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(A) Representative immunoblot of endogenous synaptophysin (Syp) <t>and</t> <t>synaptobrevin-2</t> (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.
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(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.

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