2. AIMS OF THE STUDY
3.6 LC3 regulates MT stability via ELKS1/CLASP2 function
Given that LC3 associates with ELKS1 in neurons, we next determined whether ELKS1-CLASPs function might mediate MT stability in LC3 lipidation-deficient neurons. To investigate that, first, protein levels of ELKS1 were analyzed in ATG5 KO neurons in vitro and in vivo. ELKS1 levels were found significantly upregulated in lysates from cultured neurons (Fig.28a,b) and within the brain (Fig.28d,e) of neurons lacking ATG5. Indeed, ELKS1 accumulated within axonal swellings of cultured neurons lacking the LC3 lipid conjugation machinery (Fig.28c). Since ELKS1 could be accumulated due to defective degradation via autophagy, ELKS1 proteins levels were studied upon treatment with BafilomycinA, an inhibitor of the V-type ATPase that blocks lysosomal degradation. Strikingly, we found that ELKS1 is not degraded via autophagy (Fig.28f). Thus, these data suggest that LC3 regulates ELKS1 via a non-canonical degradation independent pathway.
Figure 28. ELKS1 levels are stabilized in ATG5 KO neurons via an independent degradation mechanism.(a,b) Western blot analysis of ELKS1 levels in cultured ATG5 KO neurons compared to the
WT set to 100%. KO: 217.44±25.52%. p=0.022. N=3. (c)Cultured neurons from WT and ATG5 KO mice, carrying tdTomato allele as a reporter of Cre-recombination (red), were immunostained for ELKS1 (green). White boxes depict the magnified areas underneath. Scale bars, 10µm upper panels, 4µm lower panels. (d) Representative confocal images of cortical sections from WT and ATG5 KO mice immunostained for ELKS1 (green) and co-immunostained for β3-Tubulin (red) to reveal neuronal processes. In lower panels, ELKS1 fluorescent intensity in neurites was false color-coded with warm colors representing high intensities. Scale bars, 50µm upper panels, 5µm lower panels. (e)ELKS1 levels are significantly increased in ATG5 KO brains compared to the WT set to 100%. KO:180.12±15.20%. p=0.009. N=4. (f) ELKS1 protein levels upon 67µM BafilomycinA (BafA) or vehicle solution (DMSO) treatments for 16h in NSC34 neuronal- like cell line. p62 blot illustrates the inhibition of the autophagy-lysosome degradation pathway. All data shown represent the mean ± SEM from N independent experiments.
3.6.1 CLASP2 levels are increased in neuronal processes of ATG5 KO mice
Next, we investigated CLASP2 protein levels in WT and ATG5 KO neurons. In line with ELKS1 data, CLASP2 levels were upregulated in ATG5 KO mouse brain (Fig.29a,b). Moreover, superresolution STED imaging highlighted an increased abundance of CLASP2 association along MTs in ATG5 KO cultured neurons compared to WT (Fig.29c,d). Collectively, these data suggest
that ELKS1 retention via LC3I accumulation in ATG5 KO neurons alters the levels of the MT stabilizing protein CLASP2.
Figure 29. CLASP2 levels are increased in KO axonal swellings. (a) Representative confocal images
of cortical sections from WT and ATG5 KO mice immunostained for CLASP2 (green) and co- immunostained for β3-Tubulin (red) to reveal neuronal processes. In lower panels, CLASP2 fluorescent intensity in neurites was false color-coded with warm colors representing high intensities. Scale bars, 50µm upper panels, 10µm lower panels. (b) CLASP2 levels are significantly increased in ATG5 KO brains compared to the WT set to 100% (179.08±25.04%). p=0.046.N=4. (c,d) Confocal and STED images of neurons of CLAPS2 (green) and α-tubulin (red). White rectangular boxes depict images in e. Scale bars: 10µm. (e) Amplified images from d. Scale bars: 1µm. All data shown represent the mean ± SEM from N independent experiments.
3.6.2 Non-lipidated LC3 regulates MT dynamics via ELKS1-CLASP2 dependent mechanism in axons
Thus far, we hypothesized that the non-lipidated LC3 stabilizes ELKS1-CLASP2 within the axon, which in turn may alter MT dynamics in LC3 lipidation-deficient neurons. To test this hypothesis,
first, levels of ELKS1 in axonal swellings were analyzed upon LC3 KD (see Fig.13e,f for siRNA validation). In line with our hypothesis, LC3 KD not only rescued axonal swellings after 7 days post-transfection (13g,h), but also caused a 30% reduction of ELKS1 protein density within spheroids (Fig.30a,b). Interestingly, these data suggested that reduction of ELKS1 levels in axons might also rescue the formation of spheroids in ATG5 KO neurons. Thus, smart pools of siRNAs against Elks1 were used. Fig.30c,d shows ELKS1 KD validation in NSC34 cells after 72h post- transfection. In neurons, although only a 30% of reduction in ELKS1 protein levels was detected after 7 days of transfection (Fig.30e,f), this KD period was enough to significantly reduce the average size of axonal swellings in ATG5 KO neurons (Fig.30e,g). Moreover, ELKS KD was also accompanied by a significant reduction of CLASP2 protein levels within spheroids (Fig.30h,i). Ultimately, the deletion of ELKS1 in autophagy-deficient neurons was sufficient to completely rescue impaired MT dynamics, monitored by the expression of EB3-tdTomato in WT and ATG5 cultured neurons (Fig.30j,k). Taken together, these data demonstrate that dysfunctional LC3 lipidation, and its subsequent accumulation, alters MT dynamics via controlling the abundance of ELKS1-CLASP2 within axons.
Figure 30. Autophagy lipidation machinery regulates MT dynamics in neurons via ELKS1/CLASP2- dependent mechanism. (a)Immunostained for LC3 (red) cultured ATG5 KO neurons, transfected with
eGFP (green) and co-transfected either with scrambled siRNA (scr) or siRNA directed against LC3b. Scale bars, 10 µm upper panels, 4 µm lower panels. (b) ELKS1 levels are significantly decreased in ATG5 KO neurons treated with LC3B siRNA. KOsiLC3B: 70.1±4.37%. p=0.010.Protein levels in the KOsiLC3B were
normalized to the KOscr set to 100%. In total, 247 KOscr and 281 KOsiLC3B axons from N=3. (c,d)Levels of
72h. WT: 96.91±10.39%. KO: 29.45±4.58%. p=0.004. N=3 (e) Immunostained for ELKS1 (red) WT and ATG5 KO neurons transfected with eGFP (green) and co-transfected either with scr siRNA or Elks1 siRNA. Scale bars, 5µm. (f) Fluorescence analysis of ELKS1 levels in WT or ATG5 KO axons upon scramble or
Elks1 siRNA after 7 days from transfection. WTScr: 99.09±7.53%, N=19; KOScr: 129.82±8.2%, N=19; WTElks1
KD: 68.33±5.56, N=16, KOElks1 KD: 95.25±7.27%, N=17. p WTScr vs KOScr= 0.016, p WTScr vs WTElks1 KD=0.023, p KOScr vs WTElks1 KD< 0.000, p KOScr vs KOElks1 KD= 0.007. (g) The spheroid areain ATG5 KO neurons treated with ELKS1 siRNA (KOsiElks1: 5.76±0.23µm2) compared with scramble KO controls (KOscr:
4.25±0.30µm2). p=0.018. In total, 142 spheroids for KOscr and 125 spheroids for KOsiElks1, from N=3. (h,i)
Confocal analysis of CLASP2 levels in ATG5 KO axons upon scramble or Elks1 siRNA co-transfection with eGFP. KOScr= 84.82±1.38%. p=0.004. Protein levels in KOElks1 KD condition were normalized to the KOScr
set to 100%. N=3. (j) Representative fluorescent images from time-lapse videos of ATG5 KO neurons transfected with EB3-tdTomato and co-transfected with either scrambled siRNA (scr) or with siRNA directed against Elks1. Corresponding kymographs are shown to the right. Scale bars, x: 5µm, y: 30s. (k) EB3 comet density. WTscr: 0.06±0.00, KOscr: 0.03±0.01, Elks1siRNA: WTsiElks1: 0.08±0.01, KOsiElks1: 0.07±0.01.
pWTscr vs KOscr =0.025, pKOscr vs KOsiElks1=0.004, pWTscr vs WTsiElks1 =0.041.In total, 53 WT and 55 KO
axons from N=3.n.s.-non significant. All data shown represent the mean ± SEM from N independent experiments.