Journal: Nature Neuroscience

Article Title: Messenger RNA transport on lysosomal vesicles maintains axonal mitochondrial homeostasis and prevents axonal degeneration

doi: 10.1038/s41593-024-01619-1

Figure Lengend Snippet: a , Schematic representation of the coupling of lysosomes to the plus-end-directed kinesin-1 motor through the BORC–ARL8–PLEKHM2 ensemble . b , iPSCs were differentiated for 3 d in DMEM/F12 medium supplemented with NEAAs, GlutaMAX and N2A and doxycycline (DOX) and further cultured for 25–45 d in BrainPhys medium containing B27, laminin, BDNF, NT-3 and DOX. c , SDS-PAGE and immunoblot (IB) analysis of WT, BORCS5-KO, BORCS7-KO, BORCS5-KO re-expressing BORCS5-P2A-GFP (rescue) and BORCS7-KO re-expressing BORCS7-HA-P2A-GFP (rescue) iPSCs using antibodies to BORCS5, BORCS7 and GAPDH (loading control). After self-cleavage, 22 amino acid residues at the N-terminus of P2A remain linked to the upstream protein, thus the higher molecular mass in the rescue cells. The positions of molecular mass markers (in kDa) are indicated on the left. d , e , i3Neurons derived from the WT, BORCS5-KO, BORCS7-KO ( d ) and BORCS5 rescue and BORCS7 rescue ( e ) iPSCs shown in c were cultured for 25 d on glass coverslips and immunostained for endogenous MAP2 (soma and dendrites) (magenta), synaptophysin (SYP) (synaptic vesicles), TOMM20 (mitochondria), LAMP1 (lysosomes) or LAMTOR4 (lysosomes) (all grayscale). Nuclei were stained with DAPI (blue). Scale bars, 10 μm. See also Extended Data Figs. , and . Panel e additionally shows higher intensity images of MAP2 staining in grayscale (MAP2 bright). f , Quantification of the number of axonal puncta staining for LAMP1 or LAMTOR4 in various i3Neuron lines relative to WT i3Neurons (defined as 1.0) from n = 3 independent experiments such as that shown in e . Results are represented as SuperPlots showing the individual data points in different colors, the mean from each experiment and the mean of the means ± s.d. Statistical significance was calculated by one-way ANOVA with Dunnett’s multiple comparisons test. Axonal LAMP1 significance versus WT: BORCS5 KO *** P < 0.001, BORCS7 KO *** P < 0.001, BORCS5 rescue P = 0.995 and BORCS7 rescue P = 0.157. Axonal LAMTOR4 significance versus WT: BORCS5 KO *** P < 0.001, BORCS7 KO *** P < 0.001, BORCS5 rescue *** P < 0.001 and BORCS7 rescue P = 0.870. NS, not significant, relative to WT.

Article Snippet: The induction medium was changed every day for 3 d. Cells were then lifted with Accutase, counted and plated in neuronal culture medium consisting of BrainPhys medium (5790, STEMCELL Technologies) supplemented with 10 ng ml −1 neurotrophin-3 (450-03, PeproTech, Thermo Fisher Scientific), 10 ng ml −1 BDNF (450-02, PeproTech, Thermo Fisher Scientific), 1× B-27 supplement serum free (17504044, Gibco, Thermo Fisher Scientific), 2 μg ml −1 doxycycline (D9891, MilliporeSigma) and 1 μg ml −1 mouse laminin (23017015, Gibco, Thermo Fisher Scientific).

Techniques: Cell Culture, SDS Page, Western Blot, Expressing, Control, Derivative Assay, Staining