Journal: eLife

Article Title: Translatome analysis reveals cellular network in DLK-dependent hippocampal glutamatergic neuron degeneration

doi: 10.7554/eLife.101173

Figure Lengend Snippet: ( A ) Confocal images of Map3k12 (DLK) and Slc17a7 (VGLUT1) RNAscope analysis in hippocampal glutamatergic neurons at P15. Scale bar 1000 μm, 10 μm zoomed ROI. ( B ) Western blot of DLK and β-actin from protein extracts of hippocampal tissue of genotype indicated (age P60, each lane representing individual mice, N=3 mice/genotype). Arrow points to faint band of lower molecular weight in DLK(cKO) that is visible only under longer exposure, which may represent N-terminal truncated DLK produced using an alternative start codon (see C1). ( C ) IGV visual representation of RiboTag reads of Map3k12 (DLK) in Slc17a7 Cre/+ and Slc17a7 Cre/+ ;Map3k12 fl/fl , showing that mRNA for the floxed exon is reduced to ~1/3 level of control, while mRNA for other exons remained at a similar level as control. Dark blue illustration shows Map3k12 exon-intron structure, with pink triangles denoting loxP sites. Height of reads (y-axis) in gray or blue represents number of reads for the respective sequence. ( C1 ) Enlarged view of the dashed box in C, corresponding to the floxed exon and exons encoding the kinase domain shown in orange with red marking ATP binding site and green marking DLK palmitoylation site. Start ATG shown in bold, and candidate downstream alternative start ATGs labeled with arrows. ( D ) Illustration of DLK overexpression transgene. ( E,F ) Confocal z-stack images of NeuN immunostaining and DAPI of coronal sections of dorsal hippocampus in mice of genotype indicated, with enlarged view of CA1, CA3, and DG (dashed boxes, respectively). ~1-year-old control and Slc17a7 Cre/+ ;Map3k12 fl/fl mice for ( E ); 44–46 weeks old, control and Slc17a7 Cre/+ ; H11-DLK(iOE)/+ mice for ( F ). Scale bar hippocampus 1000 μm, enlarged view 50 μm. ( G, H, I ) Quantification of cross-sectional area from CA1, CA3, or DG in dorsal hippocampus, respectively (as outlined in ). Data points represent individual mice, averaged across 3 sections per mouse, N≥3 mice/genotype. Statistics: One way ANOVA with Dunnett’s multiple comparison test. ns, not significant; **** p<0.0001. Error bars represent SEM. Figure 1—figure supplement 1—source data 1. Original western blots for images shown in . Figure 1—figure supplement 1—source data 2. PDF showing original western blots for images shown in , along with relevant bands and genotypes. Original membranes corresponding to Panel B. Molecular weights shown using PageRuler Plus Prestained Protein Ladder. 10 min exposure (uncut membrane) was taken prior to 2 min exposure. Following 10 min exposure, membrane was cut and reprobed for DLK for 2 min exposure. Each lane represents a separate mouse. Lanes 1–3 show control samples, lanes 4–6 show DLK(cKO).

Article Snippet: Antibody , Rabbit polyclonal anti-Map3k12 antibody , Genetex , GTX124127; RRID: AB_11170703 , IF (1:250) tissue, (1:1000) cells, WB (1:1000); Lot #40653.

Techniques: RNAscope, Western Blot, Molecular Weight, Produced, Control, Sequencing, Binding Assay, Labeling, Over Expression, Immunostaining, Comparison, Membrane

Journal: eLife

Article Title: Translatome analysis reveals cellular network in DLK-dependent hippocampal glutamatergic neuron degeneration

doi: 10.7554/eLife.101173

Figure Lengend Snippet: ( A ) Western blot of DLK and β-actin in protein extracts of hippocampal tissue of Slc17a7 Cre/+ ;Map3k12 fl/fl and Map3k12 fl/fl littermate controls (age P60, each lane representing individual mice, N=3 mice/genotype). ( B ) Quantification of DLK protein level normalized to β-actin. Statistics: Unpaired t-test, *** p<0.001. Error bars represent SEM. ( C ) Confocal z-stack (max projection) images of NeuN immunostaining of coronal sections of the dorsal hippocampus in P15 and P60 mice of genotype indicated, respectively. Dashed boxes in CA1 pyramidal layers are enlarged below. Scale bar, 1000 μm in hippocampi; 100 μm in CA1 layer. ( D ) Quantification of CA1 pyramidal layer thickness. Each dot represents averaged thickness from 3 sections per mouse; N≥4 mice/genotype per timepoint. Statistics: Two-way ANOVA with Holm-Sidak multiple comparison test; ns, not significant. Error bars represent SEM. ( E ) Confocal z-stack (max projection) images of Tuj1 immunostaining of hippocampus CA1, CA3, and DG regions in control and Slc17a7 Cre/+ ;Map3k12 fl/fl mice (age P60). Dashed outlines mark ROI (region of interest) for fluorescence intensity quantification. Scale bar, 100 μm. ( F, G, H ) Tuj1 mean fluorescence intensity (MFI) after thresholding signals in dendritic regions in each hippocampal area. Each dot represents averaged intensity from 3 sections per mouse; N=4 control, 5 Slc17a7 Cre/+ ;Map3k12 fl/fl . Statistics: Unpaired t-test. ns, not significant. Error bars represent SEM. Figure 1—source data 1. Original western blots for images shown in . Figure 1—source data 2. PDF showing original western blots for images shown in , along with relevant bands and genotypes. Original membranes corresponding to Panel A. Molecular weights shown using PageRuler Plus Prestained Protein Ladder. See for more details. Each lane represents a separate mouse. Lanes 1–3 show control samples, lanes 4–6 show DLK(cKO).

Article Snippet: Antibody , Rabbit polyclonal anti-Map3k12 antibody , Genetex , GTX124127; RRID: AB_11170703 , IF (1:250) tissue, (1:1000) cells, WB (1:1000); Lot #40653.

Techniques: Western Blot, Immunostaining, Comparison, Control, Fluorescence

Journal: eLife

Article Title: Translatome analysis reveals cellular network in DLK-dependent hippocampal glutamatergic neuron degeneration

doi: 10.7554/eLife.101173

Figure Lengend Snippet: Shown are lower magnification confocal images of NeuN staining of coronal sections of mice of genotype and age indicated. ( A ) ~1-year-old control and Slc17a7 Cre/+ ;Map3k12 fl/fl animals. ( B ) P10, P15, and P60 control and Slc17a7 Cre/+ ; H11-DLK(iOE)/+ nimals. P60 tissue sections were stained at different time from those at P10, P15. ( C ) ~44–46 week old control and Slc17a7 Cre/+ ; H11-DLK(iOE)/+ animals. Arrows point to cortical thinning and ventricle expansion observed in some animals. Scale bar: 1000 μm.

Article Snippet: Antibody , Rabbit polyclonal anti-Map3k12 antibody , Genetex , GTX124127; RRID: AB_11170703 , IF (1:250) tissue, (1:1000) cells, WB (1:1000); Lot #40653.

Techniques: Staining, Control

Journal: eLife

Article Title: Translatome analysis reveals cellular network in DLK-dependent hippocampal glutamatergic neuron degeneration

doi: 10.7554/eLife.101173

Figure Lengend Snippet: ( A ) Confocal z-stack images of Slc17a7 and Map3k12 mRNAs at P15 in control and Slc17a7 Cre/ ; H11-DLK(iOE)/+ mice. Scale bar: 1000 μm. Inset shows single slice image of CA1 neurons, with dashed line showing individual nuclei as used for quantification. Scale bar: 10 μm. ( B ) Quantification of RNAscope puncta shown as ratio of Map3k12 to Slc17a7 within individual CA1 neuron nuclei. Data points represent individual mice, averaged across 3 sections per mouse, ≥50 cells per genotype, N=5, 3 mice. Statistics: Mann-Whitney U test. ** p<0.01. Error bars represent SEM. ( C ) Confocal z-stack images of control and Slc17a7 Cre/+ ; H11-DLK(iOE)/+ mice immunostained for DLK protein (P15, P60,~1 year) or no primary or LZK antibody control (P15) Dashed boxes are enlarged to the right. Individual strata are labeled in CA1, CA3; stratum pyramidale (SP), stratum radiatum (SR), stratum oriens (SO), stratum lacunosum-moleculare (SLM), stratum lucidum (SL). Scale bar 500 μm in hippocampi, 100 μm in CA1 and CA3. ( D, E ) Quantification of DLK mean fluorescence intensity from all layers in CA1, CA3, and DG at P10 ( D ) and P15 ( E ). N=4 mice/genotype for each timepoint. Statistics: Unpaired t-test. ns, not significant; * p<0.05. Error bars represent SEM.

Article Snippet: Antibody , Rabbit polyclonal anti-Map3k12 antibody , Genetex , GTX124127; RRID: AB_11170703 , IF (1:250) tissue, (1:1000) cells, WB (1:1000); Lot #40653.

Techniques: Control, RNAscope, MANN-WHITNEY, Labeling, Fluorescence

Journal: eLife

Article Title: Translatome analysis reveals cellular network in DLK-dependent hippocampal glutamatergic neuron degeneration

doi: 10.7554/eLife.101173

Figure Lengend Snippet: ( A ) RiboTag analysis of Stathmin family members, shown as transcripts per million (TPM). Differential expression analysis significance shown (p adj ). ns, not significant; **** p<0.0001. Stmn2 and Stmn4 appear to have comparable reads, however, this includes reads in a retained intron in Stmn2, which increases the reference gene length used for the TPM calculation for Stmn2. The significance of this intron retention may need further study. ( B, C ) Western blots of protein extracts from hippocampal tissue from P1, P8, P15, P60, and ~1-year-old mice, blotted for DLK, Flag, STMN4, STMN2, and actin in Slc17a7 Cre/+ ;Map3k12 fl/fl ( B ) and Slc17a7 Cre/+; H11-DLK(iOE)/+ ( C ), respectively. Larger molecular weight band of DLK in Slc17a7 Cre/+; H11-DLK(iOE)/+ would match the predicted molecular weight of DLK-T2A-tdTomato if T2A-peptide induced ‘self-cleavage’ due to ribosomal skipping is ineffective . ( D, E ) Relative DLK protein level normalized to actin and P1 control. N=3 mice/genotype. ( F, G ) Relative STMN4 protein level normalized to actin and P1 control. N=3 mice/genotype. Statistics: Two-way ANOVA with Sidak multiple comparisons test. ns, not significant; * p<0.05. All error bars represent SEM. Figure 4—figure supplement 1—source data 1. Original western blots for images shown in . Figure 4—figure supplement 1—source data 2. PDF showing original western blots for images shown in , along with relevant bands and genotypes. Pg.1 Original membranes corresponding to Panel B. Molecular weights shown using PageRuler Plus Prestained Protein Ladder. Each lane represents a separate mouse. Lanes 1,3,5,7,9 show control samples, lanes 2,4,6,8,10 show DLK(cKO) at P1, P8, P15, P60, and 1 year timepoints. Dotted lines indicate locations where membrane was cut and labeled with separate antibodies. Pg.2 Original membranes corresponding to Panel C. Molecular weights shown using Precision Plus Protein Dual Color Ladder. Each lane represents a separate mouse. Lanes 1,3,5,7,9 show control samples, lanes 2,4,6,8,10 show DLK(iOE) at P1, P8, P15, P60, and ~1 year timepoints. Dotted lines indicate locations where membrane was cut and labeled with separate antibodies. Samples were split, with half of each prepped sample loaded onto two membranes (membrane 1&2). Smaller molecular weight band matches expected size of DLK protein (and flag tagged DLK). Larger molecular weight band of DLK in Vglut1Cre/+;H11-DLKiOE/+ would match the predicted molecular weight of DLK-T2A-tdTomato if T2A-peptide induced ‘self- cleavage’ due to ribosomal skipping is ineffective. Pg.3 Original membranes corresponding to Panel C. Molecular weights shown using Precision Plus Protein Dual Color Ladder. Each lane represents a separate mouse. Lanes 1,3,5,7,9 show control samples, lanes 2,4,6,8,10 show DLK(iOE) at P1, P8, P15, P60, and ~1 year timepoints.

Article Snippet: Antibody , Rabbit polyclonal anti-Map3k12 antibody , Genetex , GTX124127; RRID: AB_11170703 , IF (1:250) tissue, (1:1000) cells, WB (1:1000); Lot #40653.

Techniques: Quantitative Proteomics, Western Blot, Molecular Weight, Control, Membrane, Labeling

Journal: eLife

Article Title: Translatome analysis reveals cellular network in DLK-dependent hippocampal glutamatergic neuron degeneration

doi: 10.7554/eLife.101173

Figure Lengend Snippet: ( A ) Volcano plot showing RiboTag analysis in Slc17a7 Cre/+; H11-DLK(iOE)/+ ;Rpl22 HA/+ vs Slc17a7 Cre/+ ;Rpl22 HA/+ (age P15). 260 genes (red) show differential expression with adjusted p-values <0.05 in Slc17a7 Cre/+; H11-DLK(iOE)/+, compared to control; names of genes with p<1E-10 are labeled. ( B ) Volcano plot showing RiboTag analysis in Slc17a7 Cre/+ ;Map3k12 fl/fl ;Rpl22 HA/+ vs Slc17a7 Cre/+ ;Rpl22 HA/+ (age P15). 36 genes (blue) show differential expression with adjusted p-values <0.05; names of genes with p<1E-10 are labeled. ( C ) Rank-rank hypergeometric overlap (RRHO) comparison of gene expression in DLK(cKO) and DLK(iOE) RiboTag datasets shows enrichment of similar genes when DLK is low or high, respectively. Color represents the -log transformed hypergeometric p-values (blue for weaker p-value, red for stronger p-value). ( D, E ) Gene ontology (GO) analysis of significantly up- or down-regulated genes in hippocampal glutamatergic neurons of DLK(iOE) mice compared to the control. Colors correspond to p-values; circle size represents fold enrichment for the GO term; X position shows # of genes significantly enriched in the GO term. ( F ) SynGO sunburst plot shows enrichment of 42 differentially expressed genes from hippocampal glutamatergic neurons of DLK(iOE) mice, with color corresponding to significance. ( G, H ) Pie charts show distribution of the 42 synaptic genes up- or down- regulated in DLK(iOE), respectively, in CA1, CA3, and DG in dorsal hippocampus, based on in situ data (P56) in the Allen Mouse Brain Atlas.

Article Snippet: Antibody , Rabbit polyclonal anti-Map3k12 antibody , Genetex , GTX124127; RRID: AB_11170703 , IF (1:250) tissue, (1:1000) cells, WB (1:1000); Lot #40653.

Techniques: Quantitative Proteomics, Control, Labeling, Comparison, Gene Expression, Transformation Assay, In Situ

Journal: eLife

Article Title: Translatome analysis reveals cellular network in DLK-dependent hippocampal glutamatergic neuron degeneration

doi: 10.7554/eLife.101173

Figure Lengend Snippet: ( A,B ) Confocal z-stack images of c-Jun ( A ) and p-c-Jun ( B ) immunostaining in CA1, CA3, and DG in control and Slc17a7 Cre/+ ; H11-DLK(iOE)/+ mice (P10). Dashed lines in corresponding DAPI staining outline region used for quantification. Scale bar 50 μm. Graphs below image panels show quantification of MFI of c-Jun ( A ) or p-c-Jun nuclei above intensity threshold per 100 μm of pyramidal or granule cell layer ( B ). Data points represent individual mice. 3 sections per mouse, N=4,5 mice for ( A ); N=4,4 mice for ( B ). Statistics: Unpaired t-test. ns, not significant, * p<0.05, ** p<0.01. ( C, D ) Confocal z-stack images of c-Jun ( C ) and p-c-Jun ( D ) immunostaining in CA1, CA3, and DG in control and Slc17a7 Cre/+ ; H11-DLK(iOE)/+ mice (P15). Dashed lines in corresponding DAPI staining outline region used for quantification. Scale bar 50 μm. Graphs below image panels show quantification of MFI of c-Jun ( C ) or p-c-Jun nuclei above intensity threshold per 100 μm of pyramidal or granule cell layer ( D ). Data points represent individual mice. 3 sections per mouse, N=7,8 mice for ( C ); N=3,5 mice for ( D ). Statistics: Unpaired t-test. ns, not significant; * p<0.05; ** p<0.01; **** p<0.0001. ( E ) Confocal z-stack images of c-Jun ( E ) and p-c-Jun ( F ) immunostaining in CA1, CA3, and DG in control and Slc17a7 Cre/+ ;Map3k12 fl/fl mice (P60). Dashed lines in corresponding DAPI staining outline region used for quantification. Scale bar 50 μm. Graphs below image panels show quantification of mean fluorescence intensity (MFI) of c-Jun and p-c-Jun in pyramidal or granule cell layer of each region. Data points represent MFI of individual mice. 3 sections per mouse, N=4,4 mice for ( E ); N=6, 7 mice for ( F ). Statistics: Unpaired t-test. ns, not significant; * p<0.05. All error bars represent SEM.

Article Snippet: Antibody , Rabbit polyclonal anti-Map3k12 antibody , Genetex , GTX124127; RRID: AB_11170703 , IF (1:250) tissue, (1:1000) cells, WB (1:1000); Lot #40653.

Techniques: Immunostaining, Control, Staining, Fluorescence

Journal: eLife

Article Title: Translatome analysis reveals cellular network in DLK-dependent hippocampal glutamatergic neuron degeneration

doi: 10.7554/eLife.101173

Figure Lengend Snippet: ( A ) Confocal single-slice image of RNAscope analysis of Stmn4 and Slc17a7 mRNAs in hippocampal neurons. Dashed circle outlines single nuclei. Scale bar, 10 μm. ( B, C ) Quantification of the ratio of Stmn4 to Slc17a7 RNAscope puncta in same nuclei of CA1 and CA3 neurons, respectively. N=6,3,3 mice of respective genotypes, quantified from >50 cells per genotype from 4 sections per mouse. Statistics: One way ANOVA with Dunnett’s multiple comparison test, ns, not significant; * p<0.05; ** p<0.01. ( D–F ) Confocal z-stack (max projection) images of CA1 immunostained for Tuj1, tyrosinated tubulin, and acetylated tubulin, respectively, in control and Slc17a7 Cre/+; H11-DLK(iOE)/+ mice of P15. SR: stratum radiatum. ( G, H ) Normalized mean fluorescence intensity (MFI) of tyrosinated and acetylated tubulin, respectively, after thresholding signals in SR in CA1 (dashed outlines on images in E-F). N=9, 6 mice, 3 sections averaged per mouse in ( G ); N=6, 4 mice, 3 sections averaged per mouse in ( H ). ( I–K ) Confocal z-stack (max projection) images of immunostained CA1 sections for Tuj1, tyrosinated tubulin, and acetylated tubulin, respectively, in control and Slc17a7 Cre/+; H11-DLK(iOE)/+ mice of P60. ( L, M ) Normalized MFI of tyrosinated and acetylated tubulin, respectively, after thresholding signal in SR in CA1 (dashed outlines on images in J-K). N=9, 9 mice, 3 sections averaged per mouse in L; N=6, 6 mice, 3 sections averaged per mouse in M. ( N–P ) Confocal z-stack (max projection) images of immunostained CA1 sections for Tuj1, tyrosinated tubulin, and acetylated tubulin, respectively, in control and Slc17a7 Cre/+ ;Map3k12 fl/fl mice of P60. ( Q, R ) Normalized MFI for tyrosinated and acetylated tubulin, respectively, after thresholding signal in SR in CA1 (dashed outlines on images in O-P). N=5, 7 mice, 3 sections averaged per mouse in Q; N=6, 7 mice, 3 sections averaged per mouse in R. All tubulin images shown as maximum projection of z-stack. Scale bar, 10 μm. In I-J, arrows point to apical dendrites with elevated immunostaining signal; arrowheads point to thin neurites with elevated signal. Statistics in ( G, H, L, M, Q, R ): Unpaired t-test. ns, not significant; * p<0.05. All error bars represent SEM.

Article Snippet: Antibody , Rabbit polyclonal anti-Map3k12 antibody , Genetex , GTX124127; RRID: AB_11170703 , IF (1:250) tissue, (1:1000) cells, WB (1:1000); Lot #40653.

Techniques: RNAscope, Comparison, Control, Fluorescence, Immunostaining

Journal: eLife

Article Title: Translatome analysis reveals cellular network in DLK-dependent hippocampal glutamatergic neuron degeneration

doi: 10.7554/eLife.101173

Figure Lengend Snippet: ( A ) Confocal single-slice images of Bassoon and Homer1 immunostaining in CA1 stratum radiatum (SR) of control and Slc17a7 Cre/+ ;Map3k12 fl/fl mice of P60. ( B, C ) Quantification of Bassoon and Homer1 puncta density, respectively. ( D ) Quantification of co-localization of Bassoon and Homer1. ( E, F ) Quantification of Bassoon and Homer1 puncta size. Data points represent average values per mouse from 3 sections. N=7 control, and 8 Slc17a7 Cre/+ ;Map3k12 fl/fl mice. Statistics: unpaired t-test or Mann-Whitney U test if not passing normality. ns, not significant. ( G ) Confocal single-slice images of Bassoon and Homer1 immunostaining in CA1 SR of control and Slc17a7 Cre/+ ; H11-DLK(iOE)/+ mice of P15. ( H–I ) Quantification of Bassoon and Homer1 puncta density, respectively. ( J ) Quantification of co-localization of Bassoon and Homer1. ( K, L ) Quantification of Bassoon and Homer1 puncta size. Data points represent average values per mouse from 3 sections, N=9 control, and 6 Slc17a7 Cre/+ ; H11-DLK(iOE)/+ mice. Statistics: unpaired t-test or Mann-Whitney U test if not passing normality. ns, not significant; ** p<0.01. Scale bars, 5 μm in panel images, and 1 μm in enlarged images. All error bars represent SEM.

Article Snippet: Antibody , Rabbit polyclonal anti-Map3k12 antibody , Genetex , GTX124127; RRID: AB_11170703 , IF (1:250) tissue, (1:1000) cells, WB (1:1000); Lot #40653.

Techniques: Immunostaining, Control, MANN-WHITNEY

Journal: eLife

Article Title: Translatome analysis reveals cellular network in DLK-dependent hippocampal glutamatergic neuron degeneration

doi: 10.7554/eLife.101173

Figure Lengend Snippet: ( A ) Confocal single-slice image of VGLUT1 immunostaining of CA1 SR in control and Slc17a7 Cre/+ ;Map3k12 fl/fl mice at P60. Scale bar 5 μm, inset scale bar 1 μm. ( B, C ) Quantification of VGLUT1 puncta density and size. Data points represent averages from individual mice across 3 sections per mouse. N=5 control, and 8 Slc17a7 Cre/+ ;Map3k12 fl/fl mice. ( D ) Confocal single-slice image of VGLUT1 immunostaining of CA1 SR in control and Slc17a7 Cre/+ ; H11-DLK(iOE)/+ mice at P15. Scale bar 5 μm, inset scale bar 1 μm. ( E, F ) Quantification of VGLUT1 puncta density ( E ) and size ( F ). Data points represent averages from individual mice across 3 sections per mouse, N=9 control, and 8 Slc17a7 Cre/+ ; H11-DLK(iOE)/+ mice. Statistics: unpaired t-test. ns, not significant; * p<0.05. All error bars represent SEM.

Article Snippet: Antibody , Rabbit polyclonal anti-Map3k12 antibody , Genetex , GTX124127; RRID: AB_11170703 , IF (1:250) tissue, (1:1000) cells, WB (1:1000); Lot #40653.

Techniques: Immunostaining, Control

Journal: eLife

Article Title: Translatome analysis reveals cellular network in DLK-dependent hippocampal glutamatergic neuron degeneration

doi: 10.7554/eLife.101173

Figure Lengend Snippet: ( A ) Confocal z-stack images of neuron morphology at DIV2 from mice of genotype indicated, visualized by tdTomato from Rosa26-tdTomato. Neurons with indicated genotypes are labeled by tdTomato from Cre-dependent Rosa26-tdTomato generated from the following crosses: for control: Slc17a7 Cre/+ X Rosa26 tdT/+ ; for DLK(cKO): Slc17a7 Cre/+ ;Map3k12 fl/fl X Map3k12 fl/fl ;Rosa26 tdT/+ ; for DLK(iOE): H11-DLK(iOE)/H11-DLK(iOE) X Slc17a7 Cre/+ ;Rosa26 tdT/+ . Red arrowheads point to long processes considered as axons. Scale bar 100 μm. ( B ) Co-immunostaining of DLK and STMN4 in DIV2 control cultured neuron growth cone shows non-overlapping puncta. ( C ) Confocal z-stack images of DLK and STMN2 co-immunostaining of DIV2 primary hippocampal neurons from genotypes indicated; neurons are labeled with tdTomato from Rosa26-tdTomato. Scale bar 10 μm. ( D ) Quantification of association between DLK level and STMN2 in cell soma. N=3 cultures/genotype, ≥45 cells/genotype. Spearman correlation r =0.4693. ( E ) Analysis of cell types in culture at DIV14 using Prox1 and Satb2 markers. Quantification from N=3 cultures/genotype, ≥200 cells/genotype. Statistics: Two-way ANOVA with Dunnett’s multiple comparison test. ns, not significant. Error bars represent SEM.

Article Snippet: Antibody , Rabbit polyclonal anti-Map3k12 antibody , Genetex , GTX124127; RRID: AB_11170703 , IF (1:250) tissue, (1:1000) cells, WB (1:1000); Lot #40653.

Techniques: Labeling, Generated, Control, Immunostaining, Cell Culture, Comparison

Journal: eLife

Article Title: Translatome analysis reveals cellular network in DLK-dependent hippocampal glutamatergic neuron degeneration

doi: 10.7554/eLife.101173

Figure Lengend Snippet: ( A ) Confocal images of DIV2 primary hippocampal glutamatergic neurons immunostained with DLK and STMN4. Neurons with indicated genotypes are labeled by tdTomato from Cre-dependent Rosa26-tdTomato, generated from hippocampi in P1 pups from the following crosses: for control: Slc17a7 Cre/+ X Rosa26 tdT/+ ; for DLK(cKO): Slc17a7 Cre/+ ;Map3k12 fl/fl X Map3k12 fl/fl ;Rosa26 tdT/+ ; for DLK(iOE): H11-DLK(iOE)/H11-DLK(iOE) X Slc17a7 Cre/+ ;Rosa26 tdT/+ . Orange arrows point to some of the thin neurites from neurons overexpressing DLK. Red dashes outline enlarged view of neurites. Scale bar, 10 μm neuron, 1 μm enlarged view. ( B ) Graph shows positive correlation between STMN4 immunostaining, measured as integrated density (Area X MFI) in neuronal soma, to integrated density of DLK immunostaining. N≥3 cultures/genotype,≥60 cells/genotype. Spearman correlation r =0.7454. ( C ) Quantification of percentage of neurons with no, one, or more than one axon (defined by neurites longer than 90 μm) in each genotype. Number of neurons: 47 from 3 Slc17a7 Cre (control) cultures, 49 from 3 DLK(cKO) cultures, 42 from 4 DLK(iOE) cultures. Statistics: Fisher’s exact test shows significance (p<0.0001) between genotype and number of axons. Pairwise comparisons with Fisher’s exact test: Axon formation in control vs DLK(cKO): p=0.1857. Formation of multiple axons in control vs DLK(cKO): p>0.9999. Axon formation in control vs DLK(iOE): p=0.0042. Formation of multiple axons in control vs DLK(iOE): p=0.0001. ( D ) Quantification of number of primary neurites, which include both branches and filopodia, per neuron. Number of neurons: 55 from 4 Slc17a7 Cre (control) cultures, 70 from 4 DLK(cKO) cultures, 45 from 5 DLK(iOE) cultures. Statistics, Kruskal-Wallis test with Dunn’s multiple comparison test. **** p<0.0001. Error bars represent SEM. ( E ) Confocal z-stack images of tyrosinated tubulin immunostaining from DIV2 cultures of genotypes indicated, showing that filopodia structures (arrows) around the soma and axons of neurons with high expression of DLK have tyrosinated tubulin. ( F ) Confocal z-stack images of acetylated tubulin immunostaining from DIV2 cultures of genotypes indicated, showing that filopodia structures (arrows) around the soma and axons of neurons with high expression of DLK do not have acetylated tubulin. Asterisks indicate stable branches containing acetylated tubulin. Scale bar in E, F, 10 μm. Tyrosinated tubulin and acetylated tubulin staining shows saturated appearance to visualize staining in thin neurites.

Article Snippet: Antibody , Rabbit polyclonal anti-Map3k12 antibody , Genetex , GTX124127; RRID: AB_11170703 , IF (1:250) tissue, (1:1000) cells, WB (1:1000); Lot #40653.

Techniques: Labeling, Generated, Control, Immunostaining, Comparison, Expressing, Staining

Journal: eLife

Article Title: Translatome analysis reveals cellular network in DLK-dependent hippocampal glutamatergic neuron degeneration

doi: 10.7554/eLife.101173

Figure Lengend Snippet: ( A ) Confocal images of axons of DIV14 neurons of indicated genotype, co-stained with Bassoon and DLK. Neurons with indicated genotypes are labeled by tdTomato from Cre-dependent Rosa26-tdTomato generated from the following crosses: for control: Slc17a7 Cre/+ X Rosa26 tdT/+ ; for DLK(cKO): Slc17a7 Cre/+ ;Map3k12 fl/fl X Map3k12 fl/fl ;Rosa26 tdT/+ ; for DLK(iOE): H11-DLK(iOE)/H11-DLK(iOE) X Slc17a7 Cre/+ ;Rosa26 tdT/+ . Scale bar, 1 μm. ( B ) Quantification of bassoon puncta density. ( C ) Quantification of average bassoon puncta size from individual neurons. Number of neurons: 30 from 3 Slc17a7-cre (control) cultures, 41 from 3 DLK(cKO) cultures, 46 from 4 DLK(iOE) cultures. Statistics: One way ANOVA with Dunnett’s multiple comparison test. ns, not significant; * p<0.05. ( D ) Confocal z-stack images of DIV14 neurons of indicated genotype, labeled by Rosa26-tdTomato. Dashed boxes outline dendrites enlarged below for dendritic spines. Asterisks provide some examples of spine types; long thin with purple; thin with blue; mushroom with white; stubby with yellow. Scale bar, 10 μm top, 5 μm bottom. ( E ) Quantification of dendritic spine density. ( F ) Quantification of mushroom spine density. ( G ) Distribution of spine types. ( E–G ) Number of neurons: 35 from 3 Slc17a7-cre (control) cultures, 31 from 3 DLK(cKO) cultures, 31 from 3 DLK(iOE) cultures. Statistics: One way ANOVA with Dunnett’s multiple comparison test. * p<0.05; ** p<0.01. All error bars represent SEM.

Article Snippet: Antibody , Rabbit polyclonal anti-Map3k12 antibody , Genetex , GTX124127; RRID: AB_11170703 , IF (1:250) tissue, (1:1000) cells, WB (1:1000); Lot #40653.

Techniques: Staining, Labeling, Generated, Control, Comparison

Journal: eLife

Article Title: Translatome analysis reveals cellular network in DLK-dependent hippocampal glutamatergic neuron degeneration

doi: 10.7554/eLife.101173

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit polyclonal anti-Map3k12 antibody , Genetex , GTX124127; RRID: AB_11170703 , IF (1:250) tissue, (1:1000) cells, WB (1:1000); Lot #40653.

Techniques: Knock-Out, Over Expression, Sequencing, RNAscope, TUNEL Assay, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Multiplex Assay, Software