Journal: Nature Communications

Article Title: Remyelination protects neurons from DLK-mediated neurodegeneration

doi: 10.1038/s41467-024-53429-5

Figure Lengend Snippet: a Schematic of the viral CRISPR/Cas9 approach to disrupt DLK and/or LZK in retinal cells. b Retinae stained with phosphorylated c-Jun and mCherry at 10 weeks post tamoxifen following treatment with sgLacZ/sgGFP, or sgDLK/sgLZK on the opposite eye. Inlays are of boxed areas and scale bar is 5 µm in boxed areas. c Representative images of phosphorylated c-Jun immunostaining in retinal flatmounts following administration of sgLacZ/sgGFP, sgDLK/sgDLK, sgLZK/sgLZK, or sgDLK/sgLZK. d Overview of retinas with locations of cleaved caspase-3+ cells in the GCL indicated by black X in viral-treated eyes. e Density of phosphorylated c-Jun cells within the GCL following sgDLK/sgDLK administration. ** p = 0.0080. Myrf fl/fl n = 3, Myrf ΔiSox10 n = 4 mice. f Density of phosphorylated c-Jun positive cells in the GCL following sgLZK/sgLZK administration. ns p = 0.0766. Myrf fl/fl n = 4, Myrf ΔiSox10 n = 5 mice. g Density of phosphorylated c-Jun cells within the GCL following sgDLK/sgLZK administration. * p = 0.0148. Myrf fl/fl n = 3, Myrf ΔiSox10 n = 5 mice. h Cleaved-caspase-3+ cells within the GCL following sgDLK/sgDLK administration. * p = 0.0254. Myrf fl/fl n = 2, Myrf ΔiSox10 n = 5 mice. i The density of cleaved-caspase-3+ cells within the GCL after sgLZK/sgLZK administration. ns P = 0.8530. Myrf fl/fl n = 3, Myrf ΔiSox10 n = 5 mice. j Cleaved-caspase-3+ cells within the GCL following sgDLK/sgLZK administration. * p = 0.0125. Myrf fl/fl n = 3, Myrf ΔiSox10 n = 5 mice. Scale bars are 50 µm in ( b , c ) and 500 µm in ( d ). Connected lines indicate retinae from the same mouse in ( e – j ). Paired Student’s t test with Holm-Šidák correction for multiple comparisons was used from ( e – j ). All statistical tests are two-sided. ns not statistically significant. Error bars are SEM. Source data for this Figure are provided as a Source Data file. a created in BioRender. Duncan (2023) BioRender.com/e99a533.

Article Snippet: Plasmids generated in this study for DLK or LZK knockout have been deposited at Addgene (Plasmid #208834, 208835, 208836, 208837) and AAV2-hSyn1-Cre is available upon request.

Techniques: CRISPR, Staining, Immunostaining

Journal: bioRxiv

Article Title: Phosphorylation regulates auto-inhibition of kinesin KIF3A

doi: 10.1101/503680

Figure Lengend Snippet: (A) Domain organization of the mouse KIF3A. Mouse and human KIF3A show high sequence homology. Serine-689 of mouse KIF3A is equivalent to serine-690 of the human KIF3A. The conserved CaMKII recognition sequence RXXS/T is marked by green characters and the conserved serine residue is highlighted in red in both human and mouse sequences. (B) Activated CaMKII phosphorylates KIF3A tail domain. Inactive FLAG-CaMKII or active FLAG-CaMKII were incubated with 5 μg of GST-WT-tail, GST-S689A-tail or GST-S689D-tail respectively for 45 min at 30 °C. The reaction mixes were separated by SDS-PAGE and subjected to Western analysis with the respective antibodies. (C) For CaMKII-activated sample, HeLa cells were treated with high [Ca 2+ ] (1.26 mM), 10 μM ionomycin and 10 nM Calyculin A for 30 min to activate CaMKII. The CaMKII-inhibited sample was treated with 10 μM of KN-62 for 30 min, and subsequently treated with high [Ca 2+ ] stimulation supplied with 10 μM of KN-62 for 30 min. Total lysates (1.5 mg) from each experimental condition was subjected to anti-KIF3A-immunoprecipitation overnight to pull down endogenous KIF3A, followed by SDS-PAGE and western blot analysis using anti-Phospho-serine/threonine antibodies to detect KIF3A phosphorylation status. 100 μg of total lysate was loaded as input. (D) HeLa cells were transfected with FLAG-KIF3A and FLAG-KIF3A-S689A respectively. The cells were treated similarly as 1(C) to activate or inhibit CaMKII. Total cell lysate (1 mg) was incubated with FLAG-M2 beads overnight to pull down the KIF3A constructs, followed by SDS-PAGE and western blot analysis using anti-Phospho-serine/threonine antibodies to detect KIF3A phosphorylation status. 100 μg of total lysate was loaded as input.

Article Snippet: Mus musculus full-length KIF3A (Addgene plasmid 13742) construct was purchased from Addgene (Cambridge, MA, USA).

Techniques: Sequencing, Residue, Incubation, SDS Page, Western Blot, Immunoprecipitation, Phospho-proteomics, Transfection, Construct

Journal: bioRxiv

Article Title: Phosphorylation regulates auto-inhibition of kinesin KIF3A

doi: 10.1101/503680

Figure Lengend Snippet: (A) Mixtures of bacterial purified His 6 -KIF3A-motor (5 μg) and 5 μg of purified GST-KIF3A WT-Tail, GST-S689A-Tail or GST-S689D-Tail were incubated with Glutathione Sepharose 4B. GST-fusion proteins were captured and the co-precipitated complex was separated by SDS-PAGE and analyzed by western analysis using anti-His and anti-GST antibodies. (B) FLAG-KIF3A-motor construct was co-transfected with GST, GST-KIF3A-Tail, GST-S689A-Tail and GST-S689D-Tail constructs in HEK293 cells. Total lysates were incubated with anti-FLAG M2 Affinity Gel and the eluents were detected by western blot using anti-FLAG or anti-GST. (C) SPR approach showed that the interaction between KIF3A-motor and S689A-Tail is stronger than WT-Tail, and S689D-Tail. GST-tail domains were captured on the chips by the immobilized GST antibody. Different concentrations of KIF3A motor were then injected into the Biacore system. GST-WT-Tail and GST-S689D-Tail show low binding intensity with KIF3A-motor. The sensograms are presented.

Article Snippet: Mus musculus full-length KIF3A (Addgene plasmid 13742) construct was purchased from Addgene (Cambridge, MA, USA).

Techniques: Purification, Incubation, SDS Page, Western Blot, Construct, Transfection, Injection, Binding Assay

Journal: bioRxiv

Article Title: Phosphorylation regulates auto-inhibition of kinesin KIF3A

doi: 10.1101/503680

Figure Lengend Snippet: (A) Schematic diagram showing the KIF3A FRET construct. When KIF3A is in an autoinhibited state, EYFP and ECFP come close enough to cause FRET. (B) Phosphorylation levels of KIF3A and CaMKII at different time points. HeLa cells co-transfected with FLAG-KIF3A and FLAG-CaMKII were treated with high [Ca 2+ ] buffer for 10, 20, 30, 45 and 60 min to activate CaMKII. The cells were then lysed and subjected to FLAG-IP. The phosphorylation levels of KIF3A and CaMKII were detected by anti-phospho-serine/threonine antibody. The graphs showing the band intensity of CaMKII and KIF3A at each time point were generated. (C) HeLa cells were transfected with pEYFP-ECFP as positive control, pEYFP-ΔTail-ECFP as negative control, and co-transfected with pXJ-GST-CaMKII and pEYFP-KIF3A-ECFP , which were further treated with low [Ca 2+ ] buffer (non-phosphorylated KIF3A) and high [Ca 2+ ] buffer (phosphorylated KIF3A). The graph shows the FRET efficiency of all samples. The number of cells analyzed (N) and statistical significance are indicated. The average FRET efficiency for each sample is in red.

Article Snippet: Mus musculus full-length KIF3A (Addgene plasmid 13742) construct was purchased from Addgene (Cambridge, MA, USA).

Techniques: Construct, Phospho-proteomics, Transfection, Generated, Positive Control, Negative Control

Journal: bioRxiv

Article Title: Phosphorylation regulates auto-inhibition of kinesin KIF3A

doi: 10.1101/503680

Figure Lengend Snippet: (A) U2-OS cells were transfected with pEYFP-ECFP as positive control, pEYFP-ΔTail-ECFP as negative control, and co-transfected with pXJ-GST-CaMKII and pEYFP-KIF3A-ECFP . Control and experimental cells were treated with low or high [Ca 2+ ] buffers. The graph shows the FRET efficiency of all samples was developed by combing the FRET efficiency of all the cells collected. The number of cells analyzed (N) and statistical significance are indicated. (B) FRET efficiency of cells expressing KIF3A-motor-ECFP and EYFP-tail of KIF3A WT and mutants. U2-OS cells were transfected with pEYFP-ECFP or co-transfected with pEYFP and pECFP, pEYFP-KIF3A and motor-pECFP, pEYFP-S689A and motor-pECFP, pEYFP-S689D and motor-pECFP , respectively. Three independent experiments were performed and the graph is developed by combing the FRET efficiency of all the cells collected. The number of cells analyzed (N) and statistical significance are indicated.

Article Snippet: Mus musculus full-length KIF3A (Addgene plasmid 13742) construct was purchased from Addgene (Cambridge, MA, USA).

Techniques: Transfection, Positive Control, Negative Control, Control, Expressing

Journal: bioRxiv

Article Title: Phosphorylation regulates auto-inhibition of kinesin KIF3A

doi: 10.1101/503680

Figure Lengend Snippet: U2-OS cells (A) or HeLa cells (B) were co-transfected with pXJ-GST-CaMKII and pEYFP-KIF3A-ECFP . Control or experimental cells treated with low or high [Ca 2+ ] (indicated by “+”) buffers were lysed and sent for western-blotting (left). The phosphorylation levels of the total lysates were detected by phospho-serine/threonine antibody. The corresponding sizes of GST-CaMKII (80 kD) and EYFP-KIF3A-ECFP (125 kD) were indicated with asterix on the blots. Three independent experiments using U2-OS cells and two independent experiments using HeLa cells were performed. In all experiments, cells treated with high [Ca 2+ ] buffer showed more intense phospho-KIF3A bands.

Article Snippet: Mus musculus full-length KIF3A (Addgene plasmid 13742) construct was purchased from Addgene (Cambridge, MA, USA).

Techniques: Transfection, Control, Western Blot, Phospho-proteomics

Journal: bioRxiv

Article Title: Phosphorylation regulates auto-inhibition of kinesin KIF3A

doi: 10.1101/503680

Figure Lengend Snippet: (A) Left panel: the profile of UV absorbance of NADH at 340 nm (ATPase assays) of purified His 6 -KIF3A motor domain (black), nucleotide-free His 6 -KIF3A-motor domain (red) and His 6 -KIF3A-motor domain incubated with Mg 2+ -ADP (blue) over 5 min; right panel: the ATP hydrolysis activities of the three samples. (B) Left panel: the profile of UV absorbance of NADH at 340 nm (ATPase assay) of nucleotide-free His 6 -KIF3A-motor domain (black), His 6 - KIF3A-motor domain incubated with Mg 2+ -ADP (green) and His 6 -KIF3A-motor domain incubated with quercetin (red), resveratrol (blue) and NBD-Cl (pink) over 5 min; right panel: the ATP hydrolysis activity of the five samples. (C) Left panel: the profile of UV absorbance of NADH at 340 nm (ATPase assay) of nucleotide-free His 6 -KIF3A motor domain (black, line); His-KIF3A-motor domain incubated with GST-KIF3A-WT-tail (red, line), GST-KIF3A-S689A-tail (blue, line) and GST-KIF3A-S689D-Tail (orange, line); GST-KIF3A-WT-Tail (green, dash line), GST-KIF3A-S689A-Tail (blue, dash line) and GST-KIF3A-S689D-Tail (purple, dash line) over 5 min; right panel: the ATP hydrolysis activities of the five samples. The data in each graph are expressed as the mean of three replicates with error bars representing standard deviations. The statistical significances are indicated.

Article Snippet: Mus musculus full-length KIF3A (Addgene plasmid 13742) construct was purchased from Addgene (Cambridge, MA, USA).

Techniques: Purification, Incubation, ATPase Assay, Activity Assay

Journal: bioRxiv

Article Title: Phosphorylation regulates auto-inhibition of kinesin KIF3A

doi: 10.1101/503680

Figure Lengend Snippet: (A) Sequence alignment of KIF3A isoforms (Mm, Mus Musculus; Hs, Homo sapiens; Rn, Rattus norvegicus; Pp, Poeciliopsis prolifica) and Kinesin-1 (Dm, Drosophila melanogaster) . Amino acid sequence “AKP” are conserved between KIF3A and Kinesin-1. (B) Complex state molecular model of the mouse KIF3A peptide, residues 686-695 (shown in red cartoon backbone and cyan stick/sphere side-chain representation), and its motor dimer (gray cartoon representation) The “AKP” sequence is labelled and Serine-689 is specifically presented in sphere. ADP (cyan sticks) and Mg 2+ ion (orange sphere), are also shown in the model. (C, D, E) Representative structure from molecular dynamic simulations showing the interaction between the motor domain dimer (gray surface representation) and the tail peptide (red cartoon) of WT (C), S689A (D) and phosphorylated S689 (E) state of the tail domains. Serine-689, Alanine-689 and serine-689 PO 3 2- (pS689) is shown in sphere representation. The residues in the motor domain which form the hydrophobic pocket is shown in stick representation and labelled. Also see supplemental movies 1 to 3 for more details.

Article Snippet: Mus musculus full-length KIF3A (Addgene plasmid 13742) construct was purchased from Addgene (Cambridge, MA, USA).

Techniques: Sequencing

Journal: bioRxiv

Article Title: Phosphorylation regulates auto-inhibition of kinesin KIF3A

doi: 10.1101/503680

Figure Lengend Snippet: When KIF3A is in an autoinhibited state, the motor and tail domains interact with each other and prevent KIF3A from binding to microtubules. Phosphorylation relieves KIF3A from autoinhibition. The active KIF3 motor can then bind to microtubules and transport cargoes along the microtubules.

Article Snippet: Mus musculus full-length KIF3A (Addgene plasmid 13742) construct was purchased from Addgene (Cambridge, MA, USA).

Techniques: Binding Assay, Phospho-proteomics

Journal: Cell reports

Article Title: Trigeminal innervation and tactile responses in mouse tongue

doi: 10.1016/j.celrep.2024.114665

Figure Lengend Snippet: (A) Left: schematic of unilateral viral injection into the mandibular branch of the left TG in Vglut2 Cre mice. Right: neurons at the mandibular branch of the TG were labeled by tdTom. Scale bar: 300 μm. (B) tdTom-labeled trigeminal afferents at the surface of the tongue. Scale bar: 800 μm. Inset: An enlarged view of the tip of the tongue. Scale bar: 300 μm. (C) Single TG afferent terminals sparsely labeled by tdTom. Scale bars (left to right): 50, 100, and 50 μm. (D) Multiple TG afferents simultaneously labeled by tdTom. Top: two clusters of nerve fibers (green and magenta) co-innervating a single fungiform papilla. Scale bar: 100 μm. Center: two independent afferents (green and magenta) innervating two separate fungiform papillae. Scale bar: 50 μm. Bottom: two independent afferents (green and magenta) innervating several filiform papillae had overlap in their terminal fields. Arrowheads: endings in the same filiform papillae. Scale bar: 100 μm. (E) Clusters of filiform papillae innervated by separate TG afferent aggregates (blue arrows). Red arrowheads: fungiform papillae. Scale bar: 100 μm. (F) The terminal field size of TG afferents innervating fungiform (0.0036 ± 0.0015, n = 20 from 5 mice) or filiform papillae (0.0435 ± 0.0096, n = 14 from 6 mice). *** p = 9.59 × 10 −10 ; two-sided Welch’s t test.

Article Snippet: For sparse to dense labeling of mandibular branch neurons, the pipette containing AAVPHP.S-CAG-FLEX-tdTomato virus (Addgene 28306-PHP.S, 1.8×10 13 vg/mL) was targeted to 1–4 injection sites spaced 150–200 μm apart in the mandibular branch (lateral 2.1–2.4 mm, posterior 2.2–2.5 mm relative to bregma).

Techniques: Injection, Labeling

Journal: Scientific reports

Article Title: Glucagon-like peptide-1 reduces pancreatic β-cell mass through hypothalamic neural pathways in high-fat diet-induced obese rats.

doi: 10.1038/s41598-017-05371-4

Figure Lengend Snippet: Figure 5. Chronic administration of liraglutide increases c-fos expression and BDNF content in the hypothalamus, restores β-cell mass and improves glucose metabolism through hepatic afferent nerves. (a) Representative c-fos staining performed on brain sections derived from each group. Red circle indicates the VMH area in the hypothalamus. 3 V; third ventricle. Scale bar = 100 μm. (b and c) Quantification of c-fos expression in the VMH (b) and BDNF content in the hypothalamus (c) of each group (n = 6). (d) Representative insulin staining performed with pancreas sections and quantification of the insulin-positive area from each group (n = 6). Scale bar = 100 μm. (e) Blood glucose (upper) and plasma insulin (lower) levels during the glucose tolerance tests in each group (n = 6). *p < 0.05 vs. HFD (Sham, PBS) group, #p < 0.05 vs. HFD (Sham, Liraglutide) group. Treatments: PBS; intraperitoneal administration of PBS, Liraglutide; intraperitoneal administration of liraglutide, Sham; sham operation, VgX; hepatic afferent vagotomy. HFD; fed with high-fat diet.

Article Snippet: On the day of the experiment, GLP-1 (7–36) (Sigma, St. Louis, MO, USA) or liraglutide (Peptides International Inc., Louisville, KY, USA) was freshly dissolved in 0.1 M phosphate-buffered saline (PBS), adjusted to pH 7.0.

Techniques: Expressing, Staining, Derivative Assay, Clinical Proteomics