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. 2022 Jun 1;19(2):544–550. doi: 10.1080/15548627.2022.2083857

A prion-like domain of TFEB mediates the co-aggregation of TFEB and mHTT

Junsheng Yang 1,✉, Huilin Xu 1, Chaoyue Zhang 1, Xiaotong Yang 1, Weijie Cai 1, Xiaoli Chen 1
PMCID: PMC9851196  PMID: 35635192

ABSTRACT

The aggregation of mutant HTT (huntingtin; mHTT) is a hallmark of Huntington disease (HD). mHTT aggregates interact and sequester dozens of proteins and affect diverse key cellular functions. Here we report that TFEB (transcription factor EB), a master regulator of lysosome biogenesis and autophagy, is yet another protein that co-aggregates with mHTT. We also found the mHTT-TFEB co-aggregation is mediated by a prion-like domain (PrLD) near the N terminus of TFEB. Our findings point out a possible limitation for therapeutic strategies targeting TFEB to clear mHTT, and also provided a possible explanation for controversies that TFEB overexpression lowered soluble mHTT in some HD models but failed to reduce mHTT aggregates or HD pathology in others. Moreover, we found that TFE3, another MiT family transcription factor that shares overlapping functions with TFEB, lacks PrLD and does not co-aggregate with mHTT, and thus might serve as an alternative drug target for HD.

KEYWORDS: Aggregate, HD, mHTT, prion-like domain, TFEB

Introduction

Huntington disease (HD) is an inheritable neurodegenerative disease caused by expanded CAG trinucleotide repeats in the HTT gene and the production of corresponding mutant HTT (huntingtin; mHTT) with an expanded poly-glutamine (polyQ) stretch [1]. mHTT is prone to form intracellular aggregates, which further develop to inclusion bodies that can sequester and affect normal functions of hundreds of other proteins [2]. It is widely thought that the accumulation of mHTT is the ultimate cause of HD, therefore a main direction in HD research is to find strategies to reduce mHTT levels, either by reducing mHTT production or by increasing mHTT clearance [3].

The autophagy-lysosomal pathway (ALP) is the major pathway used by the cells to degrade protein aggregates, thus has been one of the most extensively studied pathways in clearing mHTT. The normal functions of ALP take multiple steps and require the cooperation of autophagosomes and lysosomes coordinated by TFEB (transcription factor EB). TFEB is a member of the MiT family of transcription factors, and also the first protein in the family found to bind the coordinated lysosomal expression and regulation (CLEAR) element and regulate a corresponding network of genes (the CLEAR network) that are involved in diverse lysosomal functions including autophagy, lysosome biogenesis, lysosomal exocytosis and plasma membrane repair [4,5]. TFEB’s crucial role as a master regulator of ALP has made it a potential therapeutic target for diseases manifesting protein misfolding and aggregation. In line with this notion, accumulating evidence indicate activation of TFEB can provide beneficial effects in models of Alzheimer disease (AD) and Parkinson disease (PD) [6]. Studies from different groups also reported that TFEB overexpression can lower soluble mHTT in both cellular and mice models of HD, as well as lower mHTT aggregation in cell models of HD [4,7,8]. However, a recent study reported that the overexpression of TFEB did not decrease mHTT aggregation in a mice model [9]. It is thus crucial to elucidate the effects and limitations of TFEB activation in clearing mHTT.

Results

When we used cell models to test whether TFEB overexpression could reduce mHTT aggregation, to our surprise, we found that TFEB, when overexpressed, formed aggregates that are co-localized with mHTT (EGFP-HTTex1-74Q) aggregates (Figure 1A). The aggregation of TFEB was not observed when it was co-expressed with wild-type (WT) HTT (EGFP-HTTex1-23Q) (Figure 1A). Considering sequestration of protein into mHTT aggregates is dependent on protein expression levels [2], we checked the distribution of endogenous TFEB as well and found it also co-aggregated with mHTT (Figure 1B). This mHTT-induced aggregation of TFEB was also observed in a more relevant cell line, the mouse neuroblastoma neuro-2a (N2a) cells (Figure 1C). We also checked the TFEB aggregation with a previously reported biochemical approach that can separate soluble and insoluble mHTT [10]. After being overexpressed for 72 h, about 30% of total mHTT-EGFP was found in the insoluble pellet, while EGFP or WT HTT-EGFP control was mostly in the soluble fraction (Figure 1D, 1E). Similarly, most endogenous TFEB was soluble in the control cells that overexpressed EGFP or WT HTT-EGFP, but about 20% of endogenous TFEB was found in the pellet in cells that overexpressed mHTT-EGFP (Figure 1D, 1E).

Figure 1.

Figure 1.

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TFEB co-aggregates with mHTT. (A) mCherry-TFEB formed aggregates colocalizing with mHTT-EGFP aggregates (upper row) when co-expressed in HeLa cells (96 h after transient transfection); but did not form any aggregate when co-expressed with WT HTT-EGFP (lower row). Scale bar: 10 μm; (B) Endogenous TFEB formed aggregates colocalizing with mHTT-EGFP aggregates (upper row) (72 h after transient transfection of mHTT-EGFP); but did not form any aggregate when WT HTT-EGFP was expressed (lower row). Scale bar: 10 μm; (C) mCherry-TFEB formed aggregates co-localizing with mHTT-EGFP aggregates (upper row) when co-expressed in N2a cells (72 h after transient transfection); but did not form any aggregate when co-expressed with WT HTT-EGFP (lower row). Scale bar: 10 μm; (D) mHTT-EGFP induced TFEB aggregation. In N2a cells overexpressed EGFP or WT HTT for 72 h, both EGFP, WT HTT and the endogenous TFEB were mostly in the soluble fraction (S). In N2a cells overexpressed mHTT-EGFP for 72 h, about 30% of the mHTT-EGFP was found in the insoluble pellet (P). Similarly, about 20% of the endogenous TFEB was also found in the P fraction. This experiment has been repeated for three times and quantified. The relative proportion of S and P fractions (means ± SEMs) of the indicated proteins were shown as stacked bars in (E). (F) mHTT-EGFP interfered with TFEB activation. TFEB activity detected by 4xCLEAR dual luciferase assay on N2a cells transfected with mCherry-TFEB plus EGFP (TFEB + EGFP), WT HTT-EGFP (TFEB + WT HTT-EGFP) or mHTT-EGFP (TFEB + mHTT-EGFP) was normalized to non-transfected control cells (6 independent experiments). Statistical comparisons were made using ANOVA analysis. **P < 0.01; ***P < 0.001.

To test whether the aggregation of TFEB affects its function, we applied a 4x-CLEAR luciferase assay to monitor TFEB’s transcription activity as previously reported [11]. Since the CLEAR element is not solely controlled by TFEB [12,13], it is difficult to monitor endogenous TFEB activity accurately. We therefore co-transfected N2a cells with TFEB-mCherry and EGFP control, TFEB-mCherry and WT HTT-EGFP control, or TFEB-mCherry and mHTT-EGFP, to check whether TFEB activity can be elevated by its overexpression and whether this elevation will be affected by mHTT. We found that in N2a cells co-overexpress TFEB-mCherry and EGFP or TFEB-mCherry and WT HTT-EGFP, TFEB activity increased for about 2-fold compared with non-transfected cells 72 h after transfection. In contrast, in N2a cells that co-overexpressed TFEB and mHTT-EGFP, TFEB activity was at a similar level as non-transfected control (Figure 1F), suggesting mHTT-EGFP interfered with TFEB activation. Although we know mHTT-EGFP and mCherry-TFEB do co-aggregate 72 h after transfection, however, we still could not rule out the possible interfering effect of soluble mHTT on TFEB. We did look at TFEB activity at an earlier time point (24 h after transfection) but did not observe a significant TFEB activity increase, thus was not able to get a hint whether soluble mHTT interfere with TFEB activity.

To explore the mechanisms underlying TFEB co-aggregation with mHTT, we checked the amino acid sequence of TFEB for prion-like domains, another known factor that can increase the possibility of a protein to be sequestered to mHTT aggregates [2]. When analyzed with PLAAC, a web application that scans proteins for prion-like amino acid composition [14], we found that TFEB contains a Q-rich prion-like domain (PrLD) near the N terminus (Figure 2A). In contrast, neither TFE3 nor MITF, another two transcription factors of the MiT family, contains any PrLD (Figure 2A). In line with this, endogenous TFE3 and MITF were not found in mHTT aggregates in either HeLa cells (Figure 2B-2E) or N2a cells (Figure 2F-2I). To further characterize whether the PrLD in TFEB is required for TFEB aggregation and its inclusion to mHTT aggregates, we first compared the mHTT-co-aggregation of two previously reported TFEB constructs, CA-TFEB and TFEB-AA [15]. CA-TFEB lacks the N-terminal 135 amino acids but remained the DNA binding and trans-activating domain of TFEB. CA-TFEB manifests nuclear localization and is a constitutively active form of TFEB. TFEB-AA, on the other hand, is an S142A S211A double mutant of TFEB and also a nuclear-localized constitutively active form of TFEB. When expressed in a TFEB knockout HeLa cell background, we found both constructs localized in the nucleus as predicted (Figure 3B, 3C, indicated by DAPI staining). Interestingly, TFEB-AA could still co-aggregate with mHTT (Figure 3B), but CA-TFEB, on the contrary, could not (Figure 3C). Because these two constructs are similar in subcellular localization and activity, their different behavior in their co-aggregation with mHTT is therefore most likely due to the N-terminal 135 amino acids of TFEB.

Figure 2.

Figure 2.

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TFEB contains a PrLD. (A) TFEB, but not TFE3 or MITF, contains a PrLD (in red) predicted by PLAAC; Vit and Map refers to the maximum length of consecutive PrD state analyzed by Viterbi or MAP parses; (B)(C)(D) Endogenous TFEB, but not TFE3 or MITF, co-aggregated with mHTT-EGFP in HeLa cells (72 h after transient transfection of mHTT-EGFP), scale bar: 10 μm; (E) Quantification of (B)(C)(D) from three independent experiments (means ± SEMs, at least 20 cells with aggregates were counted for each experiment), indicating the percentages of mHTT aggregates that are co-localized with TFEB, TFE3 or MITF aggregates. (F)(G)(H) Endogenous TFEB, but not TFE3 or MITF, co-aggregated with mHTT-EGFP in N2a cells (72 h after transient transfection of mHTT-EGFP), scale bar: 10 μm; (I) Quantification of (F)(G)(H) from three independent experiments (means ± SEMs, at least 20 cells with aggregates were counted for each experiment), indicating percentage of mHTT aggregates co-localizing with TFEB, TFE3 or MITF aggregates.

Figure 3.

Figure 3.

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The PrLD in TFEB is necessary and sufficient for TFEB-mHTT co-aggregation. (A) Schematics illustrating TFEB gene structure and TFEB constructs used in this study. (B)(C) TFEB-AA, but not CA-TFEB, co-aggregated with mHTT-EGFP when co-expressed (96 h after transient transfection) in TFEB KO HeLa cells. Scale bar: 5 μm; (D) Quantification of (B)(C), from three independent experiments (means ± SEMs, at least 20 cells with aggregates were counted for each experiment), indicating percentage of mHTT aggregates co-localizing with TFEB-AA or CA-TFEB aggregates; (E) Both mCherry-TFEB and mCherry-TFEB[PrLD] co-aggregated with mHTT-EGFP; but neither mCherry nor mCherry-TFEB[∆PrLD] formed any co-aggregates with mHTT-EGFP (72 h after transient transfection) in N2a cells. Scale bar: 10 μm; (F) Quantification of (E), from three independent experiments (means ± SEMs, at least 20 cells with aggregates were counted for each experiment), indicating percentage of mHTT aggregates colocalizing with mCherry, mCherry-TFEB, mCherry-TFEB[PrLD] and mCherry-TFEB[∆PrLD] aggregates.

To further test whether the PrLD in TFEB is necessary and sufficient to mediate TFEB-mHTT co-aggregation, we generated mCherry-TFEB[∆PrLD] that only lacks the PrLD and mCherry-TFEB[PrLD] that only contains the PrLD (Figure 3A). When expressed in N2a cells, both mCherry-TFEB and mCherry-TFEB[PrLD] co-aggregated with mHTT, while neither mCherry nor mCherry-TFEB[∆PrLD] formed any co-aggregates with mHTT (Figure 3E, 3F).

Take together, the above results indicated that TFEB can form intracellular co-aggregates with mHTT. The PrLD of TFEB is necessary and sufficient to mediate TFEB-mHTT co-aggregation.

To check the pathological relevance of our finding, we tried to observe the in vivo localization of TFEB in HdhQ140 mice, a well-established knock-in HD mice model [16]. In heterozygous male mice (HdhQ140/Q7), HTT aggregates were detectable in aged mice (12 months old, Figure 4B, 4C) but not in a young mouse (1.5 month old, Figure 4A). This is consistent with previously reported results [17]. Most interestingly, among the mHTT aggregates, we did observe co-aggregation of endogenous TFEB (Figure 4B), but not TFE3 (Figure 4C).

Figure 4.

Figure 4.

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TFEB co-aggregates with mHTT in vivo. (A) Young HdhQ140/Q7 mouse did not manifest detectable HTT aggregates. Immunofluorescence images of a striatum slice from a 1.5 months old HdhQ140/Q7 mouse. HTT (green), TFEB (red), DAPI (blue); (B) TFEB co-aggregated with HTT in an aged HdhQ140/Q7 mouse. Immunofluorescent images of a striatum slice from a 12 months old HdhQ140/Q7 mouse. Arrows indicates examples of co-aggregates. HTT (green), TFEB (red), DAPI (blue); (C) TFE3 did not co-aggregate with HTT in an aged HdhQ140/Q7 mouse. Immunofluorescent images of a striatum slice from a 12 months old HdhQ140/Q7 mouse. HTT aggregates can still be observed, but no co-aggregation with TFE3 was found. HTT (green), TFEB (red), DAPI (blue). All images are of the same scale. Scale bar: 20 μm.

Discussion

Previous studies where TFEB overexpression can lower soluble mHTT level both in cellular and animal models of HD are encouraging for aiming TFEB as a potential pharmaceutical target for HD. However, the recent work by Brattas et al. indicating TFEB overexpression failed to reduce either mHTT aggregation or HD pathology in an HD mice [9] showed that a thorough understanding of the outcomes/effects of TFEB activation in HD models is still needed. We here report that TFEB can be included to mHTT aggregates both in cellular model of HD and also in vivo in HD mice. More interestingly, we found TFEB contains a Q-rich PrLD that is crucial for the co-aggregation of TFEB and mHTT. A recent study reported that nuclear TFEB form liquid-like condensates via liquid-liquid phase separation (LLPS) upon starvation or mTOR inhibition [18]. PrLD is known to be a key factor for proteins undergoing phase transition. It will be interesting to test whether the PrLD of TFEB is also required for LLPS for TFEB. More importantly, it is worth exploring whether the phase transition of TFEB and mHTT (reviewed in [19]) plays a role in their co-aggregation.

Therapeutic-wise, given the central role played by TFEB in ALP coordination, our findings provided a new possible mechanism how mHTT aggregation may compromise cellular homeostasis, especially protein quality control, which might in turn aggravate mHTT accumulation that eventually lead to disease progression. Taken together with TFEB’s effect on lowering soluble mHTT, TFEB activation may be more valuable for treating early-stage HD patients but might have limited effect on late-stage patients with severe mHTT aggregates. TFE3, on the other hand, might serve as an alternative target in treating HD, considering its overlapping functions with TFEB in ALP regulation [12,13].

Materials and methods

Plasmids and cell lines

EGFP-HTTex1-74Q, EGFP-HTTex1-23Q plasmids are kindly provided by Dr. David Rubinsztein from University of Cambridge. mCherry-TFEB plasmid and TFEB-EGFP stable HeLa cell line are kind gifts from Dr. Dan Li in Zhejiang University of Technology. CA-TFEB (79,013), TFEB-AA (79,014) plasmids were obtained from Addgene, deposited by Dr. Reuben Shaw’s laboratory.

DNA subcloning

mCherry-TFEB[∆PrLD] and mCherry-TFEB[PrLD] constructs were generated via recombination cloning (ClonExpress II One Step Cloning kit; Vazyme, C112) using mCherry-TFEB as the template, primers used are listed in Table S1.

Cell culture

HeLa cells were cultured in DMEM media (Gibco, 11,995–065) with 10% fetal bovine serum (FBS; Biological industries, 04–001-1A). N2a cells cells were cultured in MEM media (Gibco, 10,370–020) with 10% fetal bovine serum (FBS), 5% Glutamax (Gibco, 35,050–061), 5% sodium pyruvate (Gibco, 11,360–70), and 5% NEAA (Gibco, 11,140–050). All media were supplemented with 5% penicillin-streptomycin liquid (Solarbio, P1400). Lipofectamine 2000 (Invitrogen, 11,668,019) and Lipofectamine 3000 (Invitrogen, L3000015) were used for all transfection.

Immunofluoresence in cultured cells

Cells were rinsed with phosphate-buffered saline (PBS; Biological industries, 04–001-1A) once and then fixed in 4% paraformaldehyde for 15 min at room temperature. Fixed cells were washed and permeabilized with 0.1% Triton X-100 (Solarbio, T8200), blocked with 2% bovine serum albumin (Sigma-Aldrich, B2064) in PBS at 4°C overnight and then incubated with anti-TFEB (Cell Signaling Technology, 4240), anti-TFE3 (Sigma, HPA023881) or anti-MITF (Abcam, ab20663) antibodies at 4°C for overnight. Cells were then washed with PBS and incubated with secondary antibodies (Abcam, ab175471 and ab150117) for 1 h at room temperature before being subjected to fluorescence imaging.

Isolation of soluble proteins and protein aggregates and western blotting

The isolation of soluble proteins and protein aggregates were carried out as previously reported [10]. Briefly, cells were lysed in “Soluble” lysis buffer (10 mM Tris, pH 7.4, 1% Triton X-100, 150 mM NaCl, 10% glycerol) in the presence of 1x protease inhibitor cocktail (Sigma-Aldrich, P8340) and 1x phosphatase inhibitor cocktail (Abcam, GR304037-28). Cell lysate was centrifuged at 15,000 g for 20 min at 4°C and the supernatant was retained as the soluble protein fraction. The pellet was washed in lysis buffer centrifuged at 15,000 g for 5 min at 4°C twice. The pellet was then resuspended in lysis buffer supplemented with 4% SDS and sonicated for 30s with a probe sonicator (Ningbo SCIETZ Biotechnology) at room temperature. Samples were boiled for 30 min and briefly centrifuged at 6000x g, 15s. Protein samples of soluble and insoluble fractions were then loaded and separated on 4–12% gradient SDS-PAGE gels (GenScript, M00654) and transferred to polyvinylidene difluoride membranes (Millipore, IPVH00010). Membranes were blocked for 1 h with 5% milk in TBST (10 mM Tris, 150 mM NaCl, pH 7.4, 0.1% Tween 20) and incubated with anti-GFP (Roche, 11,814,460,001) and anti-TFEB (Bethyl Laboratories, A303-673A) antibodies at 4°C overnight. Bound antibodies were detected with horseradish peroxidase-conjugated anti-rabbit (Abcam, ab6721) secondary antibodies and enhanced chemiluminescence reagents. Band intensities were quantified using the NIH ImageJ software.

4x-CLEAR luciferase assay

The 4x-CLEAR luciferase assay was done as previously described [11]. Briefly, N2a cells were transfected with 4x CLEAR luciferase reporter (Addgene, 66,800; deposited by Dr. Albert La Spada’s lab) and Renilla control luciferase plasmid (Promega, E2261). After 24 h, these cells were tranfected with mCherry-TFEB + EGFP, or mCherry-TFEB + mHTT-EGFP. Cells were lysed 24 h and 72 h later for luciferase activity assay. Luciferase activities were detected with a Dual-Luciferase Reporter Assay System (Promega, E1910) on FlexStation 3 multi-mode microplate reader (Molecular Devices). The activity of 4X-CLEAR luciferase was divided by that of Renilla luciferase and then normalized to non-transfected controls.

HD mouse model and immunofluorescence

All animal-related procedures were in compliance with all relevant ethical regulations and were approved by the Laboratorial Animal Center as well as the Laboratorial Animal Welfare and Ethics Committee of the Zhejiang University of Technology (Approval No. 20,200,401,032).

Mice brains were fixed with 4% PFA for 48 h at 4°C and then immersed in 30% sucrose (Shanghai Yuanye Biotechnology, S11055) for another 48 h at 4°C before being embedded with OCT (Sakura, 4583). Frozen tissues were sectioned at −20°C into 30-μm thick slices on a freezing microtome. The slices were washed with PBS and permeabilized with 0.1% Triton X-100, blocked with 1% bovine serum albumin in PBS for 1 hr at room temperature, and then incubated with anti-TFEB (Bethyl Laboratories, A303-673A), anti-HTT/huntingtin (Merck Millipore, MAB2166) antibodies at 4°C overnight. Slices were then washed with PBS and incubated with secondary antibodies for 1 hr at room temperature before being subjected to fluorescent imaging.

Fluorescence imaging and image analysis

Imaging of both live cells and fixed cells were performed on an Olympus IX73 inverted microscope equipped with a DP80 Dual-Sensor Monochrome and Color Camera. Quantification was performed with ImageJ (NIH).

For the quantification of mHTT co-aggregation, the number of all mHTT aggregates and mHTT aggregates that are colocalized with a certain protein’s aggregates were counted. The percentage of mHTT aggregates that co-aggregated with that certain protein was then calculated.

Statistical analyses

Data are presented as the means ± standard errors of the mean (SEMs). Statistical comparisons were performed using Student’s t-tests and analyses of variance (ANOVA) with Tukey’s multiple comparison test by Prism9 (GraphPad Software, LLC). P values < 0.05 were considered statistically significant.

Supplementary Material

Supplemental Material
Click here for additional data file. (16.1KB, docx)

Acknowledgements

We thank Dr. Xiuling Cao (Zhejiang A&F University) for technical discussions. We thank Dr. Boxun Lu (Fudan University) for his support on animal experiments. This work is supported by the National Natural Science Foundation of China (82071432), Natural Science Foundation of Zhejiang Province (LY20H090019) and the Young Talent grant from the Collaborative Innovation Center of Yangtze River Delta Region Green Pharmaceuticals at ZJUT.

Funding Statement

This work was supported by the National Natural Science Foundation of China [82071432]; Natural Science Foundation of Zhejiang Province [LY20H090019].

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15548627.2022.2083857

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Supplemental Material
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