mTORC1-independent TFEB activation via Akt inhibition promotes cellular clearance in neurodegenerative storage diseases

Michela Palmieri, Rituraj Pal, Hemanth R Nelvagal, Parisa Lotfi, Gary R Stinnett, Michelle L Seymour, Arindam Chaudhury, Lakshya Bajaj, Vitaliy V Bondar, Laura Bremner, Usama Saleem, Dennis Y Tse, Deepthi Sanagasetti, Samuel M Wu, Joel R Neilson, Fred A Pereira, Robia G Pautler, George G Rodney, Jonathan D Cooper, Marco Sardiello, Michela Palmieri, Rituraj Pal, Hemanth R Nelvagal, Parisa Lotfi, Gary R Stinnett, Michelle L Seymour, Arindam Chaudhury, Lakshya Bajaj, Vitaliy V Bondar, Laura Bremner, Usama Saleem, Dennis Y Tse, Deepthi Sanagasetti, Samuel M Wu, Joel R Neilson, Fred A Pereira, Robia G Pautler, George G Rodney, Jonathan D Cooper, Marco Sardiello

Abstract

Neurodegenerative diseases characterized by aberrant accumulation of undigested cellular components represent unmet medical conditions for which the identification of actionable targets is urgently needed. Here we identify a pharmacologically actionable pathway that controls cellular clearance via Akt modulation of transcription factor EB (TFEB), a master regulator of lysosomal pathways. We show that Akt phosphorylates TFEB at Ser467 and represses TFEB nuclear translocation independently of mechanistic target of rapamycin complex 1 (mTORC1), a known TFEB inhibitor. The autophagy enhancer trehalose activates TFEB by diminishing Akt activity. Administration of trehalose to a mouse model of Batten disease, a prototypical neurodegenerative disease presenting with intralysosomal storage, enhances clearance of proteolipid aggregates, reduces neuropathology and prolongs survival of diseased mice. Pharmacological inhibition of Akt promotes cellular clearance in cells from patients with a variety of lysosomal diseases, thus suggesting broad applicability of this approach. These findings open new perspectives for the clinical translation of TFEB-mediated enhancement of cellular clearance in neurodegenerative storage diseases.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1. Amelioration of disease pathology in…
Figure 1. Amelioration of disease pathology in JNCL mice fed with trehalose.
(a) Trehalose significantly extended survival of Cln3Δex7–8 mice. Treated (Tre) Cln3Δex7-8 mice: n=13. Untreated (UT) Cln3Δex7-8 mice: n=12. (b) Weight of brains from 12-month-old WT and Cln3Δex7-8 mice with or without trehalose treatment. All groups of mice, n=4 or 5. (c) Fractional anisotropy of brains from 12-month-old WT and Cln3Δex7-8 mice with or without trehalose treatment. Left panel: representative coronal images of the four groups of brains; corpus callosa are indicated by the yellow arrowhead. Right panel: quantification of callosal volume. All groups of mice, n=3 or 4. Scale bar, 2 μm. Three-dimensional reconstructions of the corpus callosum in mice from treated and control groups are reported in Supplementary Movies 1–4. (d) In the hot plate test, Cln3Δex7-8 mice respond slower when placed on a 50 °C heated metal surface compared with wild-type (WT) littermates, indicating reduced pain sensitivity. Trehalose (Tre) treatment rescued this phenotype in Cln3Δex7-8 mice. All groups of mice, n=14–19. Data represent means±s.e.m. *P<0.05, **P<0.01, ***P<0.001.
Figure 2. Assessment of storage burden.
Figure 2. Assessment of storage burden.
(a,b) Confocal images and quantification of the storage material in trehalose-treated (Tre) and untreated mice in the primary somatosensory cortex (S1BF; a), and in the interconnected thalamic relay nucleus (VPM/VPL; b) at 7 month of age. Thresholding image analysis revealed higher levels of autofluorescent storage material in the cortex and thalamus of Cln3Δex7-8 mice, which is reduced by trehalose treatment. Scale bar, 50 μm. All groups of mice, n=3 or 4. (c,d) Confocal images and quantification of the amount of storage material in 12-month-old trehalose-treated and control mice in the primary somatosensory cortex (S1BF; c) and in the interconnected thalamic relay nucleus (VPM/VPL; d). Thresholding image analysis revealed higher levels of autofluorescent storage material in the cortex and thalamus of Cln3Δex7-8 mice, which is partially rescued by trehalose treatment. All groups of mice, n=3 or 4. Scale bar, 50 μm (a–d). Data represent means±s.e.m. *P<0.05, **P<0.01, ***P<0.001. (e,f) TEM analysis of untreated (UT) Cln3Δex7-8 mouse brains show marked accumulation of electron-dense cytoplasmic material (yellow arrowheads) in both Purkinje cells (e) and cortical neurons (f). Frequency distribution of FPPs counting revealed a significant reduction of FPPs in trehalose (Tre)-treated mice. n of cells per group of mice=18. Kolmogorov–Smirnov test was applied for frequency analysis. Scale bars, 2 μm.
Figure 3. Assessment of neuroinflammation.
Figure 3. Assessment of neuroinflammation.
(a,b) Analysis and quantification of astrocytosis in trehalose-treated (Tre) and untreated (UT) WT and Cln3Δex7-8 mice at 7 months of age using immunohistochemical staining for GFAP in the primary somatosensory cortex (S1BF; a) and in the interconnected thalamic relay nucleus (VPM/VPL; b). (c,d) Analysis and quantification of microglial activation using immunohistochemical staining for CD68 in the S1BF (c) and VPM/VPL (d) brain regions. Microglial activation is evident in both S1BF and VPM/VPL region of Cln3Δex7-8 mice, which is significantly rescued by trehalose treatment in the S1BF region. All groups of mice, n=4 or 5. (e,f) Analysis and quantification of astrocytosis in trehalose-treated (Tre) and control (UT) mice at 12 months of age using immunohistochemical staining for GFAP in the S1BF (e) and in the VPM/VPL (f). Trehalose treatment decreased GFAP immunoreactivity in Cln3Δex7-8 mice by 43% in the S1BF region and by 67% in the VPM/VPL region. (g,h) Analysis and quantification of microglial activation using immunohistochemical staining for CD68, in the S1BF (g) and VPM/VPL (h) brain regions. Microglial activation is evident in both S1BF and VPM/VPL region of Cln3Δex7-8 mice, which is reduced by 48% in the VPM/VPL region by trehalose treatment. All groups of mice, n=3 or 4. Scale bars, 50 μm. Data represent means±s.e.m. *P<0.05, **P<0.01, ***P<0.001.
Figure 4. mTORC1-independent nuclear translocation of TFEB…
Figure 4. mTORC1-independent nuclear translocation of TFEB on trehalose treatment.
(a) Confocal microscopy analysis of HeLa/TFEB cells showing time-dependent nuclear translocation of TFEB (green signal) on trehalose treatment. (b) Quantification of TFEB subcellular localization (C, cytoplasmic; N, nuclear) after 24 h of trehalose treatment (Tre) or in untreated cells (UT). Scale bars in a,b is 40 μm. (c) Immunoblot analyses show expression levels of substrates downstream of mTORC1. Wild-type (WT) and TSC2 null MEF cells were treated with trehalose (Tre; 100 mM) for 24 h or left untreated. As controls, cells were treated with Torin 1 (300 nM) or rapamycin (300 nM) for 2 h before extracting the lysates. Phospho- and total S6K1 (P-S6K1 and T-S6K1), phospho- and total S6 (P-S6 and T-S6) and phospho- and total 4E-BP1 (P-4E-BP1 and T-4E-BP1) were detected as readouts of mTORC1 activity. (d) WT and (e) TSC2 null MEF cells were transiently transfected with TFEB-3xFLAG and tested for nuclear translocation of TFEB following trehalose administration. (f) HeLa cells co-transfected with TFEB-3xFLAG and mTOR or (g) TFEB-3xFLAG and constitutively active mTOR (CA-mTOR, C2419K) constructs were treated with trehalose (100 mM for 24 h) or left untreated before immunofluorescent labelling of TFEB (red) and mTOR (green) with FLAG and mTOR antibodies, respectively. Scale bar, 10 μm (d–g). Data represent means±s.e.m.
Figure 5. Activation of the CLEAR network…
Figure 5. Activation of the CLEAR network by trehalose.
(a,b) Expression analysis of control (CTRL; a) and JNCL fibroblasts (b) showing upregulation of lysosomal genes on trehalose treatment. Gene expression was normalized relative to the housekeeping gene, GAPDH. (c) Cytoscape-generated network representing genes upregulated by trehalose administration. Dots (representing genes) are connected by blue lines with colour intensity proportional to the extent of co-regulation. The network has a core of genes with tighter expression relationships containing TFEB lysosomal targets (center of network), while other genes more loosely correlated are found in the periphery of the network. (d,e) GSEA of transcriptome changes following trehalose administration to CTRL (d) and JNCL fibroblasts (e), with lysosomal genes. Upper panels show the enrichment plots generated by GSEA of ranked gene expression data (left, red: upregulated; right, blue: downregulated). Vertical blue bars indicate the position of genes in each selected gene set within the ranked lists. Lower panels show the cumulative distribution of lysosomal genes within the ranked lists. The ranking positions that include 50% of analysed genes are indicated. The analysis shows enrichment of lysosomal genes among genes that were upregulated following trehalose administration. (f,g) GSEA of transcriptome changes following trehalose administration to CTRL (f) and JNCL fibroblasts (g), with lysosomal genes and TFEB targets with a known role in lysosomal metabolism being reported. TFEB lysosomal targets have a higher ES score than general lysosomal genes, indicating that trehalose preferentially upregulated TFEB targets participating in lysosomal function in both control and JNCL fibroblasts. Data represent means±s.e.m.
Figure 6. TFEB nuclear translocation and CLEAR…
Figure 6. TFEB nuclear translocation and CLEAR network activation in vivo.
(a,b) Expression analysis of cultured cortical neurons from WT (a) and Cln3Δex7-8 embryos (b) at E17.5 shows transcriptional activation of lysosomal genes on trehalose administration. (c,d) Confocal microscopy of brain sections from WT (c) and Cln3Δex7-8 (d) mice shows prevalent nuclear distribution of TFEB in Purkinje of treated mice. C and N in bar diagram indicate cytosolic and nuclear distributions, respectively. Scale bar, 20 μm. (e,f) Expression analysis of brain homogenates from WT (e) and Cln3Δex7-8 (f) mice on trehalose administration compared to untreated mice, showing transcriptional activation of lysosomal genes. Gene expression was normalized relative to the housekeeping gene, S16. The red dashed line indicates relative gene expression in untreated mice. Data represent means±s.e.m.
Figure 7. Akt phosphorylates TFEB at Ser467.
Figure 7. Akt phosphorylates TFEB at Ser467.
(a) Confocal microscopy analysis of HeLa/TFEB cells showing nuclear translocation of TFEB on addition of trehalose and kinase inhibitors (MK2206 for Akt; LY294002 for PI3K; torin 1 and rapamycin for mTOR). Dashed boxes (upper row) show the location of the higher power inserts (lower row). (b) Subcellular fractionation of HeLa/TFEB cells incubated with the same kinase inhibitors. (c) Multi-alignment of TFEB amino-acid sequences from the following species: Ac, Anolis carolensis; Bt, Bos taurus; Dr, Danio rerio; Fc, Felix catus; Gg, gallus gallus; Hs, Homo sapiens; La, Loxodonta africana; Mm, Mus musculus; Rn, Rattus Norvegicus; Sh, Sarcophilus harrisii; Sp, Strongylocentrotus purpuratus; Xl, Xenopus laevis. A consensus logo of Akt phosphorylation sites (generated at http://weblogo.berkeley.edu/logo.cgi) is aligned with TFEB sequences. Position 467 refers to the human protein sequence. (d) Subcellular localization of TFEB and TFEB(S467A). (e) Expression analysis of lysosomal and autophagy genes in HeLa cells transfected with TFEB or TFEB(S467A). Gene expression was normalized relative to the housekeeping gene, GAPDH. The dashed line indicates relative gene expression in cells transfected with an empty vector. (f) Co-localization assay of 14-3-3 proteins and TFEB-Flag or TFEB(S467A) in HeLa cells. (g) Co-immunoprecipitation assays of TFEB or TFEB(S467A) with 14-3-3 proteins. (h) Akt in vitro kinase assay. Recombinant active AKT1 and purified TFEB-Flag or TFEB(S467A)-Flag were incubated in the presence of [32P]ATP, revealing that Akt phosphorylates TFEB and that this reaction requires S467. (i) AKT silencing mediated by three different AKT siRNAs resulted in TFEB nuclear translocation and lysosomal expansion as indicated by western blot analysis. (j) Time course analysis of HeLa cells shows trehalose-induced AKT inactivation and increase of autophagic flux as indicated by LAMP1, p62 and LC3 markers. (k) HeLa cells co-transfected with TFEB-FLAG and either AKT-GFP or AKT(DD)-GFP were treated for 24 h with trehalose before immunofluorescence labelling of TFEB (red) and AKT-GFP (green). DAPI indicates the nucleus of cells. (l) Diminished activation of AKT was observed in WT and Cln3Δex7-8 brain homogenates from trehalose-treated mice. Scale bars, 10 μm (a,e,f,k). Data represent means±s.e.m. *P<0.05.
Figure 8. Akt inhibition promotes TFEB nuclear…
Figure 8. Akt inhibition promotes TFEB nuclear translocation and activation of the CLEAR network.
(a) LC3 staining showing increased number of puncta in cells treated with trehalose or MK2206. (b) Immunoblot analysis of LC3 lipidation. (c) Micrographs of HeLa cells showing increased number of autophagic vesicles (yellow arrows) in samples treated with trehalose or MK2206. (d) Expression analysis of lysosomal and autophagy genes in HeLa cells treated with MK2206. Gene expression was normalized relative to the housekeeping gene, GAPDH. The dashed line indicates relative gene expression in untreated cells. (e–g) Intraperitoneal injection of MK2206 in Cln3Δex7-8 mice shows inactivation of Akt (e), nuclear translocation of TFEB (f) and upregulation of lysosomal and autophagy genes (g). Scale bar, 10 μm (a), 50 nm (c) and 20 μm (f).
Figure 9. Pharmacological inhibition of Akt enhances…
Figure 9. Pharmacological inhibition of Akt enhances cellular clearance in patient-derived cells.
(a–d) Confocal microscopy analysis of primary fibroblasts with defective CLN3 (c.461-677del; a), PPT1 (c.665 T>C, p.L222P; b), TPP1 (c.380G>A, p.R127Q; g.3556, IVS5-1G>C; c) or MFSD8 (c.103C>T, p.R35X; d) shows that MK2206 and trehalose induce clearance of ceroid lipopigment deposits (green). Defective proteins are indicated. More than 60 cells have been analysed for each panel. Scale bar, 30 μm. (e) Schematic diagram for Akt-dependent trehalose activation of TFEB. Data represent means±s.e.m. *P<0.05, **P<0.01, ***P<0.001.

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