TDP-43 regulates cholesterol biosynthesis by inhibiting sterol regulatory element-binding protein 2

Abstract

Dyslipidemia is considered an essential component of the pathological process of amyotrophic lateral sclerosis (ALS), a fatal motor neuron disease. Although TAR DNA Binding Protein 43 kDa (TDP-43) links both familial and sporadic forms of ALS and cytoplasmic aggregates are a hallmark of most cases of ALS, the molecular mechanism and the in vivo relation of ALS dyslipidemia with TDP-43 have been unclear. To analyze the dyslipidemia-related gene expression by TDP-43, we performed expression microarray and RNA deep sequencing (RNA-Seq) using cell lines expressing high levels of TDP-43 and identified 434 significantly altered genes including sterol regulatory element-binding protein 2 (SREBP2), a master regulator of cholesterol homeostasis and its downstream genes. Elevated TDP-43 impaired SREBP2 transcriptional activity, leading to inhibition of cholesterol biosynthesis. The amount of cholesterol was significantly decreased in the spinal cords of TDP-43-overexpressed ALS model mice and in the cerebrospinal fluids of ALS patients. These results suggested that TDP-43 could play an essential role in cholesterol biosynthesis in relation to ALS dyslipidemia.

Figure 1

Gene expression and transcriptome profiling under TDP-43 induction. (A) A heat map showing decreased expression of genes related to the cholesterol metabolic process under TDP-43-overexpressed condition in the expression microarray in HEK293T Rex cells stably expressing doxycycline (Dox)-induced DAP-TDP-43 (Dox on (+, n = 3) vs. off (−, n = 3) condition, fold change (F.C.) < − 1.2). (B) QRT-PCR showed that mRNA levels of target genes of SREBP2 were decreased under TDP-43-overexpressed condition. *p < 0.05, **p < 0.01, t-test, error bars = s.d. For each independent experiment, n = 3. (C) Deep RNA sequencing analysis (n = 3 each) of genes related to cholesterol metabolism. Tukey, *p value < 0.05. (D) RNA-seq reads under Dox on or off condition. (E) Transcript variants of SREBP2 under Dox-on or -off condition.

Figure 2

TDP-43 inhibits SREBP2 transcriptional activity via sterol-regulatory elements. (A) Immunoblots of lysates from 293 T Rex cells treated with Dox. The expression levels of N-SREBP2 were decreased at the indicated times after Dox (1.5 μM) treatment. Three panels were cropped from original blots presented by Supplementary Fig. 2. (B) Quantification of the cleaved N-terminal form of SREBP2 relative to β-actin; *p < 0.05, **p < 0.01, t-test, error bars = s.d. n = 3. (C) HEK293T Rex cells stably expressing doxycycline (Dox)-induced DAP-TDP-43 were transfected with pSyn-SRE-Luc and pRL-SV40 and treated with indicated concentrations of Dox and lysed for dual-luciferase assay to detect sterol-regulatory element (SRE)-activity; **p < 0.01, one-way ANOVA. (D) HEK293T Rex cells were transfected with pSyn-SRE-Luc and pRL-SV40 and treated with Dox and/or 10 mM methyl-beta-cyclodextrin (MbCD) and 0.4 μM Lovastatin (Lova); two-way ANOVA. (E) Amounts of free cholesterol in 293 T Rex cells (1 × 10⁶) 48 h after treatment with Dox; *p < 0.05, **p < 0.01, t-test, error bars = s.d. n = 3 for each assay performed. (F) Immunocytochemistry of SREBP2 after treatment with Dox (right panel) and without Dox (left panel). Green: SREPBP2, Blue: DAPI. Scale bar: 10 μm. (G) The ratio of the area of nuclear-localized SREBP2 (N) to that of total cell-localized SREBP2 (T); **p < 0.01, t-test, error bars = s.d. n = 3 for each assay performed.

Figure 3

Validation of interaction between TDP-43 and SREBP2. (A) Post-translational modification of SREBP2. Anchor protein SCAP induces SREBP2 to translocate from endoplasmic reticulum (ER) to Golgi apparatus. SREBP2 is cleaved by S1P/S2P protease, and N-terminal SREBP2 is translocated to bind the SRE transcriptional domain of DNA for expression of target genes. (B) Immunoblots of proteins controlling SREBP processing pathway in TDP-43 abundant condition (Dox+) in comparison with cholesterol depleted condition. S: cholesterol-sufficient condition, D: cholesterol-depleted condition. The panels were cropped from original blots presented in Supplementary Fig. 3. (C) Quantification of the protein levels of cholesterol-related factors relative to β-actin between Dox− and Dox+ condition; *p < 0.05, t-test, error bars = s.d. n = 3. (D) HEK293T cells were transfected with or without pcDNA3.1-TDP-43 and pFLAG-NSREBP2 (1–481) and the lysates were immunoprecipitated with FLAG antibody for the following immunoblots using TDP-43 antibody.

Figure 4

Decreased cholesterol level in spinal cord tissue of ALS model mouse. (A, B) The amounts of total cholesterol, free cholesterol, triglyceride and phospholipid per g of total spinal cord tissue were measured at 12 weeks (presymptpmatic stage, A) and beyond 12 weeks (symptomatic stage, B) after birth in A315T TDP-43 transgenic (Tg)/non-transgenic (non-Tg) male/female mice (n = 10, each group at presymptomatic stage, n = 5 male and n = 10 female at symptomatic stage); *p < 0.05, **p < 0.01, t-test, error bars = s.d. (C) The immunoblots of lysates of the spinal cord from non-Tg and Tg female mice. Three panels were cropped from original blots presented in Supplementary Fig. 4. (D) Quantification of the cleaved N-terminal form of SREBP2 relative to β-actin; **p < 0.01, t-test, error bars = s.d. n = 3.

Figure 5

Decreased cholesterol level in the spinal fluids of ALS patients. The levels of free cholesterol, protein, cells and glucose in spinal fluids between control (N = 20) and ALS patients (N = 20); *p < 0.05, **p < 0.01, t-test, error bars = s.d.