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. 2023 Sep 11;12:e85561. doi: 10.7554/eLife.85561

Figure 2. Tomosyns regulate the number and/or composition of neuronal dense core vesicles (DCVs).

(A) Representative images of the DCV reporter (NPY-pHluorin) in control, double knockout (DKO), and DKO re-expressing tomosyn (Stxbp5 isoform m, ‘Rescue’) neurons. Neurons were grown in mass cultures, silenced with sodium channel blocker tetrodotoxin (TTX, 1 µM) for 48 hr, and fixed on DIV14. White boxes indicate zoomed-in segments of neurites shown under every image. Scale bar 20 µm. (B) Mean intensity of NPY-pHluorin and tomosyn immunostaining in control, DKO, and rescued neurons exemplified in (A). Data were analyzed using one-way ANOVA with Tukey’s multiple comparisons post hoc test. n=29–30 neurons (plotted as dots)/ genotype. ****p<0.0001. (C) Representative images of BDNF immunostaining in control, DKO, and DKO re-expressing tomosyn neurons. Neurons were grown on glial microislands and fixed on DIV16. White boxes indicate zoomed-in segments of neurites shown under every image. Scale bar 20 µm. (D) Quantification of the mean BDNF and tomosyn intensity in control, DKO and rescued neurons exemplified in (C). Data were analyzed using the Kruskal-Wallis test with Dunn’s multiple comparisons post hoc test (BDNF) and one-way ANOVA with Tukey’s multiple comparisons post hoc test (tomosyn). n=20 neurons (plotted as dots)/ genotype. ****p<0.0001. (E) BDNF levels as detected by western blot (WB) in lysates of control and DKO neuronal cultures. Equal loading was verified by immunodetection of GAPDH. (F) Quantification of the BDNF band intensity from WB exemplified in (E). BDNF levels in DKO neurons were normalized to control levels in the corresponding culture. DKO data are plotted as mean ± SD and were analyzed using one-sample t-test. n=8 samples/genotype from four culture preparations. ****p<0.0001. (G) Re-expression of tomosyn (either full length, ‘FL,’ or a truncated mutant lacking the SNARE domain, ‘ΔSNARE’) partially restores BDNF levels in DKO neurons as detected by WB. Same WB shows that an inhibition of lysosomal proteolysis by leupeptin (50 μM for 24 hr) does not equalize BDNF levels between control and DKO neurons. Immunodetection of tomosyn was used to validate the expression and the correct size of the rescue constructs. Equal loading was verified by immunodetection of GAPDH. (H) Quantification of the BDNF band intensity from WB exemplified in (G). BDNF levels in DKO neurons are shown as the mean % of control levels. Data were analyzed using one-sample t-test comparing to 100% (control levels). n=4 samples/genotype from two culture preparations. ***p<0.001. (I) Quantification of the BDNF band intensity in leupeptin-treated samples from WB exemplified in (G). BDNF levels in all groups are shown as % of the averaged vehicle-treated control levels. Bars indicate mean values. Data were analyzed using a two-way repeated measures ANOVA. n=4 samples/genotype from two culture preparations.

Figure 2—source data 1. Uncropped western blot (WB) images for Figure 2E and G.

Figure 2.

Figure 2—figure supplement 1. Expression of enhanced green fluorescent protein (EGFP) under the control of synapsin promoter is not affected in double knockout (DKO) neurons.

Figure 2—figure supplement 1.

(A) Representative images of EGFP expressed under the control of synapsin promoter in fixed control and DKO neurons. Scale bar 20 µm. (B) Quantification of the mean EGFP intensity in control and DKO neurons from confocal microscopy images as exemplified in (A). Data are shown as mean ± SD and were analyzed using a two-tailed unpaired t-test. N=28–30 neurons/genotype. Ns: not significant.

Figure 2—figure supplement 2. Levels of a transmembrane dense core vesicle (DCV) marker, IA-2 (PTPRN), are decreased in double knockout (DKO) neurons.

Figure 2—figure supplement 2.

(A) Representative images of the IA-2 immunostaining in control and DKO neurons. Neurons were grown in mass cultures, silenced with sodium channel blocker tetrodotoxin (TTX, 1 µM) for 48 hr, and fixed on DIV14. White boxes indicate zoomed-in segments of neurites shown under every image. Scale bar 20 µm. (B) Quantification of the mean IA-2 intensity in control and DKO neurons from images exemplified in (A). Data are shown as mean ± SD and were analyzed using a two-tailed unpaired t-test. n=24 neurons/genotype. *p<0.05; ****p<0.0001. (C) Levels of IA-2 as detected by WB in control and DKO neuronal lysates. Proteolytically processed (mature) form of IA-2 was detected at 65 kDa. Equal loading was verified by the detection of total protein stained with trichloroethanol (TCE) and by immunodetection of actin. Amount of mature IA-2 in DKO neurons was normalized to control levels in the corresponding culture. DKO data are presented as mean ± SD and were analyzed using one sample t-test. n=9 samples/genotype from three culture preparations. ****p<0.0001.
Figure 2—figure supplement 2—source data 1. Uncropped western blot (WB) images for Figure 2—figure supplement 2C.

Figure 2—figure supplement 3. Loss of tomosyns does not affect levels of endo-lysosomal proteins.

Figure 2—figure supplement 3.

(A) Representative images of LAMP1 immunostaining in DIV14 neurons. Scale bar 20 µm. (B) Quantification of the mean LAMP1 intensity in control and double knockout (DKO) neurons from confocal microscopy images as exemplified in (A). Data are shown as mean ± SD and were analyzed using a two-tailed unpaired t-test. n=10 neurons/genotype. ns: not significant. (C) Levels of LAMP1 and LIMP2 as detected by western blot (WB) are normal in DKO neurons. Equal loading was verified by immunodetection of actin. (D) Quantification of LAMP1 and LIMP2 signal from WB images exemplified in (C). Levels of LAMP1 and LIMP2 in DKO were normalized to control levels in the corresponding culture. DKO data are presented as mean ± SD and were analyzed using one sample t-test. n=9 samples/genotype from three culture preparations. ns: not significant.
Figure 2—figure supplement 3—source data 1. Uncropped western blot (WB) images for Figure 2—figure supplement 2C.