Journal: Science Advances

Article Title: Phosphorylation of Doc2 by EphB2 modulates Munc13-mediated SNARE complex assembly and neurotransmitter release

doi: 10.1126/sciadv.adi7024

Figure Lengend Snippet: ( A ) Schematic diagram showing domain organization and variant fragments of Doc2B and Munc13-1. ( B ) Binding of MUN to GST-Doc2B FL or its variant fragments measured by GST pull-down experiments and quantification of the binding. ( C ) ITC-based measurements of MUN binding to GST-Doc2B 13 to 37 (Mid, left) and to GST-Doc2B 13 to 80 (Mid-L, right). ( D ) ITC-based measurements of the binding affinities between GST-Doc2B FL or its variant fragments and MUN. ( E ) 2D 1 H- 15 N HSQC spectra of 13 C/ 15 N-labeled Doc2B 1 to 80 before (black) and after (red) addition of MUN. Cross-peaks of residues that are chosen for mutation to detect MUN binding are labeled along with their corresponding residue number (cyan). ( F ) Peak intensity alteration of 13 C/ 15 N-labeled Doc2B 1 to 80 protein expressed as ratio between integrated peak volumes after ( V ) and before ( V 0 ) addition of unlabeled MUN. ( G ) Binding of Doc2B FL or its variant mutations to GST-MUN measured by GST pull-down experiments and quantification of the binding. ( H ) Binding of MUN or its variant fragments to GST-Doc2B FL measured by GST pull-down experiments and quantification of the binding. Red asterisk shows the band of bound MUN-BC. ( I ) Binding of MUN or its variant mutations to GST-Doc2B FL measured by GST pull-down experiments and quantification of the binding. Data of GST pull-down experiments are processed by ImageJ (National Institutes of Health) and presented as means ± SEM ( n = 3). Statistical significance and P values were determined by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. * P < 0.05; *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: For detecting Doc2B binding to EphB2 or Munc13-1 from brain samples, adult mouse brain was initially ground in buffer B supplied with 1% Triton X-100 and proteinase inhibitor cocktail (TargetMol) and then incubated with GST-Doc2B FL at 4°C overnight.

Techniques: Variant Assay, Binding Assay, Labeling, Mutagenesis, Residue

Journal: Science Advances

Article Title: Phosphorylation of Doc2 by EphB2 modulates Munc13-mediated SNARE complex assembly and neurotransmitter release

doi: 10.1126/sciadv.adi7024

Figure Lengend Snippet: ( A ) Illustration of the FRET assay for detecting MUN-catalyzed SNARE complex assembly starting from the Munc18-1/Syx1 (1 to 261) complex in the presence of Syb2 (29 to 96), SN25, and MUN. FRET signal between BDPY-labeled Syb2 S61C (donor) and TMR-labeled SN25 S187C (acceptor) was monitored. ( B to D ) MUN-catalyzed SNARE complex assembly by addition of Doc2B Mid, Doc2B Mid-L, or Doc2B FL, respectively (B), addition of different concentrations of Doc2B Mid (C), and addition of MUN NFAA or Doc2B I20A, which disrupts Doc2B-MUN interaction (D). Decrease of donor fluorescence at 1500 s is shown in the column at the right of the chart. ( E ) Illustration of Munc13-catalyzed lipid mixing between liposomes bearing Syb2 (1 to 116) and liposomes bearing the Munc18-1/Syx1 (1 to 288) complex in the presence of SN25, Syt1 C2AB, Ca 2+ , and Munc13-1 (C 1 C 2 BMUN). Donor (NBD) fluorescence was monitored at 538 nm. ( F to H ) Munc13-catalyzed lipid mixing by addition of Doc2B Mid, Doc2B Mid-L, or Doc2B FL, respectively (F); addition of C 1 C 2 BMUN NFAA or Doc2B I20A (G). Munc13-catalyzed lipid mixing at 1000 s is shown in (H). F 1 , fluorescence intensity observed as a function time; F 0 , initial fluorescence intensity. Data are presented as means ± SEM ( n = 3). Statistical significance and P values were determined by one-way ANOVA with Tukey’s multiple comparisons test. * P < 0.05; *** P < 0.001; **** P < 0.0001.

Article Snippet: For detecting Doc2B binding to EphB2 or Munc13-1 from brain samples, adult mouse brain was initially ground in buffer B supplied with 1% Triton X-100 and proteinase inhibitor cocktail (TargetMol) and then incubated with GST-Doc2B FL at 4°C overnight.

Techniques: Labeling, Fluorescence, Liposomes

Journal: Nature

Article Title: TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A

doi: 10.1038/s41586-022-04436-3

Figure Lengend Snippet: ( a, b ) RNA-seq traces from IGV of representative samples from control (top) and TARDBP KD (bottom) in i 3 Neurons showing intron retention in UNC13A (A) (mean 4.50 ± 1.50 increased IR in KD) and UNC13B (mean 1.86 ± 0.63 increased IR in KD)(B), overlaid with published TDP-43 iCLIP peaks ( c ) Histogram showing number of basescope cryptic foci per nuclei in control (blue) and TDP-43 KD (grey) in WTC11-derived i 3 Neurons, p < 0.0001 unpaired t-test. ( d, e ) RT-qPCR levels of TARDBP and UNC13A with a non-targeting control sgRNA (sgTARDBP −), an intermediate TDP-43 KD (sgTARDBP +) or a higher TDP-43 KD (sgTARDBP ++) in WTC11-derived ( d ) and NCRM-5-derived i 3 Neurons ( e ). n = 4 biological replicates sgTARDBP − ( d ), n = 6 biological replicates sgTARDBP − ( e ), sgTARDBP + ( d, e ) and ++ ( d, e ). plotted as means ± SEM. ( f ) Representative images of UNC13A CE RT-PCR products ( g ) Quantification of the lower gel in ( f ) plotted as means ± SEM, n = 6 biological replicates non-targeting control sgRNA (sgTARDBP −), sgTARDBP +, sgTARDBP ++. Upper gel is quantified in Fig. . One-way ANOVA with multiple comparisons. ( h–k ) Expression of TDP-43 regulated splicing in UNC13A ( h, i ) and UNC13B ( j, k ) across neuronal datasets , in control (blue) and TDP-43 KD (yellow). Intron retention (IR)( i, k ) and CE and fsE PSI ( h, j ) significantly increase after TDP-43 depletion in most experiments, Wilcoxon test ( l ) Relative gene expression levels for TARDBP across neuronal datasets , . Normalized RNA counts are shown as relative to control mean. Numbers show log 2 fold change calculated by DESeq2. Significance shown as adjusted p-values from DESeq2. For (h–l) biological replicates are: iPSC MN Ctrl KD n = 12, TDP-43 KD n = 6; i 3 N Ctrl KD n = 4, TDP-43 KD n = 3; SH-SY5Y, SK-N-DZ a , and SK-N-DZ b Ctrl KD n = 3, TDP-43 KD n = 3, Significance levels reported as * (p < 0.05) ** (p < 0.01) *** (p < 0.001) **** (p < 0.0001).

Article Snippet: Gene expression analysis was performed by qPCR using Taqman Multiplex Universal Master Mix (Thermo 4461882) or Taqman Universal PCR Master Mix (Thermo 4304437) and TaqMan assays (UNC13A-Fam Hs00392638_m1, UNC13B-Fam Hs01066405_m1, TDP-43-Vic Hs00606522_m1, GAPDH-Jun assay 4485713, TDP-43-FAM Hs00606522_m1, UPF1-FAM Hs00161289_m1, HPRT1-FAM Hs02800695_m1) on a QuantStudio 5 or a QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems) and quantified using the ΔΔ C t method .

Techniques: RNA Sequencing, Control, Derivative Assay, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Expressing, Gene Expression

Journal: Nature

Article Title: TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A

doi: 10.1038/s41586-022-04436-3

Figure Lengend Snippet: a , Differential splicing analysis by MAJIQ in control ( n = 4) and CRISPRi TDP-43 depleted (KD) ( n = 3) iPS cell-derived cortical-like i 3 Neurons. Each point denotes a splice junction. b , Representative sashimi plots showing cryptic exon (CE) inclusion between exons 20 and 21 of UNC13A upon TDP-43 knockdown. c , f , Schematics showing intron retention (IR) (orange; bottom), TDP-43 binding region (green), and two ALS- and FTLD-associated SNPs (red) in UNC13A ( c ) and UNC13B ( f ). d , LocusZoom plot of the UNC13A locus in the most recent ALS GWAS ; the dashed line indicates the risk threshold used in that study. Lead SNP rs12973192 is plotted as a purple diamond, other SNPs are coloured by linkage disequilibrium (LD) with rs12973192 in European individuals from 1000 Genomes. Ref. var., reference variant. e , Representative sashimi plot of UNC13B showing inclusion of the FSE upon TDP-43 knockdown. g , BaseScope detection of UNC13A CE (white puncta) in control (top) and TDP-43-knockdown (bottom) i 3 Neurons co-stained for TDP-43 (green), neuronal processes (stained for TUBB3, pink) and nuclei (blue). Scale bar, 5 μm. h , Quantification of RT–PCR products using iPS cell-derived neurons made from an independent iPS cell line, NCRM5, with a non-targeting control short guide RNA (sgRNA) (sgTARDBP−), an intermediate TDP-43 knockdown (sgTARDBP+) or stronger TDP-43 knockdown (sgTARDBP++). Data are mean ± s.e.m. sgControl, n = 6; sgTARDBP+, n = 5; sgTARDBP++, n = 6; one-way ANOVA with multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. i , Schematic of nanopore long reads quantified in j , Extended Data Figs. . j , Percentage of targeted UNC13A long reads with TDP-43-regulated splice events that contain CE, intron retention or both in TDP-43-knockdown SH-SY5Y cells.

Article Snippet: Gene expression analysis was performed by qPCR using Taqman Multiplex Universal Master Mix (Thermo 4461882) or Taqman Universal PCR Master Mix (Thermo 4304437) and TaqMan assays (UNC13A-Fam Hs00392638_m1, UNC13B-Fam Hs01066405_m1, TDP-43-Vic Hs00606522_m1, GAPDH-Jun assay 4485713, TDP-43-FAM Hs00606522_m1, UPF1-FAM Hs00161289_m1, HPRT1-FAM Hs02800695_m1) on a QuantStudio 5 or a QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems) and quantified using the ΔΔ C t method .

Techniques: Control, Derivative Assay, Knockdown, Binding Assay, Variant Assay, Staining, Reverse Transcription Polymerase Chain Reaction

Journal: Nature

Article Title: TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A

doi: 10.1038/s41586-022-04436-3

Figure Lengend Snippet: Targeted nanopore sequencing reveals UNC13A CE and IR events occur largely independently in-vitro. ( a ) Sanger sequencing of cryptic bands in both SH-SY5Y and SK-N-DZ cells confirm the CE splice junctions. ( b, c ) Crosslink density across UNC13A (chr19) (b) and UNC13B (chr9) ( c ) genomic loci from novel iCLIP on endogenous TDP-43 in SH-SHY5Y cells. Crosslink densities for both genes show peaks at the CE/fsE (red) and retained introns (blue). Coordinates shown in hg38. ( d ) Percentage of all targeted UNC13A long reads in SH-SY5Y cells containing either neither CE nor IR, both, or either CE or IR. Most reads in both control and TDP-43 KD contain neither event, and while IR event is present in controls, CE is only detected in TDP-43 KD. ( e ) Representative trace in TDP-43 KD of UNC13A targeted long reads showing transcript containing either the CE or IR, and transcripts with neither.

Article Snippet: Gene expression analysis was performed by qPCR using Taqman Multiplex Universal Master Mix (Thermo 4461882) or Taqman Universal PCR Master Mix (Thermo 4304437) and TaqMan assays (UNC13A-Fam Hs00392638_m1, UNC13B-Fam Hs01066405_m1, TDP-43-Vic Hs00606522_m1, GAPDH-Jun assay 4485713, TDP-43-FAM Hs00606522_m1, UPF1-FAM Hs00161289_m1, HPRT1-FAM Hs02800695_m1) on a QuantStudio 5 or a QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems) and quantified using the ΔΔ C t method .

Techniques: Nanopore Sequencing, In Vitro, Sequencing, Control

Journal: Nature

Article Title: TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A

doi: 10.1038/s41586-022-04436-3

Figure Lengend Snippet: Relative gene expression levels for UNC13A ( a ) and UNC13B ( b ) after TDP-43 knockdown across neuronal cell lines , . Normalized RNA counts are shown as relative to control mean. Numbers show log fold change calculated by DESeq2. Significance shown as adjusted p-values from DESeq2. Number of replicates as in Extended data Fig. ( c, d ) RT-qPCR analysis shows TDP-43, UNC13A and UNC13B gene expression is reduced by TARDBP shRNA knockdown in both SH-SY5Y and SK-N-DZ human cell lines. Graphs represent the means ± SEM, n = 6 biological replicates, one sample t-test. ( e ) The 5’ ends of 29 nt reads relative to the annotated start codon from a representative ribosome profiling dataset (TDP-43 KD replicate B). As expected, we detected strong three-nucleotide periodicity, and a strong enrichment of reads across the annotated coding sequence relative to the upstream untranslated region. ( f ) UNC13A, UNC13B, and TDP-43 protein levels, measured by Western Blot, with varying levels of DOX-inducible TDP-43 knockdown in SH-SY5Y cells. Tubulin is used as endogenous control, n = 3. For gel source data, see Supplementary Figure 1. ( g ) Quantification of RT-PCR products from the transcripts containing UNC13A CE, UNC13A intron retention, UNC13B fsE, and UNC13B intron retention, with varying levels of DOX-inducible TDP-43 knockdown in SH-SY5Y cells. Graphs represent the means ± SEM n = 3 biological replicates. ( h ) UPF1 siRNA knock-down led to the rescue of hnRNPL (positive control), UNC13A , and UNC13B transcripts, but not STMN2 . Graphs represent the means ± SEM, n = 4 biological replicates, one-sample t-test. ( l ) UNC13A CE containing-transcript PSI is increased after UPF1 knockdown in i 3 Neurons. Graphs represent the means ± SEM, n = 6 biological replicates. ( j ) RT-PCR products from UNC13A in the setting of mild TDP-43 knockdown (“+”, as for Figure and S4G) with the addition of either DMSO (control) or CHX (NMD inhibition). ( k ) Quantification of ( j ) Graphs represent the means ± SEM, n = 4 biological replicates. Significance levels reported as * (p < 0.05) ** (p < 0.01) *** (p < 0.001) **** (p < 0.0001).

Article Snippet: Gene expression analysis was performed by qPCR using Taqman Multiplex Universal Master Mix (Thermo 4461882) or Taqman Universal PCR Master Mix (Thermo 4304437) and TaqMan assays (UNC13A-Fam Hs00392638_m1, UNC13B-Fam Hs01066405_m1, TDP-43-Vic Hs00606522_m1, GAPDH-Jun assay 4485713, TDP-43-FAM Hs00606522_m1, UPF1-FAM Hs00161289_m1, HPRT1-FAM Hs02800695_m1) on a QuantStudio 5 or a QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems) and quantified using the ΔΔ C t method .

Techniques: Gene Expression, Knockdown, Control, Quantitative RT-PCR, shRNA, Sequencing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Positive Control, Inhibition

Journal: Nature

Article Title: TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A

doi: 10.1038/s41586-022-04436-3

Figure Lengend Snippet: a , Ribosome profiling of TDP-43-knockdown i 3 Neurons shows a reduction in ribosome occupancy of STMN2 , UNC13A and UNC13B transcripts. b , Mass spectrometry-based proteomic analysis shows dose-dependent reduction in protein abundance of UNC13A and TDP-43 upon TDP-43 knockdown in i 3 Neurons. n = 6 biological replicates. Two-sample t -test. c , Protein and RNA quantification of TDP-43, UNC13A and UNC13B in SH-SY5Y with varying levels of doxycycline-inducible TDP-43 knockdown. n = 3 biological replicates. d , Transcript expression upon treatment with CHX suggests that UNC13A and UNC13B , but not STMN2 , are sensitive to NMD. HNRNPL is used as a positive control. n = 7 biological replicates ( UNC13A , HNRNPL and STMN2 ) and 8 biological replicates ( UNC13B ). One-sample t -test. Data are mean ± s.e.m. ( b – d ).

Article Snippet: Gene expression analysis was performed by qPCR using Taqman Multiplex Universal Master Mix (Thermo 4461882) or Taqman Universal PCR Master Mix (Thermo 4304437) and TaqMan assays (UNC13A-Fam Hs00392638_m1, UNC13B-Fam Hs01066405_m1, TDP-43-Vic Hs00606522_m1, GAPDH-Jun assay 4485713, TDP-43-FAM Hs00606522_m1, UPF1-FAM Hs00161289_m1, HPRT1-FAM Hs02800695_m1) on a QuantStudio 5 or a QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems) and quantified using the ΔΔ C t method .

Techniques: Knockdown, Mass Spectrometry, Quantitative Proteomics, Expressing, Positive Control

Journal: Nature

Article Title: TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A

doi: 10.1038/s41586-022-04436-3

Figure Lengend Snippet: a , UNC13A and STMN2 CE expression from a published dataset of frontal cortex neuronal nuclei from patients with ALS or FTLD sorted according to TDP-43 expression . b , UNC13A CE expression in bulk RNA-seq from the NYGC ALS Consortium data normalized by library size across disease and tissue samples. ALS cases are stratified by mutation status, FTLD cases are stratified by pathological subtype. c , CE expression throughout ALS-TDP and FTLD-TDP cases across tissue, number of tissue samples in brackets. d , BaseScope detection of UNC13A CE (red foci) in FTLD-TDP (9 individuals) but not control (5 individuals) or non-TDP FTLD (FTLD-TAU) (4 individuals) frontal cortex samples and quantification of background-corrected foci frequency between groups. Scale bar, 10 μm. Data are mean ± s.e.m. ( b – d ); Wilcoxon test.

Article Snippet: Gene expression analysis was performed by qPCR using Taqman Multiplex Universal Master Mix (Thermo 4461882) or Taqman Universal PCR Master Mix (Thermo 4304437) and TaqMan assays (UNC13A-Fam Hs00392638_m1, UNC13B-Fam Hs01066405_m1, TDP-43-Vic Hs00606522_m1, GAPDH-Jun assay 4485713, TDP-43-FAM Hs00606522_m1, UPF1-FAM Hs00161289_m1, HPRT1-FAM Hs02800695_m1) on a QuantStudio 5 or a QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems) and quantified using the ΔΔ C t method .

Techniques: Expressing, RNA Sequencing, Mutagenesis, Control

Journal: Nature

Article Title: TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A

doi: 10.1038/s41586-022-04436-3

Figure Lengend Snippet: ( a ) UNC13A expression across tissues and disease subtypes in the NYGC ALS Consortium RNA-seq dataset. Expression normalised as transcripts per million (TPM). Cortical regions have noticeably higher UNC13A expression than the spinal cord. ( b ) total RNA-seq library size (log10 scaled) ( c ) RNA integrity score (RIN) ( d ) Cell type decomposition across NYGC ALS Consortium RNA-seq dataset. While there are differences between tissues and disease-subtypes on these technical factors, specificity of UNC13A CE detection to tissues presumed to contain TDP-43 proteinopathy cannot be explained by these technical factors. Box plots ( a–d ): boundaries 25–75th percentiles; midline, median; whiskers, Tukey style. Wilcoxon test, significance levels reported as * (p < 0.05) ** (p < 0.01) *** (p < 0.001) **** (p < 0.0001).

Article Snippet: Gene expression analysis was performed by qPCR using Taqman Multiplex Universal Master Mix (Thermo 4461882) or Taqman Universal PCR Master Mix (Thermo 4304437) and TaqMan assays (UNC13A-Fam Hs00392638_m1, UNC13B-Fam Hs01066405_m1, TDP-43-Vic Hs00606522_m1, GAPDH-Jun assay 4485713, TDP-43-FAM Hs00606522_m1, UPF1-FAM Hs00161289_m1, HPRT1-FAM Hs02800695_m1) on a QuantStudio 5 or a QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems) and quantified using the ΔΔ C t method .

Techniques: Expressing, RNA Sequencing

Journal: Nature

Article Title: TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A

doi: 10.1038/s41586-022-04436-3

Figure Lengend Snippet: Targeted long reads in FTLD frontal cortex show that UNC13A CE and IR occur independently in-vivo. ( a ) Detection rate of UNC13A CE across tissues by RNA sequencing platform and read length. UNC13A CE was more likely to be detected in cervical spinal cord and motor cortex when sequenced on machines with 125 bp compared to 100 bp. (b) No significant differences in total RNA-seq library size (log10 scaled). (c) RNA integrity score (RIN) was significantly lower in motor and temporal cortices in samples where UNC13A was detected. (d) Cell type decomposition revealed that samples with UNC13A CE detected had a higher proportion of neurons in cervical and lumbar spinal cord, whereas in frontal, temporal, and motor cortex samples with UNC13A CE detected had a lower proportion of neurons, and in motor and temporal cortex samples with UNC13A CE detected had a higher proportion of astrocytes. Astrocy. - Astrocytes, Endothi. - Endothelial, Microgl. - Microglia. Neur. - Neurons, Oligiodendr. - Oligiodendrycytes. P-values shown are from Fisher’s exact test ( a ) or Wilcoxon test ( b–d ). N tissue samples show below in brackets. Box plots ( a–d ): boundaries 25-75th percentiles; midline, median; whiskers, Tukey style. ( e ) Percentage of targeted UNC13A long reads with TDP-43 regulated splice events that contain either both, CE, or IR in four in FTLD frontal cortices. ( f ) Percentage of all targeted UNC13A long reads in ( a ) containing neither CE nor IR, both, or either CE or IR.

Article Snippet: Gene expression analysis was performed by qPCR using Taqman Multiplex Universal Master Mix (Thermo 4461882) or Taqman Universal PCR Master Mix (Thermo 4304437) and TaqMan assays (UNC13A-Fam Hs00392638_m1, UNC13B-Fam Hs01066405_m1, TDP-43-Vic Hs00606522_m1, GAPDH-Jun assay 4485713, TDP-43-FAM Hs00606522_m1, UPF1-FAM Hs00161289_m1, HPRT1-FAM Hs02800695_m1) on a QuantStudio 5 or a QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems) and quantified using the ΔΔ C t method .

Techniques: In Vivo, RNA Sequencing

Journal: Nature

Article Title: TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A

doi: 10.1038/s41586-022-04436-3

Figure Lengend Snippet: STMN2 CE PSI correlates with TDP-43 regulated cryptics across NYGC RNA-seq dataset. IRratio in UNC13A exon 31−32 ( a ) and UNC13B exon 21−22 ( b ) across NYGC tissue samples. UNC13A IR was lower in ALS-TDP cases than in controls in cervical spinal, frontal and motor cortices, and higher in FTLD-TDP cases than controls in frontal and temporal cortices. Possibly this reflects differences in the effects of cell type composition in disease state. Box plots ( a, b ): boundaries 25–75th percentiles; midline, median; whiskers, Tukey style..Wilcoxon test, significance levels reported as * (p < 0.05) ** (p < 0.01) *** (p < 0.001) **** (p < 0.0001). ( c–e ) Correlation in ALS/FTLD-TDP cortex between RAP1GAP CE ( c ), PFKP CE ( d ), and UNC13A CE ( e ) with STMN2 CE PSI in patients with at least 30 spliced reads across the CE locus. Spearman’s correlation.

Article Snippet: Gene expression analysis was performed by qPCR using Taqman Multiplex Universal Master Mix (Thermo 4461882) or Taqman Universal PCR Master Mix (Thermo 4304437) and TaqMan assays (UNC13A-Fam Hs00392638_m1, UNC13B-Fam Hs01066405_m1, TDP-43-Vic Hs00606522_m1, GAPDH-Jun assay 4485713, TDP-43-FAM Hs00606522_m1, UPF1-FAM Hs00161289_m1, HPRT1-FAM Hs02800695_m1) on a QuantStudio 5 or a QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems) and quantified using the ΔΔ C t method .

Techniques: RNA Sequencing

Journal: Nature

Article Title: TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A

doi: 10.1038/s41586-022-04436-3

Figure Lengend Snippet: ( a ) UNC13A CE PSI by genotype (Wilcoxon test) Box plots: boundaries 25-75th percentiles; midline, median; whiskers, Tukey style. ( b ) Effect of CE or intronic SNP on the correlation between STMN2 and UNC13A CE PSI in ALS/FTD cortex in samples with at least 30 junction reads across the CE locus. Spearman’s correlation. ( c ) Raw tapestation gel images of UNC13A CE products in 2H and 2R minigines and quantification of the PCR products. Graphs represent the means ± SEM(n = 3 biological replicates); Two-way ANOVA ( d ) Raw tapestation gel images corresponding to Fig. . Two sets of primers were used to amplify either control (top row) or mutant minigene (bottom row). Left panel: single transfections were performed to ensure primer specificity. Right panel: three biological replicates of the double transfections. ( e ) Fractional changes at iCLIP peaks for 2R versus 2H minigene (mean and 75% confidence interval shown). Peaks that are within 50nt of each SNP are highlighted. (f) Mean crosslink density around the exonic (top) and intronic (bottom) SNPs in the 2H (red) and 2R (blue) minigenes, relative to the 5’ end of minigene (error bars = standard deviation; dashed lines show SNP positions). ( g, h ) Individual TDP-43 E-scores for the CE ( g ) and intronic ( h ) heptamers for which there was data ( i ) Average change in E-value (measure of binding enrichment) across proteins for heptamers containing risk/healthy intronic SNP allele; TDP-43 is indicated in red. Significance levels reported as * (p < 0.05) ** (p < 0.01) *** (p < 0.001) **** (p < 0.0001).

Article Snippet: Gene expression analysis was performed by qPCR using Taqman Multiplex Universal Master Mix (Thermo 4461882) or Taqman Universal PCR Master Mix (Thermo 4304437) and TaqMan assays (UNC13A-Fam Hs00392638_m1, UNC13B-Fam Hs01066405_m1, TDP-43-Vic Hs00606522_m1, GAPDH-Jun assay 4485713, TDP-43-FAM Hs00606522_m1, UPF1-FAM Hs00161289_m1, HPRT1-FAM Hs02800695_m1) on a QuantStudio 5 or a QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems) and quantified using the ΔΔ C t method .

Techniques: Control, Mutagenesis, Transfection, Standard Deviation, Binding Assay

Journal: Nature

Article Title: TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A

doi: 10.1038/s41586-022-04436-3

Figure Lengend Snippet: a , Ratio of UNC13A and STMN2 CE Ψ in ALS-TDP and FTLD-TDP cortex, split by genotype for UNC13A risk alleles. In box plots, the centre line shows the median, box edges delineate 25th and 75th percentiles and Tukey whiskers are plotted. b , Unique cDNAs from targeted RNA-seq in ten patients with FTLD-TDP who are heterozygous for the risk SNP within the UNC13A cryptic exon. Single-tailed binomial tests. Patients 1, 5 and 7 carry the C9orf72 hexanucleotide repeat. c , Schematic of UNC13A minigenes containing exon 20, intron 20 and exon 21 and combinations of UNC13A alleles. d , e , Representative image ( d ) and quantification ( e ) of RT–PCR products from UNC13A minigenes in SH-SY5Y cells with or without TDP-43 knockdown. Data are mean ± s.e.m. Each variant was compared with the healthy minigene with which it was co-transfected and results were compared with an unpaired t -test ( n = 3 biological replicates). f , TDP-43 iCLIP of SH-SY5Y cells containing 2R and 2H minigenes. Top, average crosslink density. Middle, average density change 2R for − 2H (20-nt rolling window, units are crosslinks per 1,000). Bottom, predicted TDP-43 binding footprints (UGNNUG motif). g , Average change in E-value (measure of binding enrichment) across proteins for heptamers containing risk or healthy CE SNP alleles. TDP-43 is indicated in red. h , Binding affinities between TDP-43 and 14-nt RNA containing the CE ( n = 4) or intronic ( n = 3) healthy or risk sequences measured by isothermal titration calorimetry. Data are mean ± s.d.; two-sample t -test. i , j , Representative image ( i ) and quantification ( j ) of RT–PCR products from UNC13A minigenes with mutated UGNNUG TDP-43 binding motifs as shown in f . Data are mean ± s.e.m.; n = 3 biological replicates; statistical analysis as in ( d , e ).

Article Snippet: Gene expression analysis was performed by qPCR using Taqman Multiplex Universal Master Mix (Thermo 4461882) or Taqman Universal PCR Master Mix (Thermo 4304437) and TaqMan assays (UNC13A-Fam Hs00392638_m1, UNC13B-Fam Hs01066405_m1, TDP-43-Vic Hs00606522_m1, GAPDH-Jun assay 4485713, TDP-43-FAM Hs00606522_m1, UPF1-FAM Hs00161289_m1, HPRT1-FAM Hs02800695_m1) on a QuantStudio 5 or a QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems) and quantified using the ΔΔ C t method .

Techniques: RNA Sequencing, Reverse Transcription Polymerase Chain Reaction, Knockdown, Variant Assay, Transfection, Binding Assay, Isothermal Titration Calorimetry

Journal: Nature

Article Title: TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A

doi: 10.1038/s41586-022-04436-3

Figure Lengend Snippet: ( a ) Expression of splice junction reads supporting the UNC13A CE across tissues and disease subtypes. Junction counts are normalised by library size in millions (junctions per million). The long novel acceptor junction is expressed across all disease subtypes in the cerebellum. Box plots: boundaries 25–75th percentiles; midline, median; whiskers, Tukey style. ( b ) Example RNA-seq traces from IGV showing UNC13A cerebellar exon which shares the long novel acceptor junction as the UNC13A CE.

Article Snippet: Gene expression analysis was performed by qPCR using Taqman Multiplex Universal Master Mix (Thermo 4461882) or Taqman Universal PCR Master Mix (Thermo 4304437) and TaqMan assays (UNC13A-Fam Hs00392638_m1, UNC13B-Fam Hs01066405_m1, TDP-43-Vic Hs00606522_m1, GAPDH-Jun assay 4485713, TDP-43-FAM Hs00606522_m1, UPF1-FAM Hs00161289_m1, HPRT1-FAM Hs02800695_m1) on a QuantStudio 5 or a QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems) and quantified using the ΔΔ C t method .

Techniques: Expressing, RNA Sequencing

Journal: The Journal of Cell Biology

Article Title: Homeostatic scaling of active zone scaffolds maintains global synaptic strength

doi: 10.1083/jcb.201807165

Figure Lengend Snippet: BRP, Unc13A, and Cac abundance scale in endo and rab3 mutants, while total levels per NMJ remain constant. (A) Representative type Ib boutons immunostained with anti-BRP, anti-Unc13A, and endogenously tagged Ca 2+ channels (Cac sfGFP-N ) in the indicated genotypes (wild type: cac sfGFP-N ; endo : cac sfGFP-N ; endo 1 /endo Δ4 ; vGlut-OE: cac sfGFP-N ; OK371-Gal4 / UAS-vGlut ; rab3 : cac sfGFP-N ; rab3 rup ). Note that BRP and Unc13A are costained at the same NMJ, while Cac images were acquired from a different NMJ of the same genotype. (B) Quantification of mean fluorescence intensity of individual BRP, Unc13A, and Cac sfGFP-N puncta shows no change in endo and vGlut-OE, with a slight but significant increase in rab3 , consistent with no major difference in the density of material within each punctum. (C) Quantification of BRP, Unc13A, and Cac sfGFP-N puncta sum fluorescence intensity reveals a significant reduction in endo , an increase in rab3 , and no change in vGlut-OE, consistent with the observed changes in puncta area for each genotype ( n ≥ 15; one-way ANOVA; Table S1). (D) The total fluorescence intensity of each individual BRP, Unc13A, and Cac sfGFP-N puncta summed across the entire muscle 4 NMJ terminal is unchanged in endo , rab3, and vGlut-OE compared with wild type. Error bars indicate ± SEM ( n ≥ 8; one-way ANOVA; Table S1). *, P < 0.05; ***, P < 0.001; ****, P < 0.0001; ns, not significant. See Materials and methods for more details on mean, sum, and total intensity measurements.

Article Snippet: For STED imaging, BRP and Unc13A primary antibodies were used and goat anti-guinea pig star635 (1:100; 1–0101002; Abberior) and goat anti-mouse Alexa Fluor 594 (1:500; A11032; Thermo Fisher Scientific) were used for secondary antibodies.

Techniques: Fluorescence

Journal: The Journal of Cell Biology

Article Title: Homeostatic scaling of active zone scaffolds maintains global synaptic strength

doi: 10.1083/jcb.201807165

Figure Lengend Snippet: Elastic modularity of AZ nanostructure revealed by STED imaging. (A) Representative STED images of AZs in wild-type, endo RNAi , and rab3 mutant NMJs labeled with anti-BRP and anti-Unc13A. (B–D) BRP (B) and Unc13A (C) ring diameters are reduced in endo RNAi and enhanced in rab3 mutants, while the BRP:Unc13A ratio remains the same (D). (E) Quantal modules of BRP clusters within single AZs are revealed by STED analysis using an averaging algorithm . The three modules of peak frequency are shown for wild type (4, 5, and 6 modules), endo RNAi (2, 3, and 4), and rab3 (8, 10, and 12). (F–H) Quantification of the mean number of BRP (F) and Unc13A (G) modules per AZ is shown, with a reduction in endo RNAi and enhancement in rab3 , while the ratio is largely unchanged (H). (I and J) Cumulative probability curve (I) and binned histogram (J) of BRP modules per NMJ are shown, with a leftward distribution observed in endo RNAi and rightward shift in rab3 mutants. Error bars indicate ± SEM ( n ≥ 53; one-way ANOVA, K-S test for I; Table S1). *, P < 0.05; ****, P < 0.0001; ns, not significant.

Article Snippet: For STED imaging, BRP and Unc13A primary antibodies were used and goat anti-guinea pig star635 (1:100; 1–0101002; Abberior) and goat anti-mouse Alexa Fluor 594 (1:500; A11032; Thermo Fisher Scientific) were used for secondary antibodies.

Techniques: Imaging, Mutagenesis, Labeling

Journal: The Journal of Cell Biology

Article Title: Homeostatic scaling of active zone scaffolds maintains global synaptic strength

doi: 10.1083/jcb.201807165

Figure Lengend Snippet: Manipulating presynaptic cargo transport to NMJ terminals by arl8 modulates the gain of synaptic strength at endo and rab3 NMJs. (A) Representative images of type Ib NMJ boutons immunostained with antibodies that recognize AZ components (BRP, Unc13A), neuronal membrane (HRP), and SV markers (vGlut, Synaptotagmin [SYT], and Synapsin [SYN]) in wild type, arl8 mutants ( arl8 : w ; arl8 e00336 ), and neuronal overexpression of arl8 (arl8-OE: w;OK6-Gal4,UAS-arl8-GFP/+ ). Note that the number of AZs and the intensity of SV markers at NMJ terminals are reduced by loss of arl8 (C; Table S1), while arl8-OE enhances the intensity of AZ and SV markers (C; Table S1). (B) EPSP traces in the indicated genotypes demonstrate that synaptic strength is reduced by loss of arl8 and increased by arl8-OE. (C) Quantification of the indicated values in the three genotypes normalized to wild-type values ( n ≥ 12; one-way ANOVA; Table S1). (D–F) Representative images (D), EPSP traces (E), and quantification (F) for NMJs of endo RNAi and endo RNAi combined with arl8-OE (endo RNAi +arl8-OE: w;OK6-Gal4,UAS-arl8-GFP/+;UAS-endo-RNAi ). arl8-OE enhances synaptic strength and BRP and vGlut intensity above endo RNAi baseline values (note that values are normalized to endo RNAi ; n ≥ 7; Student’s t test; Table S1). (G–I) Representative images (G), EPSP traces (H), and quantification (I) for NMJs of rab3 RNAi , rab3 , and arl8 double mutants ( rab3 ; arl8 : w;rab3 rup ; arl8 e00336 ), and rab3 RNAi combined with arl8-OE (rab3 RNAi +arl8-OE: w;OK6-Gal4,UAS-arl8-GFP/+;UAS-rab3-RNAi ). Note that loss of arl8 reduces BRP puncta number and intensities of BRP and vGlut below rab3 RNAi baseline values, while arl8-OE enhances these values. Values are normalized to rab3 RNAi ( n ≥ 9; one-way ANOVA; Table S1). Error bars indicate ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

Article Snippet: For STED imaging, BRP and Unc13A primary antibodies were used and goat anti-guinea pig star635 (1:100; 1–0101002; Abberior) and goat anti-mouse Alexa Fluor 594 (1:500; A11032; Thermo Fisher Scientific) were used for secondary antibodies.

Techniques: Membrane, Over Expression