TRIM16-mediated lysophagy suppresses high-glucose-accumulated neuronal Aβ

Abstract

Aβ: amyloid β; AD: Alzheimer disease; AMPK: 5' adenosine monophosphate-activated protein kinase; CTSB: cathepsin B; CTSD: cathepsin D; DM: diabetes mellitus; ESCRT: endosomal sorting complex required for transport; FBXO27: F-box protein 27; iPSC-NDs: induced pluripotent stem cell-derived neuronal differentiated cells; LAMP1: lysosomal-associated membrane protein 1; LMP: lysosomal membrane permeabilization; LRSAM1: leucine rich repeat and sterile alpha motif containing 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; p-MAPT/tau: phosphorylated microtubule associated protein tau; ROS: reactive oxygen species; STZ: streptozotocin; TFE3: transcription factor E3; TFEB: transcription factor EB; TRIM16: tripartite motif containing 16; UBE2QL1: ubiquitin conjugating enzyme E2 Q family like 1; VCP: valosin containing protein.

Keywords: Amyloid β; TFEB; TRIM16; autophagy; diabetes; hippocampal neuron.

Figure 1.

High glucose induces lysosomal dysfunction resulting in Aβ accumulation and neuronal cell death. (A-C, and E) iPSC-NDs were exposed to high glucose (HG; 25 mM) for 24 h. (D and F) Hippocampal neurons were exposed to HG for 24 h at DIV 21. (A) Immunofluorescence staining of LAMP1, MAP2, and LysoTracker were visualized. DAPI was used to stain nuclei. (B) The mean fluorescence intensities of LysoTracker were measured by flow cytometric analysis. (C and D) Double immunofluorescence staining of LAMP1 and CTSB were analyzed in iPSC-NDs and hippocampal neurons. (E and F) iPSC-NDs and hippocampal neurons were immunostained with LAMP1 and CTSD. (G and H) iPSC-NDs and hippocampal neurons at DIV 21 were exposed to HG for 48 h. Immunofluorescence staining of LAMP1 and Aβ were visualized. (I and J) iPSC-NDs were pretreated with NAC (2 mM) or curcumin C1 (1 μM) for 30 min prior to high glucose (25 mM) exposure for 72 h. (I) The percentage of apoptotic cells (ANXA5 and PI positive) were measured by flow cytometric analysis. (J) LDH from cell supernatant were measured with LDH assay kit. n = 5. Scale bars: 8 μm. All data are representative. Quantitative data are represented as mean ± SD. *P < 0.05; **P < 0.01 versus control; ^#P <0.05 versus HG.

Figure 2.

High glucose suppresses lysophagy. (A) iPSC-NDs were pretreated with NAC (2 mM) for 30 min before HG exposure for 24 h. LysoTracker was visualized by confocal microscope. (B and C) Hippocampal neurons were transfected with pEGFP-LGALS3 at DIV 4 and exposed to HG for 24 h at DIV 21. (B) Representative immunofluorescence images showing LC3 and GFP-LGALS3. (C) The cells were immunostained with GFP-LGALS3, SQSTM1, and ubiquitin. Arrow indicates GFP-LGALS3 puncta with autophagic machinery. (D) iPSC-NDs were treated with LLOMe (1 mM) for 1 h after HG exposure for 24 h and then washed away. The cells were further incubated for 3 or 12 or 24 h in the incubator. Immunofluorescence staining of LGALS3 was visualized. *P < 0.05 versus control at 0; ^$P <0.05 versus HG at 0. (E) SH-SY5Ys were transfected with tfGal3 prior to HG exposure for 24 h followed by LLOMe (1 mM) washout assays for 3 or 12 h. Bafilomycin A₁ (10 nM) was pretreated for 30 min before incubation for 12 h. mRFP-LGALS3 and GFP-LGALS3 were visualized. *P < 0.05 versus control at 3 h. (F) SH-SY5Ys were exposed to HG for 24 h prior to treatment of LLOMe (1 mM) for 1 h. The cells were then fixed and observed by transmission electron microscopy. black arrow: lysosomal membrane; white arrow: autophagosome. n = 5. Scale bars: 8 μm. All data are representative. Quantitative data are represented as mean ± SD. *P < 0.05 versus control; ^#P <0.05 versus HG.

Figure 3.

MTORC1-mediated TFEB inhibition downregulates TRIM16 under high glucose conditions. (A) iPSC-NDs were exposed to HG for 24 h. The mRNA expression levels of TRIM16, FBXO27, LRSAM1, VCP, and UBE2QL1 were investigated. (B and C) iPSC-NDs and hippocampal neurons at DIV 21 were exposed to HG for 24 h. The protein levels of TRIM16, FBXO27, LRSAM1, and VCP were detected by western blotting. (D) Representative immunohistochemistry images of the hippocampus of vehicle-or STZ-injected mice showing TRIM16, FBXO27, LRSAM1, and VCP. Scale bars: 20 μm. (E and F) iPSC-NDs were exposed to HG for 12 h. (E) LAMP1, MTOR, and DAPI were subjected to immunofluorescence analysis. (F) The protein levels of p-MTOR (Ser2448), MTOR, p-PRKAA (Thr172), AMPK, and ACTB were detected by western blotting. (G) iPSC-NDs were pretreated with rapamycin (200 nM) or BAPTA-AM (5 μM) for 30 min before HG exposure for 12 h. TFEB was immunostained. (H and I) SH-SY5Ys were exposed to HG for 12 h. DNA was immunoprecipitated with IgG, POLR/RNA polymerase, and TFEB antibody. The samples of immunoprecipitation and input were amplified with primers of GAPDH and TRIM16. Data were analyzed by conventional PCR and qPCR, respectively. (J and K) iPSC-NDs were pretreated with rapamycin (200 nM) or curcumin C1 (1 μM) for 30 min before HG treatment for 24 h. The TRIM16 mRNA expression and protein levels of TRIM16 were analyzed by qPCR and western blotting, respectively. n = 5. Scale bars: 8 μm. All data are representative. Quantitative data are represented as mean ± SD. *P < 0.05; **P < 0.01 versus control; ^#P <0.05 versus HG.

Figure 4.

Upregulation of TRIM16 recovers high-glucose-inhibited lysophagy. (A, B, and D) Hippocampal neurons were transfected with pcDNA3.1-GFP or pcDNA3.1-Trim16-GFP at DIV 4 and exposed to HG for 24 h at DIV 21. (A) Representative immunofluorescence images showing LC3 and LGALS3. (B) The cells were immunostained with ubiquitin, SQSTM1, and LGALS3. (C and E) SH-SY5Ys were transfected with pcDNA3.1-GFP or pcDNA3.1-TRIM16-GFP before HG exposure for 24 h. (C) LLOMe (1 mM) washout assay was performed for 12 h followed by immunostaining with LGALS3. (D) LysoTracker was visualized and DAPI was used to stain nuclei. (E) The mean fluorescence intensities of LysoTracker were analyzed by flow cytometric analysis. n = 5. Scale bars: 8 μm. All data are representative. Quantitative data are represented as mean ± SD. *P < 0.05 versus control; ^#P <0.05 versus HG.

Figure 5.

Upregulation of TRIM16 facilitates Aβ and p-MAPT degradation. (A) SH-SY5Ys were transfected with pcDNA3.1-GFP or pcDNA3.1-TRIM16-GFP prior to HG exposure. The protein levels of C99, p-MAPT (Thr181), p-MAPT (Thr212), t-MAPT, and ACTB were detected by western blotting. (B) Hippocampal neurons were transfected with pcDNA3.1-GFP or pcDNA3.1-Trim16-GFP at DIV 4 and exposed to HG for 48 h at DIV 21. Representative immunofluorescence images showing LysoTracker and Aβ. (C and D) SH-SY5Ys were transfected with pcDNA3.1-GFP or pcDNA3.1-TRIM16-GFP prior to HG exposure for 48 h. Cellular or secreted Aβ 1–42 were measured, respectively. (E) LDH from cell supernatant were measured after exposure to HG for 72 h. n = 5. All data are representative. Quantitative data are represented as mean ± SD. *P < 0.05 versus control; ^#P <0.05 versus HG.

Figure 6.

Pharmacological enhancement of TFEB activation inhibited Aβ and p-MAPT accumulation and cognitive impairment in diabetic mice. (A-G) Mice injected with vehicle or STZ were orally administrated vehicle or curcumin C1 (10 mg/kg) once daily for 8 weeks after diabetes induction (A) Protein levels of the hippocampal TRIM16, FBXO27, LRSAM1, VCP, and ACTB were determined by western blotting. (B) Representative immunohistochemistry images showing LGALS3 and ubiquitin. (C) The hippocampal slides were immunostained with LAMP1 and CTSB. (D) Immunofluorescence staining of LAMP1 and CTSD in the hippocampus. (E) Hippocampal C99, p-MAPT (Thr181), p-MAPT (Thr212), t-MAPT, and ACTB were subjected to western blotting. (F) Hippocampal Aβ 1–42 were measured. (G-I) The mice were subjected to open field test, NOR test, and Y-maze test, respectively. n = 5. Scale bars: 20 μm. All data are representative. Quantitative data are represented as mean ± SD. *P < 0.05 versus vehicle-injected mice; ^#P <0.05 versus STZ-injected mice.

Figure 7.

The schematic model for effects and molecular mechanism of action of high glucose on neuronal lysopahgy and subsequent accumulation of Aβ and p-MAPT. High glucose induces dysfunction of neuronal lysosomes through ROS-mediated LMP and lysophagy impairment. The expression of TRIM16 is downregulated by MTORC1-inhibited TFEB activity under high glucose conditions, but its overexpression recovered lysophagy, which in turn degrades Aβ and p-MAPT and ameliorates cognitive impairment. Conclusively, lysophagy promotion through TRIM16 targeting is a promising strategy for the modulation of DM-associated AD.