Abstract

   Alzheimer’s disease (AD) is a complex neurodegenerative disease that
   affects elderly individuals, characterized by senile plaques formed by
   extracellular amyloid beta (Aβ). Autophagy dysfunction is a
   manifestation of protein homeostasis imbalance in patients with AD, but
   its relationship with Aβ remains unclear. Here, we showed that in Aβ
   transgenic Caenorhabditis elegans, Aβ activated the TOR pathway and
   reduced the nuclear entry of HLH-30, leading to autophagy dysfunction
   characterized by autophagosome accumulation. Then, utilizing RNA-seq,
   we investigated the regulatory mechanisms by which HLH-30 modulates
   autophagy in C. elegans. We found that HLH-30 elevated the transcript
   levels of v-ATPase and cathepsin, thus enhancing lysosomal activity.
   This led to an increase in autophagic flux, facilitating more
   pronounced degradation of Aβ. Moreover, HLH-30 reduced the level of ROS
   induction by Aβ and enhanced the antioxidant stress capacity of the
   worms through the gsto-1 gene. Additionally, we identified two
   HLH-30/TFEB activators, saikosaponin B2 and hypericin, that improved
   autophagic flux, thereby enhancing protein homeostasis in C. elegans.
   Overall, our findings suggested that HLH-30/TFEB plays a key role in
   modulating autophagy and can be considered a promising drug target for
   AD treatments.

   Keywords: HLH-30, amyloid beta, autophagy, HLH-30/TFEB activators,
   Caenorhabditis elegans

Introduction

   Alzheimer’s disease (AD) is characterized by the accumulation of
   amyloid beta (Aβ) peptides and hyperphosphorylated tau (p-Tau) protein
   in senile plaques and neurofibrillary tangles, respectively, which
   cause neurotoxicity and neuron loss, leading to memory and learning
   impairments ([48]Silva et al., 2019). Therefore, the removal of
   misfolded proteins plays a significant role in maintaining protein
   homeostasis in the treatment of AD ([49]Lukiw, 2022). The
   autophagy–lysosome pathway (ALP) is a cellular pathway in which cells
   utilize lysosomes to degrade long-lived proteins and organelles. The
   inability of the ALP to fully clear misfolded proteins has been
   recognized as important in the pathophysiology of AD ([50]Cheng et al.,
   2023). Correspondingly, autophagy dysfunction is prevalent in AD
   patients ([51]Ghavami et al., 2014); however, the mechanisms underlying
   this dysfunction are poorly understood.

   The transcription factor EB (TFEB, known as HLH-30 in Caenorhabditis
   elegans), a member of the MiT/TFE family of transcription factors, is a
   key regulator of ALP ([52]Martini-Stoica et al., 2016). It is
   translocated into the nucleus to coordinate the expression of autophagy
   and lysosomal target genes ([53]Napolitano and Ballabio, 2016). The
   subcellular localization and activity of TFEB are regulated by
   mammalian target of rapamycin (mTOR)-mediated phosphorylation, which
   occurs at the lysosome surface ([54]Zhu et al., 2019a). Phosphorylated
   TFEB is retained in the cytoplasm, whereas dephosphorylated TFEB
   translocates to the nucleus to induce the transcription of target genes
   ([55]Puertollano et al., 2018). In AD patients, the level of TFEB in
   the nucleus is significantly reduced in cells in the hippocampus
   ([56]Wang et al., 2016), which is the main brain region where autophagy
   dysfunction occurs ([57]Nixon and Yang, 2011). Therefore, inhibition of
   TFEB nuclear translocation may be closely related to autophagy
   dysfunction. In our previous work, we reported impaired autophagic flux
   and abnormally elevated LET-363 (a mammalian ortholog of TOR) in Aβ
   transgenic Caenorhabditis elegans (C. elegans) ([58]Lin et al., 2022).
   It has been reported that LET-363 can affect the nuclear localization
   of HLH-30 ([59]Nakamura et al., 2016). However, the correlation between
   the aberrant upregulation of LET-363 expression and impaired autophagic
   function in Aβ transgenic worms has not yet been tested.

   Accumulating studies have suggested that targeting TFEB is promising
   for the treatment of AD ([60]Gu et al., 2022). However, there are very
   few effective and specific small molecule TFEB activators at present.
   The main known activators of TFEB are mostly mTOR inhibitors ([61]Song
   et al., 2016). mTOR is well-known as a major regulator of cell growth
   and metabolism and is involved in a wide range of biological functions.
   Therefore, identifying TFEB-specific activators would be an important
   step in developing better therapeutics.

   In this study, we determined that Aβ could activate TOR activity,
   inhibit the nuclear localization of HLH-30, and block the fusion of
   autophagosomes with lysosomes, thus leading to autophagy dysfunction in
   C. elegans. In addition, RNA sequencing to investigate the molecular
   mechanisms by which HLH-30 regulates autophagy revealed a close
   association between lysosomal function and autophagy dysfunction.
   Finally, we searched for HLH-30/TFEB-specific activators by molecular
   docking and found two active compounds, saikosaponin B2 and hypericin,
   that could specifically promote the nuclear entry of HLH-30 without
   inhibiting TOR activity, thereby enhancing autophagy and facilitating
   the degradation of Aβ.

Materials and methods

Strains and maintenance conditions

   All strains used in this study are detailed in [62]Supplementary Table
   S1. All strains were maintained and cultured under standard conditions
   at 20°C using E. coli OP50 as a food source, except CL4176, which was
   maintained at 16°C according to standard procedures.

Transgenic strain construction

   The plasmid pMH878 (lgg-1p::mcherry::gfp::lgg-1) was donated by Malene
   Hansen ([63]Chang et al., 2017). PHX3392, PHX3636 and PHX6883 were
   generated by SunyBiotech at our request. We knocked out the first four
   exons of the hlh-30 gene to construct PHX6883. Other strains were
   obtained by hybridization.

Gene expression analysis by quantitative PCR (qPCR)

   Total RNA was extracted using TRIzol A+ and reverse transcribed into
   cDNA. The expressed genes were amplified in triplicate and quantified
   by PCR using SYBR Green PCR Mix ([64]B21702, Bimake, China) with a
   Roche LightCycler system. The data were analyzed using the 2^−ΔΔCT
   method ([65]Lin et al., 2022). The primer list has been added to
   [66]Supplementary Table S2.

Paralysis assay

   Aβ transgenic CL4176 worms were maintained on NGM at 16°C and
   synchronized. L1 animals were treated with drugs or RNAi-engineered
   bacteria (or left untreated) for 36 h and then transferred to 23°C for
   transgene induction. This temperature shift stimulated Aβ expression,
   causing Aβ aggregation and leading to paralysis. Scoring began 27 h
   after the temperature shift, and the worms were considered paralyzed if
   they failed to move their bodies when touched and produced a “halo” of
   cleared bacterial lawn because they moved only their heads while
   feeding. We used PT[50] (the time interval from the onset of paralysis
   at which 50% of the worms were paralyzed) as an index ([67]Wu et al.,
   2006). For example, the PT[50] of 4.2 h for the control was obtained by
   subtracting the time at which paralysis was first observed (29 h) from
   the time when 50% of the worms were paralyzed (33.2 h). The assay was
   performed at least three times. Each individual group contained more
   than 30 worms. Statistical analysis was conducted with GraphPad Prism
   6.0 software, and p values were calculated using the log-rank test.

LysoTracker red staining

   The acidophilic dye LysoTracker Red (C1046; Beyotime Biotechnology,
   Shanghai, China) was used at a final concentration of 15 μM.
   Synchronized strains maintained at 20°C were treated with LysoTracker
   from the L1 to the young adult stage and then transferred to 25°C for
   transgene induction. After 24 h, the worms were washed twice with fresh
   M9 (without LysoTracker) and imaged using a Revolve microscope
   ([68]Imanikia et al., 2019).

RNA interference (RNAi)

   RNAi experiments were performed according to a previously reported
   protocol. In brief, RNA was delivered to the worms by feeding, gravid
   adults were bleached, and the eggs were placed on NGM dishes
   supplemented with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). An
   HT-115(DE3) bacterial colony containing L4440 or the target gene
   plasmid was inoculated in LB broth supplemented with 100 μg/mL
   ampicillin and 12.5 μg/mL tetracycline and grown for 8 h in a 37°C
   shaker. The bacteria were plated on an NGM dish containing IPTG 1 h
   prior to the addition of the worms. The synchronized strain was grown
   on RNAi NGM plates from the egg stage to the adult stage for two
   generations. Synchronized L1 worms were treated with or without drugs
   for paralysis assays as described above ([69]Zhang et al., 2016).

Measurement of reactive oxygen species (ROS)

   Endogenous ROS levels were measured using 2′,7′-dichlorofluorescein
   diacetate (H2DCF–DA), which reacts with endogenous ROS to generate a
   fluorescent product ([70]Yang et al., 2018). Worms were treated as in
   the paralysis assay, and ROS were measured at the point of paralysis.
   The cells were then incubated with 50 μM H2DCF–DA for 30 min at 37°C,
   and the fluorescence intensity was measured at excitation and emission
   wavelengths of 485 and 535 nm, respectively. The assay was performed in
   triplicate.

Oxidative stress resistance assay

   Synchronized worms were cultured to the young adult stage. Then, the
   worms were transferred to a new 96-well plate containing 1 mM H[2]O[2.]
   The worms were monitored every 12 h and scored as dead when they no
   longer moved through the liquid medium ([71]Chen et al., 2018).

HLH-30 translocation assay

   The transgenic strain was used to observe the intracellular
   localization of HLH-30. Age-synchronized L1 worms were treated with or
   without 100 μM rapamycin for 3 days, mounted on 2% agarose pads and
   visualized under a fluorescence microscope (Revolve FL, Echo
   Laboratories, United States) for GFP localization. The nuclear
   accumulation of HLH-30::GFP was quantified by ImageJ ([72]Visvikis et
   al., 2014). We use 4% paraformaldehyde as anesthetic. The assay was
   performed three times independently.

Western blotting

   The worms were synchronized, and eggs were allowed to hatch and develop
   to the L4 stage on NGM plates at 20°C. Then, the temperature was
   increased to 25°C for 24 h. The worms were then collected from the
   plates with M9 buffer and washed twice to remove bacteria. Nuclear
   protein extraction was performed using a nuclear extract kit (Active
   Motif). The samples were heated at 100°C in sample loading buffer for
   10 min and centrifuged at 10,000 × g for 10 min ([73]Sangha et al.,
   2015). The collected supernatant was boiled with loading buffer at
   100°C for 5 min before being loaded into the gel, which also included a
   10–180 kDa protein molecular weight marker ([74]PR1910, Solarbio,
   China) ([75]Zhu et al., 2019b). The samples were run at 40 V for 40 min
   on a stacking gel and at 80 V for 120 min on a separating gel. The gel
   was then transferred to a polyvinylidene fluoride membrane using 20%
   methanol transfer buffer at 100 V for 1 h. The membranes were then
   blocked in Tris-buffered saline with Tween 20 + 5% skim milk for 1 h.
   The level of phosphorylated RSKS-1 protein was detected with an
   anti-phospho-p70 S6 kinase antibody (dilution 1:2,000; 9205, Cell
   Signaling Technology), with an anti-β-actin antibody (dilution 1:2,000;
   60008, Proteintech) as a control. The RSKS-1 protein level was detected
   with an anti-p70 S6 kinase antibody (1:2,000 dilution; 9202, Cell
   Signaling Technology). The Aβ protein level was detected with the 6E10
   monoclonal antibody (dilution 1:500; 803014, BioLegend). The protein
   level of mCherry-GFP-LGG-1 was detected with an anti-GFP antibody
   (dilution 1:500 Beyotime Technology). Histone H3 (1:2,000 dilution;
   ab1791 dilution, Abcam) served as an internal reference protein for the
   nuclear protein experiments. The membrane was incubated with the
   primary antibody overnight at 4°C and then with the secondary antibody
   for 2 h at room temperature. Images were captured using a Chemiscope
   3400 mini Western blot imaging system (Amersham Imager 600, GE, United
   States). The mean densities of the Aβ bands were analyzed using ImageJ
   software.

Hybridization experiment

   Five to eight worms in the L3 stage were selected and heat shock
   cultured for 3.5 h at 33°C, after which they were transferred to 20°C
   for further culture. A number of males could be found among the
   offspring. The male worms were hybridized with hermaphroditic worms,
   and homozygous worms were identified by agarose electrophoresis
   screening ([76]Fay, 2006).

Molecular docking

   Core targets (large molecular receptors) and the corresponding active
   compounds (small molecule ligands) were selected for molecular docking
   analysis. The 3D structures of the core targets were downloaded from
   the PDB database ([77]https://www.rcsb.org/). The 2D structures of the
   corresponding active compounds were downloaded from the PubChem
   database ([78]https://pubchem.ncbi.nlm.nih.gov/). The raw files of the
   core targets and the corresponding active components were processed
   with the AutoDock (version 4.2) tool ([79]Morris et al., 2009) and
   converted to PDBQT format for molecular docking. Binding activity was
   expressed as binding energy, with lower bonding energy corresponding to
   more stable docking ([80]Morris et al., 2008). In general, a docking
   energy < −5 kcal/mol was considered to indicate good docking.

RNA sequencing (RNA-seq)

   The worms were synchronized ([81]Fabian and Johnson, 1994), and their
   eggs were allowed to hatch and develop to the young adult stage on NGM
   plates at 20°C. Then, the temperature was increased to 25°C to induce
   Aβ expression for 24 h. Gene expression was assessed by Novo Gene
   Corporation (Beijing, China). Sequencing libraries were generated using
   the NEBNext^® UltraTM RNA Library Prep Kit for Illumina^® (NEB, United
   States). To preferentially select cDNA fragments 250∼300 bp in length,
   the library fragments were purified with the AMPure XP system (Beckman
   Coulter, Beverly, United States). Then, 3 µL of USER Enzyme (NEB,
   United States) was incubated with size-selected adaptor-ligated cDNA at
   37°C for 15 min and then 5 min at 95°C. Then, PCR was performed with
   Phusion High-Fidelity DNA polymerase, universal PCR primers and Index
   (X) Primer. Finally, the PCR products were purified (AMPure XP system),
   and library quality was assessed on an Agilent Bioanalyzer 2100 system.
   Clustering of the index-coded samples was performed on a cBot Cluster
   Generation System using the TruSeq PE Cluster Kit v3-cBot-HS (Illumina)
   according to the manufacturer’s instructions. After cluster generation,
   the libraries were sequenced on an Illumina NovaSeq platform to
   generate 150 bp paired-end reads. Up- or downregulated genes were
   identified by filtering RNA-seq data with the following criteria:
   twofold change in expression level and a false discovery rate analog of
   p < 0.05. KEGG pathway enrichment analysis was performed using
   “clusterProfiler” with the criterion of p < 0.05.

Statistical analysis

   All quantified data are presented as the mean ± SD. The data were
   analyzed by Student’s t test to determine statistical significance with
   GraphPad Prism 6.0 software (GraphPad, La Jolla, United States). For
   multiple comparisons, the results were analyzed by one-way analysis of
   variance (ANOVA). p < 0.05 was considered to indicate statistical
   significance.

Results

Aβ expression affects TOR-mediated HLH-30 nuclear localization

   In previous studies, we found that both the transcription and protein
   levels of let-363 were significantly increased in Aβ transgenic C.
   elegans ([82]Lin et al., 2022). Thus, we hypothesized that Aβ
   expression might affect TOR activity in C. elegans. To test this
   hypothesis, we evaluated the transcript levels of lmtr-2 and ragc-1,
   which regulate TOR activation ([83]Charar et al., 2021; [84]Blackwell
   et al., 2019), and found that they were obviously upregulated
   ([85]Figure 1A), suggesting that Aβ expression induced TOR activation.
   To further explore the regulatory role of Aβ expression in TOR
   activation, we examined TOR activity by measuring the phosphorylation
   level of its substrate RSKS-1 (an ortholog of S6K). The results showed
   that RSKS-1 phosphorylation in the Aβ strain GMC101 was significantly
   greater than that in the control strain CL2122, while the total protein
   level of RSKS-1 remained unchanged ([86]Figures 1B, C). These results
   suggested that Aβ expression could activate the TOR pathway. TOR
   activation prevents the nuclear translocation of HLH-30 ([87]Blackwell
   et al., 2019). The strain JIN1821 is often used to observe the nuclear
   localization of HLH-30 ([88]Visvikis et al., 2014). We crossed JIN1821
   with CL4176 (a transgenic strain expressing Aβ) to observe the nuclear
   localization of HLH-30 in the presence of Aβ. Compared with that in the
   control group, the nuclear entry of HLH-30::GFP was obviously reduced
   in the Aβ expressing group ([89]Figures 1D, E). Moreover, the nuclear
   localization of HLH-30::GFP was significantly restored after the worms
   were treated with the mTOR inhibitor rapamycin at 100 μM ([90]Figures
   1D, E). We also observed a significant decrease in the nuclear level of
   the HLH-30 protein in the Aβ group compared to that in the control
   group ([91]Figures 1F, G). The above results suggested that Aβ affected
   the entry of HLH-30 into the nucleus by activating TOR and that this
   may be one of important factor causing the abnormal autophagy in Aβ
   transgenic worms.

FIGURE 1.

   [92]FIGURE 1
   [93]Open in a new tab

   Aβ affects the nuclear localization of HLH-30 by activating mTOR. (A)
   mRNA expression of genes related to mTOR (strains GM101 and CL2122).
   (B) Representative Western blot showing phospho-RSKS-1 and total RSKS-1
   in the CL2122 and GMC101 strains, three biological replicates. (C)
   Quantified Western blot gel intensities, as determined by ImageJ
   software. (D) GFP fluorescence images of HLH-30 entering the nucleus (n
   = 10 for each group, repeated three times). The dose of rapamycin was
   100 μM. (E) Fluorescence signal ratio quantification. RPM, rapamycin.
   The HLH-30::GFP accumulation in JIN1821 was defined as 100% for
   quantitative comparisons. (F) Representative Western blot showing the
   content of HLH-30::GFP in the nucleus. Each experiment was repeated
   three times. (G) Quantified Western blot gel intensities, as determined
   by ImageJ software. **p < 0.01; ***p < 0.001. Detailed genotypic
   descriptions of strains CL2122, GMC101, and JIN1821 are listed in
   [94]Supplementary Material.

The stability of autophagic flux in Aβ transgenic worms depends on HLH-30

   Previous studies have shown that Aβ causes the accumulation of
   autophagosomes in C. elegans ([95]Florez-McClure et al., 2007);
   however, the cause of this phenomenon is unknown. The transcription
   factor HLH-30 plays a crucial role in the autophagy‒lysosome pathway
   ([96]Napolitano and Ballabio, 2016). To explore the relationship
   between HLH-30 and autophagy dysfunction in Aβ transgenic worms, we
   performed hlh-30 RNAi on the PHX3392 (Aβ) and PHX3636 (no Aβ) strains,
   which express mCherry::GFP::LGG-1 (genotypes are shown in
   [97]Supplementary Table S1). Thus, the autophagosomes in these strains
   are labeled by both mCherry and GFP ([98]Chang et al., 2017). The
   number of autophagosomes in the PHX3636 (no Aβ) group was significantly
   greater than that in the control group after hlh-30 RNAi treatment
   ([99]Figures 2A, B). In addition, we found that the protein level of
   mCherry::GFP::LGG-1-Ⅱ was significantly increased ([100]Figures 2C, D).
   This suggests that hlh-30 RNAi may result in autophagy dysfunction in
   worms. To further investigate the mechanism by which HLH-30 regulates
   autophagy, we generated hlh-30-overexpressing (hlh-30 ox) strains and
   hlh-30 (syb6883) knockout mutants on the basis of the Aβ strains. The
   use of GFP labeling in the construction of transgenic nematodes is
   necessary for the microscopic observation of mCherry::GFP::LGG-1.
   Therefore, we detected the expression level of mCherry::GFP::LGG-1 to
   evaluate autophagosome accumulation in worms. The results showed that
   overexpression of hlh-30 alleviated the accumulation of autophagosomes
   induced by Aβ, while hlh-30 knockout did not increase the accumulation
   of autophagosomes in Aβ worms ([101]Figures 2E, F). Then, we compared
   the paralysis rates of hlh-30-overexpressing and hlh-30 knockout worms.
   Overexpression of hlh-30 obviously decreased the paralysis rate, while
   hlh-30 knockout accelerated the paralysis rate ([102]Figure 2G).
   Finally, we found that the protein level of Aβ was significantly
   decreased in hlh-30-overexpressing worms ([103]Figures 2H, I). These
   results indicated that the overexpression of hlh-30 could lead to the
   degradation of Aβ by enhancing autophagy.

FIGURE 2.

   [104]FIGURE 2
   [105]Open in a new tab

   The stability of autophagic flux in Aβ transgenic worms depends on
   HLH-30. (A) Puncta formation in the PHX3636 strain as shown by
   fluorescence microscopy (strain PHX3636). (B) Scoring of punctae (n =
   10 for each group, repeated three times). (C) Immunoblot analysis to
   evaluate the effects of hlh-30 RNAi on mCherry::GFP::LGG-1 expression
   (strain PHX3636). (D) Quantified Western blot gel intensities, as
   determined by ImageJ software. (E) Effects of hlh-30 overexpression or
   knockout on mCherry::GFP::LGG-1, as measured by immunoblot analysis
   (strains HBW003 and HBW004). “ox”, overexpression; “ko”, knockout. Each
   experiment was repeated three times. (F) Quantified Western blot gel
   intensities, as determined by ImageJ software. (G) Paralysis assay of
   hlh-30-overexpressing or hlh-30 knockout worms (n > 30 for each group,
   repeated three times). (H) Immunoblot analysis to evaluate the effects
   of hlh-30 overexpression or knockout on Aβ species. Each experiment was
   repeated three times. (I) Quantified Western blot gel intensities, as
   determined by ImageJ software. “ns”, not significant; **p < 0.01; ***p
   < 0.001. Detailed genotypic descriptions of strains PHX3636, HBW003,
   and HBW004 are listed in [106]Supplementary Material.

RNA interference of hlh-30 reduces the transcript level of syx-17 and leads
to autophagosome accumulation

   In mammals, TFEB can regulate hundreds of ALP genes at the
   transcriptional level, so we hypothesized that HLH-30 may also regulate
   the transcription levels of ALP genes in worms. The expression levels
   of rab-7 and syx-17, which are related to autophagosome–lysosome fusion
   ([107]Zhao et al., 2021), were found to be significantly decreased by
   hlh-30 RNAi ([108]Figure 3A). It was reported that syx-17 RNAi could
   block autophagosome–lysosome fusion ([109]Gkikas et al., 2023).
   Therefore, we investigated the association between the reduced
   expression of syx-17 and autophagy dysfunction caused by the decreased
   expression of HLH-30. We found that the number of autophagosomes in
   PHX3636 cells (without Aβ) increased significantly in response to
   syx-17 RNAi ([110]Figures 3B, C). However, autophagosome accumulation
   after syx-17 and hlh-30 dual gene RNAi was not significantly greater
   than that after syx-17 RNAi in PHX3636. Similarly, the accumulation of
   autophagosomes in PHX3392 (Aβ) cells was not further elevated by syx-17
   RNAi ([111]Figures 3B, C). These results indicated that the decreased
   expression of HLH-30 reduced the expression of syx-17, resulting in the
   abnormal fusion of autophagosomes and lysosomes in Aβ nematodes and
   subsequent autophagy dysfunction.

FIGURE 3.

   [112]FIGURE 3
   [113]Open in a new tab

   Downregulation of syx-17 at the transcriptional level leads to the
   accumulation of autophagosomes in worms. (A) Effects of hlh-30 RNAi on
   the mRNA expression of genes related to autophagosome‒lysosome fusion.
   (B) Effects of RNAi of syx-17 or hlh-30 on puncta formation in the
   PHX3636 (no Aβ) or PHX3392 (Aβ) strains, as shown by fluorescence
   microscopy. (C) Scoring of punctae (n = 10 for each group, repeated
   three times). “ns”, not significant; **p < 0.01. Detailed genotypic
   descriptions of strains PHX3636 and PHX3392 are listed in
   [114]Supplementary Material.

HLH-30 maintains the stability of autophagic flux by enhancing lysosomal
activity

   To further explore how hlh-30 regulates autophagy to stabilize protein
   homeostasis in worms, we conducted RNA-seq analysis on strains in which
   hlh-30 was overexpressed or knocked out ([115]Figure 4A). Through KEGG
   enrichment, we found that both the overexpression and knockout of
   hlh-30 affected the transcript levels of lysosome-related genes
   ([116]Figures 4B, C), which was verified by qPCR ([117]Supplementary
   Figure S1). The results showed that the v-ATPase and cathepsin B genes
   were significantly upregulated in the hlh-30 overexpression group
   ([118]Supplementary Figure S1). To further evaluate the effect of
   hlh-30 overexpression on lysosomal function, we utilized LysoTracker
   Red to analyze lysosome acidification. The results showed that hlh-30
   overexpression obviously enhanced lysosomal activity ([119]Figures 4D,
   E).

FIGURE 4.

   [120]FIGURE 4
   [121]Open in a new tab

   HLH-30 maintains the stability of autophagic flux by enhancing
   lysosomal activity. (A) RNA-seq differential gene volcano map. “ox”,
   overexpression; “ko”, knockout. (B) KEGG pathway enrichment of genes
   differentially expressed in the hlh-30-overexpressing strain. The x-
   and y-axes represent the GeneRatio and enriched KEGG pathways,
   respectively. The color represents the significance of the enrichment,
   and the bubble size represents the gene count. (C) KEGG pathway
   enrichment of genes differentially expressed in the hlh-30 knockout
   strain. (D) Representative fluorescence images of strains stained with
   LysoTracker Red after Aβ induction for 24 h (n = 10 for each group,
   repeated three times). The worms used in this study were overexpressed
   and knockout strains based on GMC101. (E) Quantified fluorescence
   intensities (n = 10 for each group, repeated three times). (F)
   Overexpression of hlh-30 delayed CL4176 paralysis in a cathepsin
   B-dependent manner (n > 30 for each group, repeated three times). The
   worms used in this study were overexpressed strains based on CL4176.
   (G) Statistical analysis of the half paralysis time of worms. *p <
   0.05; ***p < 0.001.

   Given the observed upregulation of cathepsin B transcription and
   enhancement of lysosomal activity, we hypothesized that HLH-30 promotes
   Aβ degradation by enhancing lysosomal activity and hydrolase
   expression, thereby improving protein homeostasis in worms. To further
   confirm the above results, we evaluated the impact of CA-074, a
   cathepsin-specific inhibitor ([122]Bai et al., 2021), on the paralysis
   rate in hlh-30 Aβ-overexpressing worms. The results showed that the
   ability of hlh-30 overexpression to prolong paralysis was offset by the
   administration of CA-074 ([123]Figures 4F, G). The above results
   suggested that lysosomes may be involved in the maintenance of protein
   homeostasis through the overexpression of hlh-30.

HLH-30 maintains protein homeostasis by clearing ROS through gsto-1

   In previous studies, we found that Aβ can cause severe oxidative
   stress, manifested by a large increase in reactive oxygen species (ROS)
   ([124]Lin et al., 2022). Through an ROS assay, we found that ROS levels
   were significantly decreased by hlh-30 overexpression and increased in
   hlh-30(syb6883) mutants ([125]Figure 5A). Furthermore, the survival
   time of hlh-30-overexpressing worms in an oxidative stress environment
   induced by hydrogen peroxide was increased by 10.3% compared with that
   in the control group ([126]Figures 5B, C). It has been reported that
   the levels of glutathione within brain neurons are reduced in AD
   patients, which leads to increased sensitivity of the neurons to
   oxidative damage ([127]Allen et al., 2012). Our RNA-seq analysis
   indicated that the transcription level of gsto-1, a glutathione
   transferase that is primarily involved in the metabolism of glutathione
   within cells, was significantly downregulated in hlh-30 mutants.
   Paralysis experiments revealed that the paralysis delaying and RO
   reducing effects of hlh-30 overexpression were reversed by gsto-1 RNAi
   ([128]Figures 5D, E). The augmented antioxidant capacity following
   hlh-30 overexpression was also closely associated with gsto-1
   ([129]Figure 5F). These findings indicate that gsto-1 could potentially
   contribute to the regulation of oxidative stress and the maintenance of
   protein homeostasis in worms by hlh-30.

FIGURE 5.

   [130]FIGURE 5
   [131]Open in a new tab

   HLH-30 maintains protein homeostasis by clearing ROS through gsto-1.
   (A) Levels of ROS in transgenic worms (n > 30 for each group, repeated
   three times). “ox”, overexpression; “ko”, knockout. The worms used in
   this study were overexpressed strains based on GMC101. (B) Evaluation
   of the antioxidant capacity of transgenic worms (n > 30 for each group,
   repeated three times). The worms used in this study were overexpressed
   strains based on CL4176. (C) Survival time histogram under oxidative
   stress induced by hydrogen peroxide. (D) Paralysis assay of RNAi worms
   (n > 30 for each group, repeated three times). (E) Levels of ROS in
   transgenic worms (n > 30 for each group, repeated three times). (F)
   Evaluation of the antioxidant capacity of the transgenic worms (n > 30
   for each group, repeated three times). “ns”, not significant; *p <
   0.05; **p < 0.01; ***p < 0.001.

Saikosaponin B2 and hypericin ameliorate the proteostasis imbalance caused by
Aβ by promoting HLH-30 translocation to the nucleus and reducing
autophagosome accumulation

   TFEB/HLH-30 regulators that do not inhibit the mTOR pathway are
   preferred as they may have less harmful effects on cells ([132]Song et
   al., 2016). The curcumin analog C1, a TFEB-specific activator, has been
   found to significantly reduce Aβ and tau aggregation and improve
   synaptic and memory functions in different AD animal models ([133]Song
   et al., 2020). We used C1 as a ligand for molecular docking to identify
   the active site of TFEB ([134]Figure 6A). Then, based on the predicted
   active site, we screened thousands of small molecule compounds derived
   from natural products. Subsequently, we evaluated the impact of the top
   ten small molecular compounds ([135]Supplementary Table S3), as
   determined by docking scores, on the paralysis rate of the Aβ strain
   CL4176 ([136]Supplementary Figure S2).

FIGURE 6.

   [137]FIGURE 6
   [138]Open in a new tab

   Saikosaponin B2 and hypericin improve protein homeostasis by reducing
   autophagosome accumulation and alleviating oxidative stress. (A)
   Possible binding sites of the curcumin analog C1. (B) GFP fluorescence
   images of HLH-30 in the nucleus (n = 10 for each group, repeated three
   times, strain HBW001). (C) Fluorescence signal ratio quantification.
   The HLH-30::GFP accumulation in the control group was defined as 100%
   for quantitative comparisons. (D) Possible sites of action of
   saikosaponin B2. (E) Possible sites of action of hypericin. (F)
   Immunoblot analysis to evaluate the effects of C1 (C1), saikosaponin B2
   (SAB2), and hypericin (HYP) on the phosphorylation of RSKS-1 and total
   RSKS-1. (G) Fluorescence microscopy analysis to evaluate the effects of
   the C1, saikosaponin B2, and hypericin on puncta formation in the
   PHX3392 strain. (H) Scoring of punctae (n = 10 for each group, repeated
   three times). (I) Immunoblot analysis to evaluate the effects of
   saikosaponin B2 (SAB2) and hypericin (HYP) on Aβ species (strain
   GMC101). **p < 0.01; ***p < 0.001. Detailed genotypic descriptions of
   strains HBW001, PHX3636 and GMC101 are listed in [139]Supplementary
   Material.

   By combining the results of the HLH-30::GFP nuclear localization and
   paralysis assays, we identified two active compounds, saikosaponin B2
   and hypericin ([140]Figures 6B, C). Molecular docking revealed that the
   binding sites of saikosaponin B2, hypericin, and the curcumin analog C1
   on TFEB were relatively similar ([141]Figures 6A, D, E). By detecting
   the phosphorylation levels of TOR and its downstream target protein
   RSKS-1, we confirmed that neither saikosaponin B2 nor hypericin could
   inhibit TOR activity ([142]Figure 6F). Further, both saikosaponin B2
   and hypericin could reduce autophagosome accumulation, maintain the
   stability of autophagic flux ([143]Figures 6G, H) and promote Aβ
   degradation in worms ([144]Figure 6I). Our results suggested that
   targeting TFEB/HLH-30 presents a promising alternative for discovering
   new treatments for AD.

Discussion

   Previous studies have documented autophagy dysfunction in the brains of
   Alzheimer’s disease (AD) patients, characterized by the accumulation of
   autophagosomal vesicles ([145]Yu et al., 2005). However, the underlying
   causes of this phenomenon remain elusive. Abnormal phosphorylation of
   the tau protein prevents the fusion of autophagosomes with lysosomes
   ([146]Hamano et al., 2021). Protein misfolding, immune abnormalities,
   and loss of lysosomal activity can also lead to impaired autophagy
   ([147]Cozzi and Ferrari, 2022). In this work, we used transgenic C.
   elegans to explore the association between Aβ and autophagy
   dysfunction. We found that the expression of Aβ in C. elegans activates
   the mTOR pathway, which inhibits the entry of the transcription factor
   HLH-30 into the nucleus ([148]Figures 1B–D) and ultimately leads to the
   accumulation of autophagosomes ([149]Figures 2A–C). These observations
   provide insight into the mechanisms underlying autophagy dysfunction in
   the brains of Alzheimer’s disease (AD) patients.

   Autophagy is a double-edged sword in neurodegenerative diseases
   ([150]Zhou and Soukas, 2019). Under normal conditions, autophagy is
   important for the degradation of misfolded proteins ([151]Chua et al.,
   2022). However, when autophagy is abnormal, its induction alone
   increases the metabolic burden on brain cells ([152]Zhang et al.,
   2021). As a transcription factor that can simultaneously induce
   autophagy and increase lysosomal activity, TFEB/HLH-30 is a worthy
   target for study. Here, we discovered that hlh-30 is the key gene
   involved in regulating protein homeostasis through our observations of
   an hlh-30-overexpressing strain and an hlh-30 (syb6883) mutant strain
   ([153]Figures 2E, H). Through RNA-seq, we found that hlh-30
   overexpression can significantly increase the level of cathepsin B
   transcripts and promote lysosomal activity. Cathepsin B has been
   reported to degrade Aβ ([154]Hook et al., 2020). We identified the
   critical role of cathepsin B in the regulation of protein homeostasis
   mediated by HLH-30 through the use of specific cathepsin B inhibitors
   ([155]Figures 4F, G). The lysosome, a key organelle that degrades
   misfolded proteins, plays a necessary role in maintaining protein
   homeostasis. With increasing age, lysosome function gradually weakens,
   resulting in waste materials and aging organelles in the cell not being
   effectively removed, which in turn accelerates the aging process of the
   cell ([156]Colacurcio and Nixon, 2016). In this study, we found that
   overexpressing hlh-30 could enhance lysosomal acidification and the
   expression of cathepsins, which contribute positively to protein
   homeostasis. It is necessary to consider age-related lysosomal
   dysfunction when developing treatment strategies for TFEB-related
   Alzheimer’s disease.

   Furthermore, we discovered that the regulation of protein homeostasis
   by hlh-30 is dependent on the involvement of gsto-1 ([157]Figures 5D,
   F). The gene gsto-1 encodes glutathione transferase, which is involved
   in the synthesis and transport of glutathione within cells
   ([158]Burmeister et al., 2008). Glutathione transferase is also an
   important factor in the crosstalk between apoptosis and autophagy
   ([159]Mazari et al., 2023). However, there is insufficient research on
   the relationship between hlh-30 and gsto-1. Moreover, the interactions
   among apoptosis, autophagy dysfunction and severe oxidative stress
   contribute greatly to the difficulty of studying the disease mechanisms
   of AD ([160]Twarowski and Herbet, 2023); therefore, finding suitable
   models and research methods is the key to advancing the study of these
   factors.

   The known activators of TFEB/HLH-30 are mainly inhibitors of mTOR, such
   as rapamycin. As a major regulator of cell growth and metabolism, mTOR
   is involved in a wide range of biological functions ([161]Saxton and
   Sabatini, 2017). Therefore, identifying TFEB-specific activators is a
   promising strategy, as it may offer reduced cytotoxicity to cells
   ([162]Song et al., 2021). We found that two natural products,
   saikosaponin B2 and hypericin, have potential as TFEB activators. The
   binding sites of saikosaponin B2, hypericin, and the curcumin analog C1
   are located at the N-terminus of TFEB ([163]Figures 6A, D, E),
   suggesting that the development of activators targeting TFEB should
   focus on the N-terminal protein structure of TFEB. Furthermore, both
   saikosaponin B2 and hypericin improved autophagy dysfunction and
   promoted the degradation of Aβ ([164]Figures 6G, I) without inhibiting
   TOR activity ([165]Figure 6F). In summary, our findings support a close
   association between inhibited nuclear entry of HLH-30/TFEB and
   autophagy dysfunction in AD and indicate that identifying TFEB-specific
   activators is a promising strategy for treating AD.

Conclusion

   We report that Aβ-induced autophagy dysfunction in C. elegans may be
   associated with the inhibition of nuclear translocation of the
   transcription factor HLH-30. This could lead to a reduction in the
   expression of the membrane fusion gene syx-17, which is a key molecular
   event in Aβ-induced autophagy dysfunction. Overexpression of the hlh-30
   gene appears to enhance autophagy activity and may decrease
   autophagosome accumulation, potentially promoting Aβ degradation. In
   addition, two potential HLH-30/TFEB activators, saikosaponin B2 and
   hypericin, were found by molecular docking and demonstrated to promote
   the nuclear entry of HLH-30, thereby enhancing autophagy and
   facilitating the degradation of Aβ.

Acknowledgments