD95 bands obtained from 9 blots of 3 mice.saralad) Re-examination of exon skipping by an independent group of Syn1-Cre/PQBP1-cKO mice (N=3), HFD-1W mice (N=3), HFD-6W mice (N=3) and C57BL/6J mice (N=3). Odds ratios of each mice vs C57BL/6J mice are shown with p-value by Fisher’s exact test and FDR by Benjamini-Hochberg procedure.

Article Snippet: Immunohistochemistry was performed using the following primary antibodies: mouse anti-PQBP1 (1:400, sc-374260, Santa Cruz Biotechnology, Dallas, TX, USA); goat anti-Iba1 (1:500, 011-27991, Wako, Osaka, Japan); rabbit anti-S100B (1:500, ab52642, Abcam, Cambridge, UK); mouse anti-MAP2 (1:500, sc-32791, Santa Cruz Biotechnology, Dallas, TX, USA); mouse anti-VAMP2 (1:200, 13647S, Cell Signaling Technology, Danvers, MA, USA); rabbit anti-PSD95 (1:200, 3409S, Cell Signaling Technology, USA). mouse GAD67 (1:200, ab26116, Abcam, Cambridge, UK). mouse GPHN (1:200, ab177154, Abcam, Cambridge, UK) Secondary antibodies were as follows: Cy3-AffiniPure Donkey Anti-Goat IgG (H + L) (1:500, 705165003 Jackson ImmunoResearch); Cy3-AffiniPure Donkey Anti-Rabbit IgG (H + L) (1:500 711165152 Jackson ImmunoResearch); donkey anti-mouse IgG Alexa488 (1:1000, A21202, Molecular Probes, Eugene, OR, USA).

Techniques: Injection, Microscopy, Western Blot, Marker

Journal: bioRxiv

Article Title: PQBP1-dependent alternative RNA splicing underlies high calorie diet-induced cognitive impairment

doi: 10.1101/2025.06.02.657545

Figure Lengend Snippet: a) Protocol of the rescue experiment for HFD-induced synapse pathology by pioglitazone. 6 mouse groups were prepared.saralab) Results of Morris water maze-test in the 6 mouse groups.saralac) Results of Y maze-test in the 6 mouse groups.saralad) Results of fear conditioning test in the 6 mouse groups.saralae) Western blot analysis of PQBP1, PSD95 (post-synapse marker) and VAMP2 (pre-synapse marker) in total cerebral cortex tissues of the 6 mouse groups. Right graphs show quantitative analyses of the results.saralaf) Immunohistochemistry of PQBP1 in MAP2-positive neurons and of VAMP2-PSD95 co-staining for mature synapses in 6 mouse groups. Right graphs show quantitative analyses of signal intensities of neuronal PQBP1 and of numbers of mature synapses. The mean value of three cortex areas was used as a representative value for a mouse.saralag) Immunohistochemistry of GAD67 (pre-synapse marker of inhibitory synapses) and GPHN (post-synapse marker of inhibitory synapses). Right graphs show quantitative analyses of signal intensities.

Article Snippet: Immunohistochemistry was performed using the following primary antibodies: mouse anti-PQBP1 (1:400, sc-374260, Santa Cruz Biotechnology, Dallas, TX, USA); goat anti-Iba1 (1:500, 011-27991, Wako, Osaka, Japan); rabbit anti-S100B (1:500, ab52642, Abcam, Cambridge, UK); mouse anti-MAP2 (1:500, sc-32791, Santa Cruz Biotechnology, Dallas, TX, USA); mouse anti-VAMP2 (1:200, 13647S, Cell Signaling Technology, Danvers, MA, USA); rabbit anti-PSD95 (1:200, 3409S, Cell Signaling Technology, USA). mouse GAD67 (1:200, ab26116, Abcam, Cambridge, UK). mouse GPHN (1:200, ab177154, Abcam, Cambridge, UK) Secondary antibodies were as follows: Cy3-AffiniPure Donkey Anti-Goat IgG (H + L) (1:500, 705165003 Jackson ImmunoResearch); Cy3-AffiniPure Donkey Anti-Rabbit IgG (H + L) (1:500 711165152 Jackson ImmunoResearch); donkey anti-mouse IgG Alexa488 (1:1000, A21202, Molecular Probes, Eugene, OR, USA).

Techniques: Western Blot, Marker, Immunohistochemistry, Staining

Journal: bioRxiv

Article Title: PQBP1-dependent alternative RNA splicing underlies high calorie diet-induced cognitive impairment

doi: 10.1101/2025.06.02.657545

Figure Lengend Snippet: a) Protocol of the rescue experiment for HFD-induced synapse pathology by AAAV-PQBP1. 4 mouse groups were prepared.saralab) Results of Morris water maze-test in the 4 mouse groups.saralac) Results of Y maze-test in the 4 mouse groups.saralad) Results of fear conditioning test in the 4 mouse groups.saralae) Western blot analysis of PQBP1, PSD95 (post-synapse marker) and VAMP2 (pre-synapse marker) in total cerebral cortex tissues of the 4 mouse groups. Right graphs show quantitative analyses of the results.saralaf) Immunohistochemistry of PQBP1 in MAP2-positive neurons and of VAMP2-PSD95 co-staining for mature synapses in 4 mouse groups. Right graphs show quantitative analyses of signal intensities of neuronal PQBP1 and of numbers of mature synapses. The mean value of three cortex areas was used as a representative value for a mouse.saralag) Immunohistochemistry of GAD67 (pre-synapse marker of inhibitory synapses) and GPHN (post-synapse marker of inhibitory synapses). Right graphs show quantitative analyses of signal intensities.

Article Snippet: Immunohistochemistry was performed using the following primary antibodies: mouse anti-PQBP1 (1:400, sc-374260, Santa Cruz Biotechnology, Dallas, TX, USA); goat anti-Iba1 (1:500, 011-27991, Wako, Osaka, Japan); rabbit anti-S100B (1:500, ab52642, Abcam, Cambridge, UK); mouse anti-MAP2 (1:500, sc-32791, Santa Cruz Biotechnology, Dallas, TX, USA); mouse anti-VAMP2 (1:200, 13647S, Cell Signaling Technology, Danvers, MA, USA); rabbit anti-PSD95 (1:200, 3409S, Cell Signaling Technology, USA). mouse GAD67 (1:200, ab26116, Abcam, Cambridge, UK). mouse GPHN (1:200, ab177154, Abcam, Cambridge, UK) Secondary antibodies were as follows: Cy3-AffiniPure Donkey Anti-Goat IgG (H + L) (1:500, 705165003 Jackson ImmunoResearch); Cy3-AffiniPure Donkey Anti-Rabbit IgG (H + L) (1:500 711165152 Jackson ImmunoResearch); donkey anti-mouse IgG Alexa488 (1:1000, A21202, Molecular Probes, Eugene, OR, USA).

Techniques: Western Blot, Marker, Immunohistochemistry, Staining

Journal: bioRxiv

Article Title: PQBP1-dependent alternative RNA splicing underlies high calorie diet-induced cognitive impairment

doi: 10.1101/2025.06.02.657545

Figure Lengend Snippet: a) Protocol of the rescue experiment for HFD-induced synapse pathology by AAV-Cask, AAV-Cyfip2, AAV-Cacnb1 or AAV-empty. 4 mouse groups were prepared.saralab) Results of Morris water maze-test in the 4 mouse groups.saralac) Results of Y maze-test in the 4 mouse groups.saralad) Results of fear conditioning test in the 4 mouse groups.saralae) Western blot analysis of PQBP1, PSD95 (post-synapse marker) and VAMP2 (pre-synapse marker) in total cerebral cortex tissues of the 4 mouse groups. Right graphs show quantitative analyses of the results.saralaf) Immunohistochemistry of PQBP1 in MAP2-positive neurons and of VAMP2-PSD95 co-staining for mature synapses in 4 mouse groups. Right graphs show quantitative analyses of signal intensities of neuronal PQBP1 and of numbers of mature synapses. The mean value of three cortex areas was used as a representative value for a mouse.saralag) Immunohistochemistry of GAD67 (pre-synapse marker of inhibitory synapses) and GPHN (post-synapse marker of inhibitory synapses). Right graphs show quantitative analyses of signal intensities.

Article Snippet: Immunohistochemistry was performed using the following primary antibodies: mouse anti-PQBP1 (1:400, sc-374260, Santa Cruz Biotechnology, Dallas, TX, USA); goat anti-Iba1 (1:500, 011-27991, Wako, Osaka, Japan); rabbit anti-S100B (1:500, ab52642, Abcam, Cambridge, UK); mouse anti-MAP2 (1:500, sc-32791, Santa Cruz Biotechnology, Dallas, TX, USA); mouse anti-VAMP2 (1:200, 13647S, Cell Signaling Technology, Danvers, MA, USA); rabbit anti-PSD95 (1:200, 3409S, Cell Signaling Technology, USA). mouse GAD67 (1:200, ab26116, Abcam, Cambridge, UK). mouse GPHN (1:200, ab177154, Abcam, Cambridge, UK) Secondary antibodies were as follows: Cy3-AffiniPure Donkey Anti-Goat IgG (H + L) (1:500, 705165003 Jackson ImmunoResearch); Cy3-AffiniPure Donkey Anti-Rabbit IgG (H + L) (1:500 711165152 Jackson ImmunoResearch); donkey anti-mouse IgG Alexa488 (1:1000, A21202, Molecular Probes, Eugene, OR, USA).

Techniques: Western Blot, Marker, Immunohistochemistry, Staining