Exploring the ATG9A interactome uncovers interaction with VPS13A.

ATG9A, the only transmembrane protein of the core autophagy pathway, cycles between the Golgi, endosomes and a vesicular compartment. ATG9A was recently shown to act as a lipid scramblase and this function is thought to require its interaction with another core autophagy protein ATG2A, which acts as a lipid transfer protein. Together ATG9A and ATG2A are proposed to function to expand the growing autophagosome. However, ATG9A is implicated in other pathways including membrane repair and lipid droplet homeostasis. To elucidate other ATG9A interactors within the autophagy pathway, or interactors beyond autophagy we performed an interactome analysis through mass spectrometry. This analysis revealed a host of proteins involved in lipid synthesis and trafficking, including ACSL3 and VPS13A and C. Furthermore, we show that ATG9A directly interacts with VPS13A and forms a complex distinct from the ATG9A:ATG2A complex

Sensorimotor behavioural tests after CSP-TTK21 treatment in chronic SCI with severe disability.

(A) BMS quantification of CSP (n = 15) or CSP-TTK21 (n = 14) treated mice after chronic SCI (Treatment: f(1) = 0.002, p = 0.96 (TOST: given equivalence bounds of −0.21 and 0.21, 90% confidence intervals fall −0.37 and 0.32 and p = 0.307, thus H0 is undecided); Time: f(12) = 192.1, p p = 0.215, thus H0 is rejected); Interaction (treatment × time): f(12) = 6.6, p = 0.82 (TOST: given equivalence bounds of −0.21 and 0.21, 90% confidence intervals fall 4.19 and 4.99 and p > 0.999, thus H0 is rejected). Two-way repeated measures ANOVA with Sidak post hoc test. (B) Gridwalk quantification of the percentage of slips per total number of steps per run in CSP (n = 4) or CSP-TTK21 (n = 6) treated mice with a BMS score greater than 3 after chronic SCI (Treatment: f(1) = 0.2, p = 0.69 (TOST: given equivalence bounds of −1.78 and 1.78, 90% confidence intervals fall −7.96 and 2.63 and p = 0.693, thus H0 is undecided); Time: f(12) = 7.1, p p p = 0.82 (TOST: given equivalence bounds of −1.78 and 1.78, 90% confidence intervals fall −28.59 and 41.29 and p > 0.999, thus H0 is rejected). Two-way repeated measures ANOVA with Sidak post hoc test). (C) Bar graph indicating the number of mice with a BMS score greater than 3 across postinjury time points. (D) Hargreaves test indicating average paw withdrawal latency in CSP or CSP-TTK21 (Welch two-tailed t test: CSP: 6.7 ± 1.1, n = 4; CSP-TTK21: 7.6 ± 0.5, n = 6, p = 0.51, TOST: t(8) = −0.3, p = 0.63 given equivalence bounds of −0.5 and 0.5 on a raw scale and an alpha of 0.05) treated mice with a BMS score greater than 3. (E) Von Frey test indicating average paw withdrawal threshold in CSP or CSP-TTK21 (Welch two-tailed t test: CSP: 5.5 ± 1.6, n = 4; CSP-TTK21: 6.4 ± 0.7, n = 6, p = 0.64, TOST: t(4.3) = −0.1, p = 0.52 given equivalence bounds of −0.8 and 0.8 on a raw scale and an alpha of 0.05) treated mice with a BMS score greater than 3. All data are given as mean ± SEM. n = biologically independent animals. The data can be found in S1 Data. BMS, Basso Mouse Scale; CSP, carbon nanosphere; SCI, spinal cord injury; TOST, two one-sided tests.</p

S1 Fig -

(A) Twelve weeks old mice were housed in SH or EE 1 week after a spinal cord transection. (B and C) Animals in SH remained impaired unable to step until day 42 after injury as shown by BMS (B) and Gridwalk (C). EE significantly enhanced locomotion (mean ± SEM, two-way ANOVA, Fisher LSD post hoc ** P P P S1 Data. BMS, Basso Mouse Scale; EE, enriched environment; SH, standard housing. (PDF)</p

S4 Fig -

(A) Representative micrographs of GFAP intensity (red) around the SCI site (white asterisks) and cavity size (white dotted line) in CSP or CSP-TTK21 mice. (B) Quantification of cavity size in CSP or CSP-TTK21-treated mice (CSP: 471,574.0 ± 76,631.0, n = 8; CSP-TTK21: 486,466.0 ± 45,491.0, n = 14; p = 0.87; TOST: t(20.0) = 0.5, p = 0.31 given equivalence bounds of −56,344.2 and 56,344.2 on a raw scale and an alpha of 0.05). (C) Quantification of GFAP intensity in CSP or CSP-TTK21-treated mice (CSP: 1,272.0 ± 31.3, n = 3; CSP-TTK21: 1,339.0 ± 20.8, n = 4, p = 0.12, TOST: t(3.7) = −1.4, p = 0.88 given equivalence bounds of −14.5 and 14.5 on a raw scale and an alpha of 0.05). Mean ± SEM; unpaired two-tailed Student t test or Welch t test. n = biologically independent animals. The data can be found in S1 Data. CSP, carbon nanosphere; SCI, spinal cord injury; TOST, two one-sided tests. (PNG)</p

Table summarizing the experimental design and data analysis.

Table summarizing the experimental design and data analysis.</p

Excel spreadsheets containing the quantitative data for each experiment as described in the results and figure legends.

(XLSX)</p

Graphical diagram summarizing the experimental design. <i>Made with BioRender</i>.

(PNG)</p

S5 Fig -

(A) Representative micrographs of CD68 immunofluorescence (red) and DAPI (blue) around the SCI site (white asterisks) in CSP or CSP-TTK21-treated mice. Lesion site (white dotted line). (B) Quantification of CD68 intensity in CSP or CSP-TTK21-treated mice (CSP: 472.0 ± 8.1, n = 3 CSP-TTK21: 434.5 ± 37.8 n = 4, p = 0.44, TOST: t(3.3) = 0.5, p = 0.69 given equivalence bounds of −16.3 and 16.3 on a raw scale and an alpha of 0.05). Mean ± SEM; unpaired two-tailed Student t test. n = biologically independent animals. The data can be found in S1 Data. CSP, carbon nanosphere; SCI, spinal cord injury; TOST, two one-sided tests. (PNG)</p

S3 Fig -

(A) Representative micrographs of cJUN immunostaining (green, white arrows) in layer 5 cortical neurons from CSP or CSP-TTK21-treated mice. (B) Quantification of cJUN immunostaining in layer 5 cortical neurons (CSP: 2,539.0 ± 295.4; CSP-TTK21: 2,355.0 ± 206.7, p = 0.63, TOST: t(0.1) = 0.1, p = 0.5 given equivalence bounds of −153.0 and 153.0 on a raw scale and an alpha of 0.05, n = 4). (C) Representative micrographs of SPRR1a immunostaining (green, white arrows) in layer 5 cortical neurons. (D) Quantification of SPRR1a immunostaining in layer 5 cortical neurons in CSP or CSP-TTK21 (CSP: 3,940.0 ± 491.6, n = 3; CSP-TTK21: 3,636.0 ± 350.3, n = 4; p = 0.08, TOST: t(4.8) = −1.9, p = 0.94 given equivalence bounds of −344.4 and 344.4 on a raw scale and an alpha of 0.05). Mean ± SEM; unpaired two-tailed Student t test or Welch t test. n = biologically independent animals. The data can be found in S1 Data. CSP, carbon nanosphere; TOST, two one-sided tests. (PNG)</p

Expression of regeneration-associated proteins in DRG neurons in CSP-TTK21-treated mice in a chronic SCI.

(A-N) Representative micrographs of immunostaining for regeneration-associated proteins (green, white arrows) in DRG neurons from CSP or CSP-TTK21-treated mice (A, C, E, G, I, K, M) and respective quantification of immunostaining in DRG neurons (B, D, F, H, J, L, N). (A, B) Atf3 (CSP: 2,113.0 ± 461.5; CSP-TK21: 4,614.0 ± 449.4, p C, D) Sprr1a (CSP: 4,904.0 ± 1,033.0; CSP-TK21: 9,331.0 ± 914.6, p (E, F) cJun (CSP: 3,697.0 ± 302.2; CSP-TK21: 4,904.0 ± 340.7, p (G, H) Klf7 (CSP: 4,097.0 ± 199.1; CSP-TK21: 772.00 ± 317.9, p I, J) Gap43 (CSP: 1,178.0 ± 173.2; CSP-TK21: 4,643.0 ± 163.6, p K, L) pErk (CSP: 3,433.0 ± 428.9; CSP-TK21: 3,036.0 ± 750.7, p = 0.66, TOST: t(4.8) = 0.03, p = 0.51 given equivalence bounds of −366.7 and 366.7 on a raw scale and alpha of 0.05). (M, N) pStat3 (CSP: 2,894.0 ± 470.1; CSP-TK21: 3,579.0 ± 656.8, p = 0.43, TOST: t(5.4) = −0.42, p = 0.66 given equivalence bounds of −342.7 and 342.7 on a raw scale and alpha of 0.05). Mean ± SEM; unpaired two-tailed Student or Welch t test; *p p p n = 3 or 4 biologically independent animals. The data can be found in S1 Data. CSP, carbon nanosphere; DRG, dorsal root ganglion; SCI, spinal cord injury; TOST, two one-sided tests.</p