Distinct recognition of mutant huntingtin aggregates by autophagy receptor SQSTM1/p62 versus optineurin has differential effects on cell survival

doi:10.21203/rs.3.rs-3998870/v1

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Distinct recognition of mutant huntingtin aggregates by autophagy receptor SQSTM1/p62 versus optineurin has differential effects on cell survival

https://doi.org/10.21203/rs.3.rs-3998870/v1

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Huntington's disease (HD) is a neurodegenerative disease caused by a polyglutamine (polyQ) expansion in the N-terminus of huntingtin (HTT). Mutant HTT (mHTT) undergoes misfolding and tends to aggregate, yet these aggregates are not effectively cleared by autophagy thus contributing to neurotoxicity in HD. The polyQ length of HTT in patients with HD varies from 40 to > 90; however, the precise mechanisms of autophagy dysfunction for mHTT with varying polyQ lengths remain unclear. In this study, we developed new HTT-polyQ aggregation sensors based on bimolecular fluorescence complementation (BiFC) to monitor the real-time aggregation process of mHTT with varying polyQ lengths. Using BiFC-based aggregation sensors, we demonstrated that mHTT aggregation kinetics is faster with a longer polyQ length, suggesting a correlation between polyQ length and the onset age of HD. Interestingly, we discovered that the different aggregation kinetics of mHTT may determine the physical properties of the aggregates: mHTT-polyQ43 forms liquid-like protein condensates, whereas mHTT-polyQ103 generates tightly concentrated aggregates. Furthermore, mHTT aggregates with different physical states were selectively recognized by distinct autophagy receptors, which resulted in differential effects on cell viability. The liquid-like mHTT-polyQ43 condensates were recognized by SQSTM1/p62 but failed to proceed through autophagy thereby facilitating cytotoxicity. In contrast, mHTT-polyQ103 aggregates were selectively recognized by optineurin, which led to autophagic degradation and prolonged cell survival. Therefore, our results suggest that different therapeutic strategies should be considered for the HD patients with different polyQ lengths.

Biological sciences/Cell biology/Autophagy/Macroautophagy

Biological sciences/Neuroscience/Diseases of the nervous system/Huntington's disease

autophagy

BiFC

huntingtin

Huntington disease

LLPS

optineurin

polyQ

SQSTM1/p62

Huntington’s disease (HD) is a hereditary neurodegenerative disorder characterized by motor, cognitive, and psychiatric dysfunctions ¹. HD is caused by the expansion of CAG trinucleotide repeats in the huntingtin gene that are translated into an abnormally long polyglutamine (polyQ) tract in the N-terminus of huntingtin (HTT). HTT is a large protein of 350 kDa comprising a N17 domain, a polyQ region, a proline-rich domain, and HEAT repeat domains. HTT contains the cleavage sites for caspase, and the cleaved N-terminus of mutant HTT including polyQ sequences may increase its aggregation in HD ². Other evidence suggests that the first 17 amino acids and the extended polyQ tracts in the N-terminus are crucial for the formation of mHTT aggregates, which is the major hallmark of HD ^{3, 4}.

The length of the expanded polyQ tract in HD patients influences the onset age of symptoms ⁵. The polyQ lengths typically observed in HD patients range between 40 and 55, and their disease symptoms appear in 30s and 40s ⁶. Conversely, juvenile HD (JHD) patients with the polyQ length of > 60 exhibit the first disease symptoms under the age of 20 ⁷. Although rare, patients with polyQ repeats over 90 display infantile-onset symptoms. The correlation between the polyQ length of mHTT and age of HD onset may be explained by the tendency of polyQ sequences to aggregate. A longer polyQ length in mHTT generally leads to faster formation of larger aggregates ⁸, and can be converted into fibrillary assembly ⁹. Their accumulation also leads to the generation of neuronal inclusion, which is an important neuropathological hallmark of HD.

Cells utilize a lysosomal degradation process autophagy for the clearance of long-lived proteins, protein aggregates, and dysfunctional organelles ^{10, 11}. The autophagy process consists of the following stages: formation of phagophores and autophagosomes, fusion with lysosomes (autolysosomes), and degradation by lysosomal enzymes. During the stages of phagophore and autophagosome, isolation membranes from the endoplasmic reticulum can engulf diverse substrates such as misfolded proteins and damaged organelles ¹². These substrates can be recognized by autophagy receptors, for example SQSTM1/p62 and optineurin (Optn), which recruit the substrates to LC3-containing phagophores or autophagosomes via the LC3-interacting region (LIR) motif ^{13, 14}. The autophagosomes subsequently fuse with lysosomes where the substrates can be degraded by lysosomal enzymes.

The accumulation of mHTT aggregates is observed in HD, suggesting failure of the autophagy process, however, the dysregulated steps and factor of autophagy for mHTT with different polyQ lengths remain unclear. Several previous studies have suggested that impaired autophagy of mHTT aggregates in HD may be caused by defective substrate recognition or incomplete lysosomal degradation ^{15, 16}. Protein aggregates are recognized by autophagy receptors during substrate recognition step of autophagy ¹³; hence absence or changes in these receptors under pathological conditions may accelerate the accumulation of the aggregates ¹⁷. SQSTM1/p62 and Optn have been identified as autophagy receptors for mHTT aggregates, but it is unclear how these receptors interact with mHTT aggregates containing different lengths of polyQ tract. Furthermore, the pathological mechanisms of the defects in substrate recognition and/or subsequent steps of autophagy remain unclear.

To investigate autophagy stages for the mHTT with polyQ tract of different lengths, we developed HTT-polyQ aggregation sensors based on bimolecular fluorescence complementation (BiFC). HTT-polyQ tracts of different lengths (10, 30, 43, 61, and 103) were fused to the N- and C-terminal fragments of a yellow fluorescent protein (FP) Venus (VN and VC), which can report the aggregation of HTT-polyQ tract by yellow fluorescent signals in live cells. These HTT-polyQ sensors were employed to demonstrate the differential aggregation kinetics of the mHTT aggregates containing the polyQ tract of different lengths. Furthermore, we investigated which steps of autophagy are dysregulated for the clearance of mHTT aggregates with different polyQ lengths using the previously developed autophagy sensor RB-LC3 and various autophagy biomarkers. Surprisingly, our study revealed that the mHTT aggregates with different polyQ lengths are selectively recognized by distinct autophagy receptors, SQSTM1/p62 or optineurin, resulting in differential effects on their degradation and cell viability. These results may aid in providing a careful assessment of the type of personalized treatment required for JHD and other patients with HD.

Cell culture and transfection

The HEK293A cell line was maintained in DMEM (Hyclone, SH30243.01) supplemented with 10% fetal bovine serum (Hyclone; 11668019), 1 unit/mL penicillin, 100 µg/mL streptomycin (Corning; 30-002-Cl) and 100 µM MEM non-essential amino acid solution (Gibco; 31985-070). Cell culture reagents were purchased from Hyclone. The cells were cultured in a humidified 95% air, 5% CO2 incubator at 37°C. The striatal cell line STHdhQ7/7 (Q7) was maintained in DMEM (HyClone; SH30243.01) supplemented with 10% fetal bovine serum (Hyclone; 11668019), 1 unit/mL penicillin, and 100 µg/mL streptomycin. The cell culture reagents were purchased from HyClone. The cells were cultured in a 95% humidity, 5% CO2 incubator at 33°C. Cells were transiently transfected using LipofectamineTM 2000 reagent (Invitrogen; 11668019) according to the manufacturer’s protocol.

Antibodies and reagents

Antibodies against LC3 (ab48394), SQSTM1/p62 (ab109012), and Optn (ab151240, ab264242) were purchased from Abcam. Anti-GFP (sc-9996), anti-cMyc (sc40) and anti-GAPDH (sc-47724) antibodies were purchased from Santa Cruz Biotechnology. For immunofluorescence (IF), primary antibody was used 1:1000 in 1% BSA in PBS. Anti-PtdIns(3, 5)P₂ antibody (Z-P035) was purchased from Echelon. Goat anti-mouse antibody conjugated to Alexa Fluor 594 was used 1:1000 in 1% BSA in PBS. Rapamycin was achieved from Sigma. LysoTracker™ Red DND-99 (L7528) was purchased from Thermo Fisher Scientific. Optineurin siRNA (sc-390540) was purchased from purchased from Santa Cruz Biotechnology. In the HEK293A cells, Optn siRNA (10 nM) was delivered by Lipofectamine™ 2000 (Invitrogen) according to the manufacturer’s protocol.

Plasmid constructs

Huntingtin constructs, HTT Q2, mHTT Q62, HTT Q25-GFP, mHTT Q103-GFP and mHTT Q103-mTagBFP2 were previously described ¹⁸. BiFC-based HTT polyQ sensors were constructed from mHTT Q103-GFP construct. First, the GFP is replaced with the PCR products of N- or C-terminal fragments of Venus, VN or VC, as previously described ¹⁹. Different polyQ lengths were generated by PCR and inserted in the BiFC-based HTT polyQ sensors. The P-rich-HEAT domains of cMyc-mHTT-polyQ-P-rich-HEAT were constructed from HTT Q2. The components were amplified by PCR and fused by In-Fusion (Clontech) technique in the BiFC-based HTT polyQ sensors prepared by EcoRI/KpnI digestion. The RB-LC3 sensor composed of mApple, mTagBFP2, and LC3 in the pRK5 vector was previously generated ²⁰. The SEP-LC3 and mScarlet-LC3 sensor were generated by fusion of SEP or mScarlet with LC3 in the pRK5 vector. The plasmids containing SQSTM1/p62, Optn, and Lamp1 were achieved from Addgene. In-Fusion (Clontech; 639650) technique was used to generate the constructs. New constructs were verified by sequencing.

Co-immunoprecipitation

For co-immunoprecipitation experiment, we transfected myc-polyQ43-PRD-HEAT with mRFP1-SQSTM1/p62 or myc-polyQ103-PRD-HEAT with Optn-mTagBFP2 in HEK293A cells for 48 hours. The harvested samples were centrifuged (16,000 g, 4°C, 15 min), and the pellets were incubated at -80°C for overnight. RIPA buffers with protease inhibitor were added to the frozen pellets of the samples, the suspension was on ice for 10 min, and then centrifuged (16,000 g, 4°C for 15 min) to achieve the supernatants. Pearson protein A/G agarose (Thermo Scientific; 20421) were washed three times with PBS and RIPA buffer (3,300 g, 4°C for 1 min). The beads were incubated with 1.5 µg of anti-p62 antibody (Abcam; ab109012) or anti-Optn antibody (Abcam; ab264242) for overnight at 4°C under gentle agitation. For immunoprecipitation (IP), the supernatants were incubated on bead with antibodies for overnight at 4°C under gentle agitation. To remove the unbound proteins, samples were centrifuged (500 g, 1 min), and the supernatant was discarded. After the overnight incubation, the samples were washed three times with ice-cold RIPA buffer and the prepared IP samples were ready for Wetstern blotting experiments.

Western blotting

Protein concentration was measured using the BCA Protein Assay Kit (Thermo Fisher Scientific; 23225). The samples were subjected to SDS-PAGE and blotted with an LC3, SQSTM1/p62, Optn or GFP antibody (1% BSA in PBS; 1:1000 dilution). The equal amount of protein loading was assessed and normalized with GAPDH (1% BSA in PBS; 1:1000 dilution) on the same membrane. Western blot membranes were developed with an enhanced chemiluminescence (ECL) solution, using SuperSignal™ West Pico PLUS (Thermo Fisher Scientific; 34577) or SuperSignal™ West Femto Substrate (Thermo Fisher Scientific; 34095). Images were captured with Amersham ImageQuant 800 systems (Cytiva) and the quantification of band intensity was performed with Image Lab 5.2.1 (Bio-Rad).

Image Acquisition

Live cell imaging was performed in humidified 95% air, 5% CO2 and a 37°C temperature-controlled chamber (Live Cell Instrument). The cells expressing various constructs were prepared on cover glass-bottom dishes (SPL; 100350) coated with 10 µg/ml of fibronectin (Gibco; 33010-018). Images were collected by a Nikon Ti-E inverted microscope with a cooled charge-coupled device camera (Andor, iXon 888), and analyzed with NIS software (Nikon). Red fluorescent images were collected by a 562DF40 excitation filter, a 593DRLP dichroic mirror, and a 641DF40 emission filter with a neutral density 16 (ND16) filter for 200 ms of exposure time. Green fluorescent images were collected by a 482DF40 excitation filter, a 506DRLP dichroic mirror, and a 536DF40 emission filter with a ND16 filter for 100 ms. Blue fluorescent images were collected using a 377DF40 excitation filter, a 409DRLP dichroic mirror, and a 447DF40 emission filter with a ND8 filter for 400 ms. A 100x objective lens was used for the detailed observation of the subcellular localizations of mHTT aggregates or the autophagic vesicles. The background of each image was subtracted by NIS program.

Immunofluorescence

For the immunostaining, HEK293A cells were fixed with 4% paraformaldehyde for 5 min and permeabilized with 0.1% Triton X-100 for 10 min. The cells were maintained in 1% BSA in PBS for 1 h for blocking, and then incubated with mouse anti-PtdIns(3, 5)P₂ antibody (10 µg/ml, Echelon; Z-P035) or anti-cMyc (2.0–3.0 mg/ml, Santacruz; sc40) overnight at 4°C. The cells were washed three times with PBS and then incubated with goat anti-mouse antibody conjugated to Alexa Fluor 594 (1% BSA in PBS diluted; 1:1000) for 2 h. The stained cells were observed under a Nikon Ti-E fluorescence microscopy.

Super‑resolved structured illumination microscopy (SR‑SIM)

For SR-SIM imaging, HEK293A cells expressing autophagy receptors and mHTT aggregates were fixed with 4% paraformaldehyde for 10 min. The cells were observed under an Elyra 7 microscope (Zeiss) with a 60 × objective. The 3D SIM images were obtained every 1 µm along the z-axis and then aligned (3D) and reconstructed with the Zeiss Zen 3.0 SR (black) program. Dual iterative SIM (SIM2), a nonlinear iterative reconstruction algorithm developed by Zeiss, was used to reconstruct the images.

Fluorescence recovery after photobleaching (FRAP)

The STHdhQ7/Q7 cells expressing mRFP-p62 and mHTT aggregates were cultured on cover glass-bottom dishes. The FRAP assay was performed using a Nikon Ti-E fluorescence microscope. To initiate bleaching, a circular region of interest with a diameter of approximately 20 µm was temporally focused by adjusting the aperture of the field diaphragm and manually pushing the F slider arm on the left side of the Nikon Ti-E microscope. The bleaching of mRFP-p62 and polyQ VN + NC were carried out using a neutral density 1 (ND 1) filter for a duration of 2 min, and time-lapse images were subsequently acquired. The RFP fluorescence was bleached using a 531/40 nm beam. The Venus fluorescence was bleached using a 482/35 nm beam. The fluorescence intensity was quantified using the NIS program.

pH titration of RB-LC3

HEK293A cells expressing RB-LC3 and HTT-Q103-GFP constructs were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.00025% Triton X-100 for 5 min. The pH buffer solutions were prepared according to the Carmody buffer system ²¹. In brief, the buffer is composed of 0.05 M citrate and 0.2 M boric acid. To make the buffer solutions at pH 5.5, 6.0, 6.5, 6.75, 7.0, 7.3 and 8.0, 0.1 M tertiary sodium phosphate was added. Each fluorescence image was monitored under a Nikon Ti-E inverted microscope, and the background-subtracted fluorescence signals were analyzed by NIS program (Nikon). The sizes of HTT aggregates were measured from the HTT-Q103-GFP images by NIS program.

Protein Purification and In Vitro pH Titration of FPs

For protein expression, the E. coli strain BL21 was transformed and cultured overnight on agar plates containing LB and kanamycin. A single colony was picked and grown for 3 h in 5 mL of LB supplemented with kanamycin at 37°C, and 100 mM of isopropyl β-d-1-thiogalactopyranoside was added to the LB media. The bacteria were grown at 180 rpm at room temperature for 18 h. The protein was purified from the supernatant by the Chelating Excellose Spin Kit His-tagged Protein Purification (Takara) according to the manufacturer’s instructions. Fluorescence intensity as a function of pH was determined by dispensing 2 µL of the purified protein solution (1 µg/µL) into 50 µL of the desired pH buffer in quintuplicate into a 384-well cell culture microplate (Greiner) and measured in a fluorescence spectrometer (BioTek, Synergy H1). The pH buffer solutions from pH 3 to 11 were prepared according to the Carmody buffer system ²¹.

Computational analysis

The intensity level of each autophagic vesicle was calculated by the software program that we developed based on openCV library. The input of the software program is a sequence of pairs of the reference image (mTagBFP2) and the test image (mApple) that were taken at the same time point. In each pair of the images, the vesicles are detected by the Watershed algorithm from the reference image. To improve the accuracy of vesicle detection, the software finds a set of local maxima, where each maximum point is separated from another maximum point by a specified distance. When the mean intensity of a detected vesicle is lower than a threshold, the software discards the vesicle from consideration. The ratio intensity was computed from the test image using the coordinates of the vesicles extracted from the paired reference cell image.

Cell Viability Assay

The Quanti-MAX WST-8 cell viability assay kit (Biomax; QM1000) was used to measure cell viability. Briefly, the cells were seeded in a 96-well plate at a density of 5 × 10⁴ cells/mL in a volume of 100 µL/well. Following 24 h of incubation, 10 µL of WST (water-soluble tetrazolium salt) reagent solution was added to each well and the plates were incubated for 1 h at 37°C. The absorbance of the living cells was revealed at 450 nm using a microplate reader.

Statistical analysis

All data are shown as the mean ± standard error of the mean (s.e.m.). The significance of differences between groups was calculated using two-tailed Student’s t-test or One-way ANOVA followed by post hoc Sidak’s multiple comparisons test (GraphPad Prism 8). A significant difference was determined by p < 0.05.

Autophagy process for mutant HTT is impaired

We previously developed an autophagy sensor named RB-LC3 ²⁰, which consists of a pH-sensitive red FP (mApple), a pH-stable blue FP (mTagBFP2) and LC3 (an autophagy biomarker) (Fig. 1a). RB-LC3 can be located in the autophagic vesicles throughout the autophagy process. When autophagosomes fuse with lysosomes to become autolysosomes, the pH-sensitive red FP, but not the pH-stable blue FP, decreases its fluorescent intensity. Therefore, real-time progression of autophagy can be monitored by measuring the red/blue (R/B) intensity ratios of RB-LC3 in live cells ^{20, 22}.

To confirm the defects in autophagy progression in HD, we expressed the RB-LC3 autophagy sensor together with normal or mutant HTT (HTT-Q25-EGFP or mHTT-Q103-EGFP) in HEK293A cells (Supplementary Fig. 1a). We observed the formation of mHTT-Q103-EGFP aggregates, while HTT-Q25-EGFP was evenly distributed throughout the cell (Supplementary Fig. 1b). The R/B ratios in the autophagic vesicles of HTT-Q25-expressing cells significantly decreased (Supplementary Fig. 1c), indicating the acidification of the autophagic vesicles during autophagy progression. In contrast, the R/B ratios in vesicles colocalized with mHTT-Q103-EGFP aggregates remained unchanged (Supplementary Fig. 1c), suggesting impaired autophagy progression.

To determine the pH values in autophagic vesicles, we established a correlation equation between pH and the R/B intensity ratios of RB-LC3 by plotting the measured R/B intensity ratios under different pH conditions (Fig. 1b) ²⁰. We confirmed that the R/B ratios in autophagic vesicles were independent of their size (Supplementary Fig. 1d). Using the equation (Fig. 1b), we found that the pH in autophagic vesicles in cells expressing HTT-Q25-EGFP changed from 7.4 to 6.4 (Fig. 1c), indicating normal autophagy progression. However, the pH of autophagic vesicles in the cells expressing mHTT-Q103-EGFP remained constant as 7.5 (Fig. 1c), confirming impaired autophagy in HD. Furthermore, the levels of SQSTM1/p62 increased in mHTT-expressing cells (Fig. 1d). These results indicate that autophagy is impaired in cells expressing mHTT-Q103-EGFP.

Visualization of autophagy process for HTT-polyQ tract of different lengths

The length of polyQ tract in mHTT varies among patients with HD, and this variation is correlated with the age of onset of associated symptoms. The polyQ length in HD patients typically ranges from 40 to 55, whereas it exceeds 60 and 90 in patients with juvenile HD and infantile HD patients, respectively ^{6, 7}. It is anticipated that longer polyQ length in mHTT leads to more rapid formation of aggregates. To demonstrate the aggregation kinetics of mHTT with different lengths of polyQ tract, we designed the BiFC-based HTT-polyQ aggregation sensors by fusing the N- or C-terminal fragments of a yellow FP Venus (VN and VC) with N17 and different lengths of polyQ tract (Q10, Q30, Q43, Q61, and Q103) (Fig. 1e). When the HTT-polyQ tracts in the BiFC sensors aggregate, the attached VN and VC can be reconstituted thereby resulting in increased yellow fluorescence. Thus, the BiFC-based HTT-polyQ aggregation sensors can visualize the real-time progression of HTT-polyQ aggregation in live cells.

We first validated that the BiFC-based HTT-polyQ sensors do not fluoresce when only VN or VC-containing part is expressed (Supplementary Fig. 1e). Additionally, we tested different amounts of VN and VC constructs for transfection and determined the experimental conditions for the BiFC-based HTT-polyQ sensors. The yellow HTT-polyQ sensors and the red/blue colored RB-LC3 sensor were used to investigate the autophagy process induced by the aggregates with varying polyQ lengths. At 24 h post-transfection, we observed the formation of Q103 VN + VC aggregates, while Q61 VN + VC began to accumulate at 48 h (Fig. 1f), suggesting different aggregation kinetics of mHTT. We calculated the pH values of autophagic vesicles in these groups based on the correlation equation. In cells expressing Q10 VN + VC and Q30 VN + VC, the pH in the autophagic vesicles changed from 7.4 to 6.5 at 48 h post-transfection (Fig. 1g). This decrease in the pH levels of autophagic vesicles was not detected under the treatment of bafilomycin A1, which blocks the acidification of autophagosomes (Supplementary Fig. 1f). In contrast, the pH levels in groups of Q43, Q61, and Q103 VN + VC remained constant at 48 h (Fig. 1g), indicating the failure of autophagy for the aggregates with over 43 of polyQ length.

To check whether normal autophagy machinery for other cellular substrates is also affected, we compared the pH levels in autophagy vesicles that are colocalized with or without mHTT-polyQ VN + VC aggregates (Fig. 1h). Interestingly, when Q61 or Q103 VN + VC were expressed, autophagic vesicles without mHTT aggregates were also less acidified (Fig. 1h), indicating that mHTT with longer polyQ lengths may interfere with normal autophagy process of other cellular substrates. We confirmed the levels of SQSTM1/p62 are increased in the cells expressing mHTT with longer polyQ lengths (Fig. 1i). These results suggest that mHTT with different polyQ lengths has differential effects on the autophagy process.

Different kinetics of mHTT aggregates with varying polyQ lengths

As the onset ages of the patients with HD depend on the lengths of polyQ, the length of the polyQ tracts plays a key role in the progression of mHTT aggregation ^{23, 24}. The differential effects of mHTT-polyQ lengths on autophagy progression may be attributed to their aggregation kinetics. To investigate this, we visualized the real-time aggregation process of polyQ43, Q61, and Q103 using the BiFC-based mHTT-polyQ aggregation sensors (Fig. 2a). Q43 VN + VC aggregates began to be accumulated near the nucleus at 58 h post-transfection, and eventually entered inside the nucleus at 72 h. The nuclear localization of mHTT aggregates in the nucleus induces severe cytotoxicity ²⁵. The aggregation kinetics of Q61 VN + VC was faster than that of Q43 VN + VC, with assembly starting at 36 h, accumulation observed at 52 h, and the presence of Q61 VN + VC aggregates within the nucleus at 58 h. The aggregation kinetics of Q103 VN + VC was significantly faster than the others, with compact aggregates observed at 6 h and presence in the nucleus at 24 h. We confirmed similar expression levels between a series of polyQ VN + VC (Fig. 2b). Thus, our results demonstrated in live cells that the aggregates with longer polyQ lengths exhibited more rapid aggregation kinetics. We also generated the HTT constructs containing an N-terminal myc tag, N17, polyQn (n = 30, 43, 61, 103), PRD, and HEAT domains (Fig. 2c), and confirmed the faster aggregation kinetics of mHTT with longer polyQ lengths (Fig. 2d, e). Using the RB-LC3 autophagy sensors and the correlation equation (Fig. 1a, b), we further validated that autophagic vesicles containing Q43, Q61, and Q103 fail to undergo acidification until later time points in their aggregation kinetics (Supplementary Fig. 2). These results suggest that the progression of autophagy for mHTT with Q43, Q61, and Q103 is impaired with different kinetics.

mHTT-Q103 aggregates cannot be recognized by SQSTM1/p62 thereby fail to be recruited to autophagosomes

We next investigated which steps of autophagy process are dysregulated for the aggregates with different polyQ lengths. In the initial step of autophagy, substrates are recognized by autophagy receptors and then recruited to LC3-containing phagophores ^{26, 27}. Subsequently, the phagophore is closed to form an autophagosome that matures and finally fuses with lysosomes for substrate degradation. Hence, we first examined whether the mHTT aggregates of Q43 or Q103 VN + VC colocalized with the major autophagy receptor SQSTM1/p62 (Fig. 3a, b). Specifically, we assessed these colocalizations at 58 or 6 h, when the aggregates of Q43 or Q103 VN + VC are clearly appeared, respectively (Fig. 2a). The results showed that the aggregates containing Q43 VN + VC are generally colocalized with mTagBFP2-p62 (Fig. 3a, c-e), whereas Q103 VN + VC aggregates exhibited poor colocalization with mTagBFP2-p62 (Fig. 3b-e). The preference of SQSTM1/p62 for the Q43 aggregates comparing to Q103 aggregates was also validated in the striatal cell line STHdhQ7/Q7 (Supplementary Fig. 3a). Similar preference of endogenous SQSTM1/p62 toward Q43 aggregates was observed (Supplementary Fig. 3b). These results indicate that the Q43 aggregates, but not Q103 aggregates, are recognized by the autophagy receptor SQSTM1/p62. Thus, the Q103 VN + VC cannot be recruited to autophagosomes by the major autophagy receptor SQSTM1/p62, resulting in the failure of autophagy initiation.

We further confirmed with mHTT-Q43 or Q103 constructs (Fig. 2c) that SQSTM1/p62 can recognize mHTT-Q43 but not mHTT-Q103 aggregates (Fig. 3f, g). In particular, the co-immunoprecipitation (co-IP) experiments clearly showed the distinct recognition of mHTT aggregates depending on the polyQ length (Fig. 3h). Therefore, these results suggest that mHTT-Q103 aggregates cannot be successfully recognized by the major autophagy receptor SQSTM1/p62 thus fail to initiate the autophagy process.

mHTT-Q43 aggregates are recognized by SQSTM1/p62, but subsequent autophagy progression is hampered

mHTT-Q43 aggregates may be recognized by SQSTM1/p62 (Fig. 3), but the pH levels in the autophagic vesicles with Q43 VN + VC aggregates did not decrease (Supplementary Fig. 2) suggesting unsuccessful autophagic process for clearing Q43 VN + VC aggregates. Thus, we further examined the binding structure between mHTT-polyQ43 aggregates and SQSTM1/p62 utilizing three-dimensional super-resolved structured illumination microscopy (SR-SIM). Surprisingly, Q43 VN + VC aggregates were not enclosed by SQSTM1/p62 but they were rather mingled together (Fig. 4a). Again, SQSTM1/p62 and Q103 aggregates did not colocalize (Fig. 4b), confirming that Q103 aggregates cannot be recognized by SQSTM1/p62 ¹⁵.

As a result, the Q43 VN + VC aggregates cannot be successfully recruited to LC3-positive autophagic vesicles or lysosomes for the progression of autophagy. Our results showed that mScarlet-LC3 colocalizes with SQSTM1/p62, but not exactly with Q43 VN + VC aggregates (Fig. 4c, d). Similarly, Lamp1-mApple was not successfully recruited to the Q43 VN + VC aggregates (Fig. 4c, d). These results suggest that Q43 VN + VC aggregates are mingled together with SQSTM1/p62, which partially recruits LC3-containing phagophores via LIR motif, however, the SQSTM1/p62-Q43 complexes are failed to be enclosed in autophagosomes. Consequently, the subsequent autophagy steps for the Q43 VN + VC aggregates cannot be successfully proceeded, and therefore the levels of Q43-GFP and SQSTM/p62 were not decreased through the autophagy process (Supplementary Fig. 4a).

The mHTT-polyQ103 aggregates appeared as more compact structures, whereas the mHTT-polyQ43 aggregates were less dense and relatively larger in size (Fig. 4a, b), suggesting potential differences in their aggregation process. These findings also imply variations in the physical states and properties of these aggregates, with polyQ103 aggregates forming solid-like tight structure while polyQ43 aggregates may exist in a liquid or gel-like state. Recent studies in fact have proposed that mHTT can exist in liquid, gel, or solid-like phases ^{8, 9}. To prove the physical states of the polyQ aggregates, we applied fluorescence recovery after photobleaching (FRAP) assay ²⁸. While the fluorescent intensity of Q103 VN + VC aggregates showed negligible recovery after bleaching, we observed a significant recovery of the fluorescence of Q43 VN + VC aggregates (Fig. 4e, f), suggesting that the high mobility of the liquid-like Q43 VN + VC condensates. We conducted this FRAP assay on the Q43 or Q103 VN + VC mHTT aggregates with similar size and intensity before photobleaching (Supplementary Fig. 4b-d). Therefore, the aggregates with varying polyQ lengths may exhibit distinct physical properties, i.e. the liquid-like Q43 condensates and solid-like Q103 aggregates.

Remarkably, we also observed the enlarged and bulky SQSTM1/p62 structures (Fig. 4a). The SQSTM1/p62 contains the Phox and Bem1p (PB1) domain capable of forming oligomers and plays a crucial role in facilitating multivalent interactions between cargoes and autophagic vesicles. Due to this oligomeric property of SQSTM1/p62, it has been suggested that liquid–liquid phase separation (LLPS) may occur when the concentration of SQSTM1/p62 proteins approaches a threshold ^{29, 30}. This phenomenon can lead to the formation of liquid-like condensates known as SQSTM1/p62 bodies which are crucial for autophagic degradation ^31–34. In fact, we observed the formation of SQSTM1/p62 bodies in HEK293A and STHdhQ7/Q7 cells after the treatment with the autophagy inducer rapamycin (Supplementary Fig. 4e-h). We further confirmed the liquid-like property of the SQSTM1/p62 bodies by FRAP assay (Fig. 4g, h). The affinity of SQSTM1/p62 for the mHTT aggregates with shorter polyQ length may stem from their shared liquid-like characteristics.

Q103 aggregates are preferentially recognized by Optn, enclosed by autophagosomes and fused to lysosomes

We next investigated whether mHTT-polyQ aggregates can be recognized by another autophagy receptor Optn ^35–37 and particularly assessed whether the recognition of mHTT aggregates by Optn is also dependent on polyQ length. Interestingly, our results showed strong colocalization between Optn and Q103 VN + VC aggregates, but not Q43 VN + VC condensates (Fig. 5a, b). This preference of Optn for the longer polyQ aggregates contrasts with that of SQSTM1/p62 which efficiently recognizes the shorter polyQ (Fig. 3, 4). We also showed with mHTT-Q43 or Q103 constructs (Fig. 2c) that Optn strongly prefers to bind mHTT-Q103 (Fig. 5c, d), and the co-IP assay confirmed distinct affinity of mHTT-Q103 by Optn (Fig. 5e). Therefore, our results suggest that the two major autophagy receptors, SQSTM1/p62 and Optn, exhibit contrasting preferences and distinct association patterns with mHTT aggregates of varying polyQ lengths: SQSTM1/p62 recognizes and mingles with the liquid-like mHTT aggregates with shorter polyQ length, whereas Optn binds to solid-like mHTT aggregates with longer polyQ length.

We then explored whether Optn, which recognizes mHTT-Q103 aggregates, could subsequently recruit autophagic vesicles. First, the SR-SIM imaging revealed that Optn clearly surrounds the outer lines of the Q103 aggregates (Fig. 5f). This observation was confirmed in the striatal cell line STHdhQ7/Q7 (Supplementary Fig. 5a). The Q103 VN + VC aggregates recognized by Optn were successfully enclosed by the LC3-positive autophagosomes (Supplementary Fig. 5b), and the colocalization of LC3 and Q103 VN + VC aggregates significantly increased when Optn-mTagBFP2 was expressed (Supplementary Fig. 5c). We also confirmed that the autophagosomes containing the Q103 VN + VC aggregates can fuse with lysosomes as evidenced by colocalization with mApple-Lamp1 (Supplementary Fig. 5d). Consequently, the colocalization of Q103 VN + VC aggregates and Lamp1 significantly increased with the expression of Optn-mTagBFP2 (Supplementary Fig. 5e). The formation of autolysosomes containing Q103 VN + VC aggregates was further confirmed by staining with phosphatidylinositol 3,5-bisphosphate (PtdIns(3, 5)P₂), a major lipid component of the lysosomal membrane (Supplementary Fig. 5f). We also confirmed that mHTT-Q103 recognized by Optn is colocalized with LC3-positive autophagosomes (Fig. 5g, h) and lysosomes (Fig. 5i, j). The results suggest that Optn can recognize mHTT-Q103 aggregates and initiate the subsequent steps of autophagy.

mHTT exhibits differential preference toward SQSTM1/p62 and Optn depending on the polyQ lengths

We discovered the distinct recognition of mHTT aggregates with varying polyQ lengths by two autophagy receptors SQSTM1/p62 and Optn (Fig. 3–5). We further examined the affinities of mHTT aggregates for autophagy receptors when both SQSTM1/p62 and Optn are overexpressed. First, we confirmed that the Q43 VN + VC aggregates, which appeared to be liquid-like condensates, preferentially bound to SQSTM1/p62 (Fig. 6a). Q61 VN + VC aggregates could exist as liquid-like condensates or sphere-shaped aggregates in the cells expressing both SQSTM1/p62 and Optn (Supplementary Fig. 6a). The preference of Q61 VN + VC aggregates for SQSTM1/p62 or Optn was dependent on their physical states: the liquid-like Q61 aggregates did not show preferred colocalization (Fig. 6b, upper panels), while the sphere-shaped compact Q61 VN + VC aggregates were strongly colocalized with Optn (Fig. 6b, lower panels). Finally, Q103 VN + VC aggregates were dominantly colocalized with Optn (Fig. 6c). This distinct recognition of polyQ aggregates by SQSTM1/p62 or Optn was also confirmed in the striatal cell line STHdhQ7/Q7 (Supplementary Fig. 6b-d). Therefore, liquid-like condensates (Q43) and sphere-shaped tight aggregates (Q103) exhibit different affinities to the autophagy receptors SQSTM1/p62 and Optn (Fig. 6d).

Optn overexpression can reduce the accumulation and toxicity of mHTT aggregates

We discovered that Optn recognizes the mHTT-Q103 aggregates, which are subsequently enclosed by autophagosomes and fused with lysosomes (Fig. 5 and Supplementary Fig. 5). We further investigated whether the Optn-recognized mHTT-Q103 aggregates can be degraded in autolysosomes. To monitor the pH changes inside the autophagic vesicles containing Q103-BFP2 aggregates, we used pH-sensitive SEP-tagged LC3 (Fig. 7a, upper panel), which exhibits sharply decreased fluorescence at lower pH (Supplementary Fig. 7a) ²⁰. At 12 h post-transfection, we observed that the Q103 VN + VC aggregates are clearly enclosed by SEP-tagged LC3, suggesting the formation of autophagosome (Fig. 7a, lower panels). The ring structure of SEP-LC3 in the outer lines of the Q103-Optn complex was weakened at 48 h and disappeared at 96 h, indicating that the autophagosomes fused to lysosomes to form autolysosomes at these time points (Fig. 7a, lower panels). We also observed the colocalization of LysoTracker with these autophagosomes starting from 24 h (Fig. 7b). Finally, the levels of Optn and Q103-GFP noticeably decreased at 72 and 96 h (Fig. 7c and Supplementary Fig. 7b, c). The level of Optn was constant until 96 h without the expression of Q103-GFP (Supplementary Fig. 7d) or with the expression of Q43-GFP (Supplementary Fig. 7e). Therefore, the autophagy receptor Optn preferentially recognizes polyQ103 aggregates, leading to their autophagic clearance.

Next, we investigated the effects of Optn and SQSTM1/p62 on cytotoxicity induced by mHTT aggregates with Q30, Q43 and Q103 (Fig. 7d-f). We observed no effect of SQSTM1/p62 or Optn on the cytotoxicity in the cells expressing normal HTT-polyQ30 (Fig. 7d). The cytotoxicity caused by mHTT-Q43 aggregates was slightly enhanced by SQSTM1/p62 overexpression (Fig. 7e). The overexpression of SQSTM1/p62 itself did not induce severe cytotoxicity (Supplementary Fig. 7f). We observed significant protective effect of Optn on the cells expressing mHTT-Q103 aggregates (Fig. 7f). We also confirmed increased cytotoxicity on the cells expressing mHTT-Q103 aggregates when Optn is knockdown by siRNA (Supplementary Fig. 7g, h).

Therefore, we have discovered that mHTT aggregates are preferentially recognized by either SQSTM1/p62 or Optn, depending on their polyQ lengths, and this distinct recognition by the two autophagy receptors has differential effects on the degradation of mHTT aggregates and subsequently on cell viability.

HD is caused by the accumulation of mHTT aggregates containing abnormally long polyQ tract in the N-terminus ³⁸. The length of the polyQ tract is correlated with the age of HD onset ³⁹, however exact pathological mechanisms of mHTT with varying polyQ lengths in HD remain unclear. In our study, we developed BiFC-based polyQ aggregation sensors and visualized real-time aggregation kinetics of mHTT with varying polyQ lengths in live cells. The results showed that longer polyQ length induces faster mHTT aggregation. The mHTT aggregates were not cleared and accumulated leading to cytotoxicity, thus confirming a correlation between polyQ length and the onset age of HD.

Interestingly, we discovered that different aggregation kinetics of mHTT with varying polyQ lengths can determine the physical characteristics of the aggregates: mHTT-polyQ103 formed compact solid-like aggregates, whereas mHTT-polyQ43 appeared to be liquid-like protein condensates. These results indicate that, possibly due to slow aggregation kinetics, mHTT with polyQ43 does not ultimately adopt the same physical form as mHTT-polyQ103 aggregates despite having the same composition of polyQ sequences. In fact, recent evidence indicates that mHTT aggregates can exist in liquid-like condensates ^{8, 40}. During the mHTT aggregation process, various substances can be included in the liquid-like condensates through LLPS ^{40, 41}. Notably, LLPS also mediates the assembly of SQSTM1/p62-containing protein condensates during autophagy ^{29, 42}, because the PB2 domain of SQSTM1/p62 mediates its oligomerization and the multivalent interaction between clustered SQSTM1/p62 and cargo proteins can form liquid-like protein condensates ²⁹.

Both physical forms of mHTT aggregates were not successfully cleared and eventually caused cytotoxicity, however different steps of autophagy were failed for mHTT aggregates with varying polyQ lengths. First, Q103 aggregates failed to be recognized by the representative autophagy receptor SQSTM1/p62 in the initial step of autophagy. In contrast, Q43 condensates were recognized by SQSTM1/p62, possibly due to their liquid-like properties and LLPS, however the large and bulky complexes between polyQ43 and SQSTM1/p62 were unable to generate intact autophagosomes. The formation of autophagosomes relies on the proper functions of ATG2A, a lipid transporter linking the endoplasmic reticulum (ER) to developing phagophores, and ATG9A which balances the lipid composition in an ATP-independent manner ⁴³. SQSTM1/p62 plays important role in recruiting ATG9A to the phagophores ⁴⁴, thus the improper association of SQSTM1/p62 with polyQ aggregates may impede the formation of autophagosomes. Consequently, the interaction between SQSTM1/p62 and polyQ43 aggregates may not be beneficial for the autophagic progression of polyQ aggregates. In fact, the overexpression of SQSTM1/p62 enhances the cytotoxicity in the mHTT-polyQ43 expressing cells. This observation is consistent with previous reports that the overexpression of SQSTM1/p62 increases the cytotoxicity of mHTT aggregates ⁴⁵, whereas the depletion of SQSTM1/p62 reduces the nuclear accumulation of mHTT aggregates ameliorating mHTT-mediated cytotoxicity in HD ⁴⁶.

Remarkably, we discovered that another autophagy receptor Optn can recognize mHTT-polyQ103 aggregates, but not mHTT-polyQ43 condensates, and successfully digested them in LC3-positive autophagosomes. These findings are consistent with previous studies demonstrating that Optn facilitates the formation of LC3-positive autophagosomes by regulating double FYVE-containing protein 1 (DFCP1) ⁴⁷ and recruiting the Atg12-5-16L1 complex to Wipi2-positive phagophores ⁴⁸. We observed that the autophagosomes containing polyQ103 aggregates can be subsequently transformed into autolysosomes, thereby clearing the mHTT-polyQ103 aggregates. Therefore, the overexpression of Optn enhances cell viability through autophagy progression for Q103 aggregates. Previous studies have suggested that the C-terminus of Optn is necessary to reduce the cytotoxicity of mHTT by sequestering toxic aggregates ³⁵. This protective effect of Optn was less pronounced in cells expressing mHTT-polyQ43 aggregates, possibly because mHTT-polyQ43 preferentially forms liquid-like condensates with SQSTM1/p62, reducing the likelihood of their neat sequestration by Optn.

We propose that the distinctive recognition of mHTT aggregates with varying polyQ lengths by autophagy receptors may be attributed to different ubiquitin (Ub) modifications on the surface of the aggregates, characterized by specific inter-ubiquitin linkages. Ubiquitination has the capacity to structurally diversify target proteins based on the mode of linkage, encompassing K6, K11, K27, K33, K48, K63, and M1, thereby facilitating recognition by distinct molecules ⁴⁹. For instance, unfolded polypeptides decorated with poly-Ub K48-linked and branched K48-K11 chains are recognized and processed by the ubiquitin-proteasome system (UPS), while protein aggregates exhibit a preference for modification by K63-linked poly-Ub, making them susceptible to the autophagy process ⁵⁰. Intriguingly, p62/SQSTM1 can mediate both UPS and autophagy through its PB1 domain and LC3-interacting region (LIR), respectively.

A ubiquitin-conjugating E2 enzyme, huntingtin-interacting protein-2 (HIP2), has been reported to facilitate the assembly of poly-Ub K48/K63 branched Ub conjugates on wild-type HTT (Q16) and mHTT-polyQ44 ⁵¹. However, this phenomenon was not observed in the case of mHTT-polyQ82 ⁵². These findings imply the potential existence of differential ubiquitin modifications on mHTT aggregates with varying polyQ lengths. In fact, it has been demonstrated that mHTT with long polyQ109 is preferentially modified with polyUb-K63 and recognized by OPTN ⁵³. Therefore, our current hypothesis is that HIP2 mediates the poly-Ub K48/K63 modification of wild-type HTT and mHTT with relatively short polyQ lengths, leading to recognition by p62/SQSTM1. In contrast, mHTT aggregates with longer polyQ lengths are tagged with K63-linked polyUb, thereby recognized by OPTN. These hypotheses form the basis for our ongoing investigation, and we intend to explore these mechanisms further in our subsequent studies.

In summary, we developed new HTT-polyQ sensors based on BiFC in this study and uncovered different aggregation kinetics of mHTT with varying polyQ length, which may influece the physical properties of the mHTT aggregates. Furthermore, we identified SQSTM1/p62 and Optn as two key autophagy receptors capable of selectively recognizing mHTT aggregates in different physical states, thereby exerting distinct influences on the autophagic process. SQSTM1/p62 recognizes the liquid-like mHTT-polyQ43 condensates but does not form intact autophagosomes, thereby facilitating the cytotoxicity of mHTT aggregates. Thus, the depletion of SQSTM1/p62 may reduce the formation of toxic complexes with liquid-like polyQ43 condensates, thus it can be considered for patients with HD who have relatively shorter polyQ lengths. In contrast, Optn specifically recognizes the solid-like mHTT-polyQ103 aggregates and reduces their levels through autophagy progression. Particularly, it has been suggested that Optn levels are reduced in the striatal region of patients with HD ⁵⁴. Therefore, Optn may serve as a potential therapeutic strategy to mitigate cytotoxicity and delay the onset of disease in patients with long polyQ lengths. Therefore, our findings suggest that patients with HD with different mHTT-polyQ lengths require more sophisticated therapeutic strategies.

COMPETING INTERESTS

The authors declare no competing interests.

AUTHOR CONTRIBUTIONS

J.S. and H.K. designed research; H.K., H.L. performed experiments; J.S., H.K., H.L., K-S.I., and H.R. analyzed data; J.S. and H.K. wrote the manuscript.

ACKNOWLEDGEMENTS

This work was supported by the New Faculty Startup Fund from Seoul National University, the National Research Foundation of Korea (NRF) grant No. 2021R1A2C1093429 and RS-2023-00227950 (J.S.).

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Distinct recognition of mutant huntingtin aggregates by autophagy receptor SQSTM1/p62 versus optineurin has differential effects on cell survival

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