SMCR8 negatively regulates AKT and MTORC1 signaling to modulate lysosome biogenesis and tissue homeostasis

Abstract

The intronic hexanucleotide expansion in the C9orf72 gene is one of the leading causes of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS), two devastating neurodegenerative diseases. C9orf72 forms a heterodimer with SMCR8 (Smith-Magenis syndrome chromosome region, candidate 8) protein. However, the physiological function of SMCR8 remains to be characterized. Here we report that ablation of SMCR8 in mice results in splenomegaly with autoimmune phenotypes similar to that of C9orf72 deficiency. Furthermore, SMCR8 loss leads to a drastic decrease of C9orf72 protein levels. Many proteins involved in the macroautophagy-lysosome pathways are downregulated upon SMCR8 loss due to elevated activation of MTORC1 and AKT, which also leads to increased spine density in the Smcr8 knockout neurons. In summary, our studies demonstrate a key role of SMCR8 in regulating MTORC1 and AKT signaling and tissue homeostasis. Abbreviations: ALS: amyotrophic lateral sclerosis; C9orf72: chromosome 9 open reading frame 72; FTLD: frontotemporal lobar degeneration; GEF: guanosine nucleotide exchange factor; GTPase: guanosine tri-phosphatase; KO: knockout; MTOR: mechanistic target of rapamycin kinase; SMCR8: Smith-Magenis chromosome region, candidate 8; WDR41: WD repeat domain 41; WT: wild type.

Keywords: AKT-MTORC1; ALS/FTLD; C9orf72-SMCR8-WDR41; autophagy; inflammation; lysosome.

Figure 1.

SMCR8 deficiency in mice results in splenomegaly and autoimmunity. (a) Schematic drawing of the mouse Smcr8 gene exon 1 and 2, and the site targeted for editing by CRISPR-Cas9. Sequencing traces of the edited Smcr8 gene from genomic PCR show 128-bp deletion (highlighted with yellow) near the Cas9 cleavage site. (b) Representative images of spleens and quantification of spleen weight from 4 month-old WT and smcr8^−/- mice. n = 3, **: p < 0.01, student’s t-test. (c) H&E staining of spleen tissues from 12 month-old WT or smcr8^−/- mouse with higher magnification images of white pulp (WP) and red pulp (RP). WP, arrowheads indicate expanded germinal centers in the KO spleen; RP, arrows indicate megakaryocytes and arrowheads indicate erythroid precursors. Scale bar: 500 µm (100 µm and 50 µm in the zoomed in images for WP and RP, respectively). (d) H&E staining of kidney tissue from 12-month-old mice. Lymphocytes and macrophage infiltrates (arrowheads) are detected in the interstitium around the pelvis and multifocally in the cortex and medulla in the smcr8^−/- (KO) kidney. A dilated tubule (arrow) and other tubules with slightly basophilic cytoplasm are also observed in the smcr8^−/- kidney. Scale bar: 100 µm (50 µm in the zoomed in images for KO kidney) (e) H&E staining of liver from 12-month-old mice showing infiltrates of lymphocytes and macrophages (arrowheads) in the smcr8^−/- liver. Arrows indicate hypereosinophilic hepatocytes that might be undergoing degeneration and necrosis. Scale bar: 20 µm. (f) Immunostaining of 12-month-old liver sections of WT and smcr8^−/- mice with anti-IBA1 and GRN (granulin) antibodies. Scale bar: 10 µm. (g) ELISA to measure anti-dsDNA antibodies (Abs) in serum obtained from 4-months-old WT and smcr8^−/- (KO) mice. n = 3–7, **: p < 0.01, student’s t-test.

Figure 2.

C9orf72 and SMCR8 require each other for stability, and the C9orf72-SMCR8 heterodimer is stabilized by WDR41. (a) Western blot analysis of SMCR8 and C9orf72 proteins in WT and smcr8^−/- lysates as indicated. (b) Quantification of C9orf72 protein levels for the experiment in (a). C9orf72 levels were quantified and normalized to GAPDH (n = 3, *, p < 0.05, student’s t-test). (c) qPCR analysis of c9orf72 mRNA levels in WT and smcr8^−/- brain and MEF samples. (d) Western blot analysis of C9orf72 and SMCR8 protein in WT and c9orf72^−/- brain and MEF cell lysates. (e) SMCR8 levels were quantified and normalized to GAPDH for experiment in (D) (n = 3, *, p < 0.05, student’s t-test). (f) Western blot analysis of C9orf72 and SMCR8 proteins in WT and wdr41^−/- brain and MEF cell lysates. (g) C9orf72 and SMCR8 levels were quantified and normalized to GAPDH for the experiment in (f) (n = 3, *, p < 0.05, student’s t-test).

Figure 3.

Reduced levels of TFEB and autophagy-lysosome proteins in smcr8^−/- fibroblasts. (a, b) Western blot analysis of TFEB, SQSTM1, LC3, LAMP1 and GAPDH proteins in WT and smcr8^−/- MEF lysates. Proteins levels were quantified and normalized to GAPDH (n = 3, *, p < 0.05, student’s t-test). (c,d) Western blot analysis of ULK1 and RB1CC1 proteins in WT and smcr8^−/- MEF lysates. Proteins levels were quantified and normalized to GAPDH (n = 3, *, p < 0.05, student’s t-test).

Figure 4.

Analysis of autophagy flux and mRNA levels of autophagy-lysosome genes in smcr8^−/- fibroblasts. (a-c) Western blot analysis of SQSTM1, LC3, and GAPDH proteins in WT and smcr8^−/- MEF lysates under the indicated conditions. Cells were in 10% FBS or serum starved for 6 h with or without chloroquine treatment (CQ). Proteins levels were quantified and normalized to GAPDH (n = 3, *, p < 0.05, student’s t-test). (d) Autophagy flux was calculated by dividing the LC3-II:GAPDH in the presence of chloroquine by LC3-II:ACTB at baseline. (e) qPCR analysis of Tfeb, Lamp1, Sqstm1, Lc3b, Ulk1 and Rb1cc1 mRNA levels in WT and smcr8^−/- fibroblasts. The mRNA levels are normalized to Actb (n = 3, *, p < 0.05, student’s t-test).

Figure 5.

Reduced levels of TFEB and autophagy-lysosome proteins in smcr8^−/- neurons and brain lysates. (a,b) Western blot analysis of TFEB, SQSTM1, LC3, LAMP1 and GAPDH proteins in day in vitro (DIV)21 cortical neurons cultured from WT and smcr8^−/- pups. Proteins levels were quantified and normalized to GAPDH (n = 3, *, p < 0.05, student’s t-test). Ctx, cortical neurons. (c,d) Western blot analysis of TFEB, SQSTM1, LC3, LAMP1 and GAPDH proteins in brain lysates from 4-month-old WT and smcr8^−/- mice. Proteins levels were quantified and normalized to GAPDH (n = 3; *, p < 0.05; ns, not significant; student’s t-test).

Figure 6.

TFEB nuclear translocation is impaired in smcr8^−/- MEFs and neurons. (a, b) WT and smcr8^−/- MEFs were either grown in serum-containing medium (+serum), or starved for 4 h in DMEM (-serum) or 2 h in RPMI medium without amino acids (-a.a). Cells were fixed and stained with rabbit anti-TFEB antibodies and Hoechst. Scale bar: 10 µm. Nuclear and cytoplasmic TFEB signals were analyzed using Image J software  (n = 3; *, p < 0.05; ns, not significant; student’s t-test). At least 50 cells were analyzed in each treatment. (c, d) WT and smcr8^−/- DIV7 cortical neurons were fixed and stained with rabbit anti-TFEB antibodies, mouse anti-MAP2 antibodies and Hoechst. Scale bar: 10 µm. Nuclear and cytoplasmic TFEB signals were analyzed using ImageJ software (n = 3; *, p < 0.05; student’s t-test).

Figure 7.

Increased MTORC1 activities in smcr8^−/- cells. (a,b) Western blot analysis of p-RPS6KB/S6K, RPS6KB/S6K and GAPDH in WT and smcr8^−/- fibroblasts lysates (a) or DIV21 cortical neuron lysates (b). p-RPS6KB/S6K and RPS6KB/S6K proteins levels were quantified and normalized to GAPDH (n = 3, *, p < 0.05, student’s t-test). Ctx, cortical neurons. (c,d) Immunostaining of MTORC1 and LAMP1 and quantification of MTORC1 and LAMP1 colocalization (Manders’ Coefficient) using ImageJ in WT and smcr8^−/- MEFs with or without amino acid starvation (n = 3; *, p < 0.05; ns, not significant; student’s t-test). (e,f) Immunostaining of MTORC1 and LAMP1 and quantification of MTORC1 and LAMP1 colocalization using ImageJ in DIV21 WT and smcr8^−/- cortical neurons (n = 3; *, p < 0.05; student’s t-test). Scale bar: 10 µm.

Figure 8.

Increased AKT signaling due to SMCR8 deficiency. (a-d) Western blot analysis of p-MTOR, MTOR and GAPDH in WT and smcr8^−/- MEF lysates (a,b) and DIV21 cortical neurons (c,d). p-MTOR and MTOR proteins levels were quantified and normalized to GAPDH (n = 3; *, p < 0.05; ns, not significant; student’s t-test). (e-j) Western blot analysis of p-AKT T308, p-AKT S475, AKT and GAPDH in WT and smcr8^−/- MEF lysates (e,f), DIV21 cortical neurons (g,h) and adult brain lysates (i,j). p-AKT and AKT proteins levels were quantified and normalized to GAPDH (n = 3; *, p < 0.05; ns, not significant; student’s t-test).

Figure 9.

PtdIns3K inhibition rescues increased AKT activities in SMCR8-deficient fibroblasts. (a,b) WT and smcr8^−/- (KO) fibroblasts were infected with control lentiviruses or lentiviruses expressing GFP-SMCR8. The levels of AKT and p-AKT (308) were analyzed and normalized to GAPDH (n = 3; *, p < 0.05; ns, not significant; student’s t-test). (c,d) WT and smcr8^−/- (KO) fibroblasts were treated with DMSO or PtdIns3K inhibitor LY294002 (25 μM) for 4 h. The levels of p-MTOR, MTOR, AKT and p-AKT (308) were analyzed and normalized to GAPDH (n = 3, *, p < 0.05, ns, not significant; student’s t-test).

Figure 10.

SMCR8 deficiency results in increased spine density. (a,b) Western blot analysis of PSD95, SYN1 and GAPDH in WT and smcr8^−/- DIV21 cortical neurons (a) and brain lysates (b). DLG4/PSD95 and SYN1 (synapsin I) proteins levels were quantified and normalized to GAPDH (n = 3; *, p < 0.05; ns, not significant; student’s t-test). Ctx, cortical neurons. (c) Golgi staining of primary auditory cortexes in 6-months-old WT and smcr8^−/- mice. The number of spines along dendrites was quantified. Scale bar: 10 µm. n = 3, *, p < 0.05, student’s t-test. (d) SMCR8 negatively regulates AKT and MTORC1 signaling to modulate autophagy and lysosome activities. Increased AKT and MTORC1 activities due to SMCR8 deficiency leads to decreased autophagy activities and lysosomal biogenesis and subsequent tissue homeostasis defects. SMCR8 might also regulate lysosomal recruitment of MTOR and Tfeb transcription through other unknown mechanisms.