Enhancement of lysosome biogenesis as a potential therapeutic approach for neurodegenerative diseases

Abstract

Millions of people are suffering from Alzheimer’s disease globally, but there is still no effective treatment for this neurodegenerative disease. Thus, novel therapeutic approaches for Alzheimer’s disease are needed, which requires further evaluation of the regulatory mechanisms of protein aggregate degradation. Lysosomes are crucial degradative organelles that maintain cellular homeostasis. Transcription factor EB-mediated lysosome biogenesis enhances autolysosome-dependent degradation, which subsequently alleviates neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease. In this review, we start by describing the key features of lysosomes, including their roles in nutrient sensing and degradation, and their functional impairments in different neurodegenerative diseases. We also explain the mechanisms — especially the post-translational modifications — which impact transcription factor EB and regulate lysosome biogenesis. Next, we discuss strategies for promoting the degradation of toxic protein aggregates. We describe Proteolysis-Targeting Chimera and related technologies for the targeted degradation of specific proteins. We also introduce a group of LYsosome-Enhancing Compounds, which promote transcription factor EB-mediated lysosome biogenesis and improve learning, memory, and cognitive function in APP-PSEN1 mice. In summary, this review highlights the key aspects of lysosome biology, the mechanisms of transcription factor EB activation and lysosome biogenesis, and the promising strategies which are emerging to alleviate the pathogenesis of neurodegenerative diseases.

Introduction

The pathogenesis of Alzheimer’s disease (AD) is characterized by the accumulation of protein aggregates, including amyloid-β (Aβ) and MAPT/Tau, which subsequently results in the loss of neuronal cells and dysfunction of the human brain. According to a recent study, dysfunction of the autophagy-lysosome system aggravates the pathogenesis of AD. In Tg2576/TRGL mice, an animal model of AD, the impairment of autolysosomal acidification occurs before Aβ accumulation in the brain (Lee et al., 2022). These results suggest that autolysosomal dysfunction further aggravates toxic protein accumulation. In this review, we summary the mechanisms of transcription factor EB activation and lysosome biogenesis, and the promising strategies which are emerging to alleviate the pathogenesis of neurodegenerative diseases.

Search Strategy and Selection Criteria

PubMed and Web of Science were searched to retrieve papers. During searching, all publications reviewed were in the English language, and no filters were applied such as restricting the date, publication type, or subjects. Literature retrieval was performed using the following keywords: lysosome biogenesis, small molecule compounds, TFEB, neurodegenerative disease, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, protein aggregates, degradation, phosphorylation, acetylation, ubiquitination, SUMOylation, oxidation, glycosylation, and PARsylation. The last search was conducted on December 29, 2022.

Structure and Degradative Function of Lysosomes

Lysosomes are major catabolic organelles, which were discovered by Christian de Duve and his colleagues in the 1950s (De Duve et al., 1955). They are usually found in all mammalian cells, except mature red blood cells, and they are located in the cytoplasm with the highest concentration in the perinuclear region. The number of lysosomes in each mammalian cell is between 50 and 1000 (Ballabio and Bonifacino, 2020), and the diameter of lysosomes typically ranges from 0.2 to 0.8 μm, with a minimum of 0.05 μm and a maximum of several microns. Lysosomes contain more than 70 acid hydrolases for the degradation of proteins, nucleic acids, lipids, and other biological macromolecules (Maxfield et al., 2016). The degradative capacity of lysosomes is critical for removing damaged organelles or recycling metabolites within cells via the autophagy pathway (Levine and Kroemer, 2019; Mizushima and Levine, 2020).

Notably, lysosomes are acidic organelles. The maintenance of an acidic lumenal microenvironment, with the pH between 4.5 and 5.0, is essential for lysosomal functions (Ballabio and Bonifacino, 2020). However, this does not mean that a lower pH is better. Lysosomal hyperacidification impairs lysosomal proteolytic activity and further leads to lysosome dysfunction (Hu et al., 2022). The acidic pH of the lysosome is maintained mainly by the vacuolar ATPase in the lysosomal membrane. However, the electrochemical gradient generated by the vacuolar ATPase must be dissipated to enable sustained proton import into the lysosomal lumen. ClC-7, a Cl^–/H⁺ antiporter located in the lysosomal membrane, is responsible for lysosomal Cl^– permeability (Leray et al., 2022). It imports 2 Cl^– into the lysosomal lumen while ejecting 1 H⁺ into the cytoplasm to dissipate the electrochemical gradient across the lysosome membrane (Jentsch and Pusch, 2018). Previous studies have found that TMEM175, a genetic risk factor for PD, encodes a proton-activated/proton-selective channel in the lysosomal membrane which regulates the lysosomal H⁺ leak (Cang et al., 2015; Hu et al., 2022). Based on the highly acidic microenvironment of lysosomes, many lysosomal probes have been developed to monitor the acidity and function of lysosomes (Lukinavicius et al., 2016; Farfel-Becker et al., 2019; Lee et al., 2022). Besides proton transporters, there are other ion channels in the lysosomal membrane. For example, the transient receptor potential mucolipin 1 (TRPML1, also named as MCOLN1) and the two-pore channel proteins serve as lysosomal Ca²⁺ channels which release Ca²⁺ to the cytoplasm (Dong et al., 2008; Schieder et al., 2010). The voltage-activated BK channel, a potassium channel, delivers K⁺ into the lysosomal lumen from the cytosol. Dysregulation of the flux of these ions impairs lysosome function, which further leads to lysosome-related diseases.

Furthermore, there are many other types of transporters in the lysosomal membranes (Maxfield et al., 2016). After the damaged organelles and macromolecules are delivered to lysosomes and degraded in lysosomes, their products, including amino acids, sugars, and nucleosides, are selectively transferred from lysosomes to the cytoplasm via amino acid transporters, sugar transporters, and nucleoside transporters, respectively. These catabolites then become the building blocks for further cellular biosynthesis and metabolic processes.

Lysosomes Function as a Signaling Hub

Lysosomes also serve as nutrient sensors of amino acids, glucose, and cholesterol. Lysosomal nutrient sensing is related to the control of metabolic signal transduction via the key regulators mTORC1 and AMP-activated protein kinase (AMPK) (Zoncu et al., 2011; Wolfson et al., 2017; Ma et al., 2022; Shin et al., 2022; Zhang et al., 2022a). Lysosomes can sense amino acid concentrations and regulate mTORC1 activity. Under the condition of amino acid sufficiency, Rag guanosine triphosphatases (GTPases) recruit mTORC1 to the lysosomal surface from the cytoplasm and activate mTORC1 (Liu and Sabatini, 2020). Arginine binds to SLC38A9 and promotes the interaction of SLC38A9 with the Rag-Ragulator complex on the lysosome surface, then recruits mTORC1 anchored on Rag GTPases (Wyant et al., 2017). When leucine is sufficient, SAR1B, a small GTPase, directly binds to leucine and dissociates from GATOR2, resulting in mTORC1 activation. However, when leucine is deficient, SAR1B binds to GATOR2 and inhibits the activity of mTORC1 (Chen et al., 2021). Lysosomes can also sense the glucose concentration and regulate AMPK activity. Notably, lysosomal vacuolar ATPase functions as a crucial switch to regulate AMPK and mTORC1 activation and signal transduction via protein-protein interactions (Zhang et al., 2014; Zhang et al., 2017; Li et al., 2019). Furthermore, cholesterol transporters are also localized in the lysosomal membrane (Trinh et al., 2018). Cholesterol can be transported from the lysosomal lumen to the cytoplasm via its transporters, such as NPC1 (Niemann Pick type C1), LIMP-2 (lysosomal integral membrane protein 2), and PTCH1 (protein patched homolog 1) (Chu et al., 2015; Trinh et al., 2017; Lim et al., 2019; Meng et al., 2020; Qian et al., 2020). This subsequently drives mTORC1 recruitment and activation at the lysosomal surface via the Rag GTPases (Castellano et al., 2017; Shin et al., 2022).

Although lysosomes are located in the cytoplasm, especially in the perinuclear region, they also have crosstalk and/or contacts with other organelles, including perinuclear endoplasmic reticulum (Boutry and Kim, 2021; Tan and Finkel, 2022), peroxisomes (Chu et al., 2015), and mitochondria (Boutry and Kim, 2021). Lim et al. (2019) found that oxysterol binding protein is a key regulator that mediates cholesterol transfer from the endoplasmic reticulum to the lysosomal cholesterol pool via the endoplasmic reticulum anchor proteins VAPA and VAPB. This process subsequently activates mTORC1 on lysosome membranes (Lim et al., 2019). Lysosomes also transfer free cholesterol to peroxisomes through dynamic membrane contacts, which are regulated by the binding between lysosomal synaptotagmin VII and peroxisomal PI(4,5)P2 (Chu et al., 2015). Lysosome-mitochondrion contracts have also been found in mammalian cells and play important roles in maintaining metabolite homeostasis. For instance, the lysosomal cation channel TRPML1 mediates the efflux of intracellular calcium to mitochondria through voltage-dependent anion channel 1 (VDAC1) and mitochondrial calcium uniporter (MCU) (Peng et al., 2020). In neurons, lysosome-mitochondrion contacts exist in soma, axons, and dendrites. Dysregulation of lysosome-mitochondrion contacts may be related to multiple neurodegenerative diseases (Cioni et al., 2019; Kim et al., 2021a).

Lysosomes and Neurodegenerative Diseases

Lysosomal dysfunction aggravates many human diseases (Maxfield et al., 2016). One group of lysosome-related diseases is known as lysosomal storage disorders (LSDs) (Parenti et al., 2015; Platt et al., 2018). Lysosomal storage disorders are usually caused by mutations in the genes encoding lysosomal hydrolases or lysosomal membrane proteins. Since lysosomes are major catabolic organelles in almost all mammalian cells, they play a critical role in regulating cell homeostasis. In the nervous system, neuronal cells also contain a lot of lysosomes in both soma and neurites, and lysosomes play important roles in neural metabolic homeostasis and cargo delivery under physiological conditions (Iacoangeli et al., 2019; Roney et al., 2021). Therefore, the other group is age-related neurodegenerative diseases (Saftig and Klumperman, 2009), which also showed lysosomal function impaired. Under pathological conditions of neurodegenerative diseases lysosomal functions are impaired by many factors, including abnormality of lysosomal acidification, mutations of lysosomal genes, and calcium (Ca²⁺) dysregulations.

Alzheimer’s disease

AD is the most common neurodegenerative disease in China and is characterized by the abnormal accumulation of protein aggregates (Aβ and/or Tau). Presenilin-1 (PSEN1) and presenilin 2 (PSEN2) are catalytic subunits of the gamma (γ)-secretase enzyme complex and are responsible for the hydrolysis of Aβ. Lee et al. (2010) found that autophagy requires PSEN1, and the depletion or mutation of PSEN1 results in impaired lysosomal acidification, protease inactivation, and lysosomal proteolytic dysfunction. PSEN2 plays a critical role in modulating intracellular Ca²⁺ homeostasis independently from γ-secretase activity. In AD, mutant PSEN2 causes impaired lysosomal Ca²⁺ signaling and abnormal autolysosome degradation (Fedeli et al., 2019). Mutation of TMEM106B is also identified in AD patients, which results in hyperfunction of the encoded protein. TMEM106B, a single-pass transmembrane protein, is located on endosomal and lysosomal membranes. Its elevated expression causes lysosomal hyperacidification and trafficking disorders, which lead to the impairment of lysosome-dependent degradation (Root et al., 2021).

Parkinson’s disease

Parkinson’s disease (PD) is characterized by neurological and motor dysfunction. Several genes related to PD progression have been identified, for instance, TMEM175, GBA1, and VPS13C (Alcalay et al., 2014; Darvish et al., 2018; Wie et al., 2021). TMEM175, regulating potassium (K⁺) leak in the lysosomal membrane is also identified as mediating lysosomal H⁺ leak, as mentioned above. TMEM175 deficiency results in lysosomal hyperacidification and diminished catalytic activity, which leads to α-synuclein aggregation (Hu et al., 2022). Interestingly, Lee et al. (2022) discovered that reduced acidification of autolysosomes occurs before Aβ accumulation. This indicates that the functional impairment of autolysosomes is not completely caused by deposits of toxic proteins, and the defective autolysosomes will further aggravate the accumulation of protein aggregates. Therefore, enhancement of lysosomal function may be a better therapeutic strategy to improve PD.

Huntington’s disease

Huntington’s disease (HD) is a neurodegenerative disease characterized by striatal neurodegeneration resulting from mutation of the huntingtin (HTT) gene. The mutant HTT (mHTT) knock-in HD-like mouse model shows abnormal cargo loading into autophagosomes and abnormal fusion between autophagosomes and lysosomes (Heng et al., 2010), which lead to reduced autophagic clearance of mHTT aggregates by lysosomes. The enzymatic activity of Glutamine Synthetase 1 is reduced in HD patients. Glutamine synthetase 1 deficiency causes autophagy inactivation, subsequent accumulation of toxic Htt-Q93 aggregates, and neuron loss in a Drosophila model of HD (Vernizzi et al., 2020). In addition, the level of the protease Cathepsin D is reduced in lysosomes in HD because mHTT disrupts the interaction between optineurin and Rab8 within the Golgi, which subsequently prevents efficient trafficking of Cathepsin D to the lysosomes (Liang et al., 2011).

Amyotrophic lateral sclerosis

In recent studies, more and more mutations of lysosomal genes, such as C9orf72 (chromosome 9 open reading frame 72), have been identified to be associated with the pathogenesis of ALS. Iacoangeli et al. (2019) discovered that the C9orf72 gene contains a hexanucleotide repeat expansion in amyotrophic lateral sclerosis (ALS) patients. The hexanucleotide motif GGGGCC (G₄C₂) is repeated about 24 to 30 times (Iacoangeli et al., 2019). C9orf72 deficiency in mouse or human cells leads to lysosomal swelling, impairment of mTORC1 activation and reduced lysosomal degradation (Amick et al., 2016; Shao et al., 2020).

Lysosome biogenesis and function are deficient in neurodegenerative diseases (Bajaj et al., 2019). The impaired lysosome biogenesis and function will accelerate the accumulation of protein aggregates, which will further aggravate neurodegeneration. Enhancement of lysosome biogenesis will increase cargo degradation via lysosomes, reduce neuronal impairment, and ameliorate neurodegenerative diseases. Therefore, it is critical to uncover methods for promoting lysosome biogenesis, especially in diseases.

Transcription Factor EB and Lysosome Biogenesis

To ameliorate the pathogenesis of neurodegenerative diseases, we need to promote lysosome biogenesis and enhance the degradative functions of lysosomes. Transcription factor EB (TFEB) belongs to the microphthalmia-associated transcription factor (MiTF) family, which contains TFEB, TFE3, TFEC and MiTF. These proteins form homo/heterodimers and function as transcription factors to promote the expression of their target genes. In fed, unstressed cells, TFEB is inactive and sequestered by 14-3-3 proteins in the cytosol. During starvation or treatment with certain small-molecule compounds, TFEB becomes active and translocates into the nucleus to promote the expression of autophagic/lysosomal genes. During the activation and translocation of TFEB, many changes in post-translational modifications occur, including phosphorylation (Li et al., 2016; Puertollano et al., 2018; Yin et al., 2022), acetylation (Zhang et al., 2018; Wang et al., 2020b; Li et al., 2022), ubiquitination (Sha et al., 2017; Li et al., 2022), oxidation (Martina et al., 2021) and alkylation (Zhang et al., 2022b).

Phosphorylation

The phosphorylated sites on TFEB have been identified by recent studies (Sardiello et al., 2009; Settembre et al., 2011; Ferron et al., 2013; Li et al., 2016; Palmieri et al., 2017; Vega-Rubin-de-Celis et al., 2017; Hsu et al., 2018; Martina and Puertollano, 2018; Iacoangeli et al., 2019; Contreras et al., 2020; Yin et al., 2020, 2022; Paquette et al., 2021; Figure 1). In normal conditions, Rag GTPase recruits mTORC1 and TFEB onto lysosome membranes, where active mTORC1 phosphorylates TFEB at S122, S142, and S211 (Sardiello et al., 2009; Settembre et al., 2011; Vega-Rubin-de-Celis et al., 2017; Iacoangeli et al., 2019). Then, phosphorylated TFEB binds with 14-3-3 proteins and is sequestered in the cytosol. When amino acids are deficient, mTORC1 is inactivated. Thus, TFEB is dephosphorylated and activated, and it translocates into the nucleus to promote lysosome biogenesis. Based on a screen of small-molecule compounds, we found that HEP14 (a naturally occurring plant compound; see below) activated the PKCα/δ-GSK3β-TFEB axis to promote lysosome biogenesis independently of mTORC1 signaling (Li et al., 2016). In untreated cells, GSK3β phosphorylates TFEB at S134 and S138 on the lysosome membranes. After HEP14 treatment, activated PKCα/δ translocates onto lysosome membranes, where PKCα/δ inhibits GSK3β activity. Thus, inactivated GSK3β and dephosphorylated TFEB are released from lysosome membranes. Subsequently, the dephosphorylated TFEB translocates into the nucleus and promotes lysosome biogenesis. Recently, we also identified that inhibition of the dopamine transporter (DAT) promotes lysosome biogenesis (Yin et al., 2022). After treatment of cells with the small-molecule compound LH2-051, DAT translocates from the plasma membrane onto lysosome membranes, where DAT regulates the DAT-CDK9-TFEB axis and induces dephosphorylation of TFEB at S97, T99, S109, S144, S132, and T331. Then, dephosphorylated TFEB translocates into the nucleus and promotes lysosome biogenesis. Interestingly, CDK4/6 also phosphorylate TFEB at S142, which occurs in the nucleus and regulates TFEB export from the nucleus into the cytosol (Yin et al., 2020). Recent studies also demonstrate that TFEB phosphorylation is regulated by several kinases and phosphatases, including MAP4K3, PKCβ, Akt, c-Abl, ERK2, AMPK, PP2A and calcineurin (Settembre et al., 2011; Ferron et al., 2013; Palmieri et al., 2017; Hsu et al., 2018; Martina and Puertollano, 2018; Contreras et al., 2020; Paquette et al., 2021; Figure 1). Notably, phosphorylation of TFEB can increase or decrease its activity, especially phosphorylation at S467 in the C-terminus of TFEB.

Figure 1.

Post-translational modifications of TFEB.

The key sites of TFEB modifications are shown in the left panels, including phosphorylation, acetylation, ubiquitination, oxidation, and other modifications (SUMOylation, PARsylation, and glycosylation). The related effectors of TFEB modifications are shown in the right panels, including kinases, phosphatases, HDACs, KATs, and E3 ligases. Ac: Acetylation; AD: N-terminal transcriptional activation domain; bHLH: basic helix-loop-helix region; C: cysteine; E: glutamic acid; G: glycine; Gl: glycosylation; Gln rich: glutamine-rich domain; K: lysine; KATs: lysine acetyltransferases; NLS: nuclear localization signal; Ox: oxidation; P: phosphorylation; Pa: PARsylation; Pro rich: proline-rich domain; S: serine; Su: SUMOylation; T: threonine; TFEB: transcription factor EB; Ub: ubiquitination; V: valine; Zip: leucine zipper. Created with Adobe Illustrator CC 2018.

Acetylation

The mechanisms of TFEB acetylation have already been reported by several groups. Zhang et al. (2018) reported that treatment with SAHA, an inhibitor of histone deacetylases (HDACs), induces lysosome biogenesis via promoting TFEB acetylation at four sites, including K91, K103, K116, and K430. By screening small-molecule compounds, we found that trichostatin A (TSA), the pan-inhibitor of HDACs, also promotes TFEB activation and lysosome biogenesis (Li et al., 2022). Four acetylated lysine residues in TFEB, including K116, K236, K237, and K431, are important for TFEB nuclear translocation and lysosome biogenesis after TSA treatment. Notably, K236 and K237 localize in the NLS region of TFEB (Figure 1), suggesting that the acetylation at K236/K237 is crucial for unmasking the TFEB NLS region and interrupting the binding between TFEB and 14-3-3 proteins. We also found that TFEB acetylation is regulated by HDACs (HDAC5, HDAC6, and HDAC9) and lysine acetyltransferases, including ELP3, CREBBP, and HAT1 (Figure 1). Surprisingly, during TSA-induced TFEB activation, acetylation of TFEB is independent of TFEB dephosphorylation. On the other hand, acetyltransferase GCN5 promotes the acetylation of nuclear TFEB at K274 and K279, which subsequently disrupts nuclear TFEB dimerization and its transcriptional activity (Wang et al., 2020b).

Ubiquitination

A novel ubiquitination site on TFEB at K347 has been identified (Figure 1), which is evolutionarily conserved. After TSA treatment, a turnover between ubiquitination and acetylation occurs on K347: the ubiquitination on K347 is decreased, while the acetylation is increased. The reduced level of K347 ubiquitination allows TFEB to avoid proteasome-mediated degradation (Li et al., 2022). However, the E3 ligases which regulate K347 ubiquitination are still unknown. STUB1, an E3 ligase, has been reported to regulate TFEB protein levels and lysosome biogenesis. STUB1 can interact with phosphorylated TFEB (p-S142/p-S211), and dephosphorylation of TFEB (S142A and/or S211A) rescues the ubiquitination of TFEB and restores lysosome biogenesis (Sha et al., 2017). However, the STUB1-regulated ubiquitination sites of TFEB are still unknown. Further assays using mass spectrometry are needed to dissect the ubiquitination sites of phosphorylated TFEB (p-S142 or p-S211). STUB1 activates TFEB and its mutation is an effective method to explore the TFEB ubiquitination regulated by STUB1. However, one possibility needs to concerned: mutant STUB1 results in the accumulation of misfolded proteins, which also in turn promotes TFEB dephosphorylation and lysosome biogenesis for the autophagic clearance of these misfolded proteins. To exclude the above possibility, it will be important to mutate the STUB1-related ubiquitinated sites on TFEB after they have been found by mass spectrometry as mentioned above instead of STUB1 mutation. Functional analysis of the mutated TFEB will reveal the mechanisms of STUB1-related TFEB ubiquitination and lysosome biogenesis.

Oxidation and Other Modifications

TFEB contains a cysteine residue at site 212 (C212), which can be oxidized by reactive oxygen species. Cysteine oxidation promotes the formation of disulfide bonds and TFEB oligomers, which abolish the interaction between TFEB and Rag GTPases, thus inhibiting the phosphorylation of TFEB mediated by mTORC1 on lysosomal membranes (Wang et al., 2020a; Martina et al., 2021). Furthermore, TFEB can be alkylated at C212. Under normal conditions, mTORC1 phosphorylates TFEB at S211 and subsequently inactivates TFEB. After itaconate (an anti-inflammatory metabolite) treatment, TFEB is alkylated at C212, which attenuates the phosphorylation of TFEB at S211 by mTORC1. This in turn blocks the binding between 14-3-3 proteins and TFEB, which results in TFEB activation and enhanced lysosome biogenesis (Zhang et al., 2022b).

SUMOylation is a post-translational modification that adds a small ubiquitin-like-modifier (SUMO) to amino acid residues of target proteins. TFEB is SUMOylated at K316 by SUMO1 (Miller et al., 2005). There is no direct evidence showing whether SUMOylation affects the activity of TFEB. SUMOylation of MiTF does not affect MiTF localization, but it reduces the capacity of MiTF to bind to the promoter region of its target genes (Miller et al., 2005). Thus, an important future goal is to evaluate whether TFEB SUMOylation affects TFEB activity.

TFEB glycosylation occurs when the host is infected with Legionella pneumophila. SetA, a glucosyltransferase from Legionella pneumophila, glycosylates TFEB at S195 or S196, T201 or S203, and T208, which disrupts the binding between TFEB and 14-3-3 proteins. This molecular process promotes the nuclear translocation of TFEB (Beck et al., 2020). Meanwhile, SetA also directly glycosylates TFEB at S138 and blocks GSK3β-mediated phosphorylation of S138, which impairs TFEB nuclear export (Beck et al., 2020).

TFEB can also be modified by the addition of ADP-ribose polymers, which is called PARsylation. TNKS1 (Tankyrase 1) PARsylates TFEB at V65, G67 and E68 when the WNT/β-catenin signaling pathway is activated. The PARsylated TFEB translocates into the nucleus. However, the Wnt signaling-dependent PARsylation of TFEB does not induce the expression of lysosomal genes (Kim et al., 2021b).

LYsosome-Enhancing Compounds and Targeted Protein Degradation Technology

Toxic protein aggregates are the pathological characteristics of neurodegenerative diseases, and enhanced clearance of aggregated proteins is a direct therapeutic approach to increase the survival of neurons, restore the function of the central nervous system, and ameliorate neurodegenerative diseases. Protein quality control is dependent on two systems: the ubiquitin-proteasome system (UPS) and the autophagy-lysosome system. In the UPS system, misfolded proteins are targeted by ubiquitinating enzymes, then recognized and degraded by proteasomes. The UPS machinery is present in most mammalian cells, and the impairment of UPS has been reported during the pathogenesis of neurodegeneration. This suggests the possibility of treating neurodegenerative diseases through targeting UPS. A new technology named Proteolysis-Targeting Chimera (PROTAC) has been developed to enhance the specificity of the proteasomal pathway (Alabi and Crews, 2021). The PROTAC system consists of three parts: a ligand that binds the protein of interest (POI), a linker, and an E3 ligase-targeting ligand. Thus, the PROTAC molecule acts as a bridge between the POI and the E3 ligase, and the E3-PROTAC-POI complex increases the degradation of the POI by UPS.

Dysregulation of the autophagy-lysosome system has also been identified as a defining feature of neurodegenerative diseases, whereas enhancement of the autolysosome pathway is able to improve AD, PD, and HD. Based on knowledge of autophagosomes and lysosomes, new targeted protein degradation strategies have been developed to hijack the lysosomal degradation pathway. These include Autophagy-TArgeting Chimera (AUTAC), AuTophagosome-TEthering Compound (ATTEC), AUTOphagy-TArgeting Chimera (AUTOTAC), LYsosome-TArgeting Chimera (LYTAC), GlueTAC, and AbTAC (Ding et al., 2022) technologies. AUTAC incorporates the FBnG tag which mimics S-guanylation and binds to the POI; thus, the POI can be poly-ubiquitinated on K63. The POI is then recognized by SQSTM1/p62 and degraded by lysosomes (Takahashi et al., 2019). ATTEC directly combines POI and LC3; thus, the POI can be tethered by autophagosomes and subsequently degraded via the autophagy pathway. AUTOTAC is different from ATTEC: it combines SQSTM1/p62 and the POI, then the AUTOTAC system is recognized by LC3 rather than binding directly to LC3. Subsequently, the POI is tethered to the phagophores for degradation (Ji et al., 2022). The LYTAC system can degrade extracellular and membrane proteins via lysosomes. It is composed of an antibody or small molecule, which binds to receptors on the surface of the lysosome (e.g., cation-independent mannose-6-phosphate receptor, CI-M6PR) via a ligand (Banik et al., 2020). Notably, using the specific receptor on the surface of the lysosome can achieve tissue-specific targeting in human or mouse tissues. The POI will be degraded by lysosomes, and the receptor on the surface of the lysosome will be recycled to the plasma membrane for reuse. GlueTAC consists of three parts: a covalently-modified nanobody, a cell-penetrating peptide, and a lysosome-sorting sequence. The nanobody is responsible for recognizing and binding to the POI, and the cell-penetrating peptide promotes endocytosis of the POI-GlueTAC complex. The lysosome-sorting sequence then enhances lysosomal degradation (Zhang et al., 2021). AbTAC consists of a recombinant bispecific antibody. The recombinant bispecific antibody has two functions: one is targeting the POI and the other is targeting RNF43 (an E3 ligase). Subsequently, the POI is degraded in a lysosome-dependent manner (Cotton et al., 2021). The advantage of AbTAC is that it can target membrane proteins.

The above strategies are designed to enhance the recognition of cargos. During the pathogenesis of neurodegenerative diseases and aging, the functions of lysosomes are impaired, and the lysosomal degradative capacity is consequently reduced (Lee et al., 2022). Therefore, the enhancement of cargo degradation by lysosome biogenesis is a powerful strategy. As mentioned above, TFEB is one of the key regulators that promote lysosome biogenesis. Hence, small-molecule compounds that enhance TFEB activity and lysosome biogenesis are potential therapeutic agents for ameliorating AD (Table 1). In recent years, based on screening of small-molecule compounds, we identified a series of LYsosome-Enhancing Compounds (LYECs) that promote TFEB activation and lysosome biogenesis in cells stably expressing TFEB-EGFP. The previously published LYECs include HEP14, LH2-051, and TSA (Li et al., 2016, 2022; Yin et al., 2022). HEP14 (5β-O-angelate-20-deoxyingenol) is a natural compound isolated from Euphorbia peplus Linn. It promotes lysosome biogenesis via the PKC-GSK3β-TFEB pathway and the PKC-JNK/p38-ZKSCAN3 pathway (Li et al., 2016). Recently, we identified the LYEC LH2-051, which directly binds with and inhibits DAT. LH2-051 promotes TFEB activation and lysosome biogenesis via the DAT-CDK9-TFEB axis (Yin et al., 2022). Using APP-PSEN1 mice (an AD-like rodent model), we evaluated the effects of LYECs on cognitive and learning functions. Treatment with LYECs significantly decreased the accumulation of Aβ in APP-PSEN1 mice in a manner dependent on TFEB-mediated lysosome biogenesis. The performance of the AD mice was improved in the Morris water maze and Y maze tests (Li et al., 2016, 2022; Yin et al., 2022).

Table 1.

Small-molecule compounds enhance TFEB activity and lysosome biogenesis

Compounds	Structure	Mechanisms	Animal models of neurodegenerative diseases	Effects on neurodegenerative diseases	References
Catalpol		Activates AMPK and inhibits mTOR	APP/PS1-AD mice	Reduces Aβ load; alleviates cognitive impairments	Ren et al., 2019; Du et al., 2022
Curcumin		Inhibits mTORC1	5xFAD-AD mice; 3xTg-AD mice	Attenuates both APP and Tau pathology; improves cognitive deficits	Song et al., 2020
Compound E4		Inhibits the Akt-mTORC1 pathway	N.A.	N.A.	Wang et al., 2020c
HEP14		Directly binds to PKCα and PKCδ, thus activating PKCα and PKCδ	APP/PS1-AD mice	Reduces Aβ plaques; decreases Aβ40 and Aβ42	Li et al., 2016
Itaconate		Enhances the alkylation of TFEB at site C212	N.A.	N.A.	Zhang et al., 2022b
LH2-051		Directly binds to dopamine transporter (DAT) and inhibits DAT function	APP/PS1-AD mice	Reduces Aβ plaques; decreases Aβ40 and Aβ42; alleviates cognitive impairments	Yin et al., 2022
MSL		Directly binds to and stabilizes calcineurin A	N.A.	N.A.	Lim et al., 2018
Oblongifolin C		Inhibits mTORC1	N.A.	N.A.	Wu et al., 2019
Procyanidin B2		Potentially binds to TFEB	N.A.	N.A.	Su et al., 2018
Quercetin		Directly inhibits mTORC1	3xTg-AD mice; PD rats (rats were injected with 6-OHDA in striatum); HD rats (rats were injected with 3-NP)	Decreases Aβ and protects cognitive function; increases BDNF level and enhances cognitive effects; corrects movement disturbances and anxiety	Chakraborty et al., 2014; Huang et al., 2018; Paula et al., 2019; Naghizadeh et al., 2021
Rapamycin		Directly binds to mTOR and inhibits mTORC1	3xTg-AD mice; PD mice (C57BL/6 mice were intraperitoneally injected with MPTP)	Reduces Aβ plaques and Tau pathology; protects substantia nigral neurons	Malagelada et al., 2010; Majumder et al., 2011; Battaglioni et al., 2022
SAHA		Enhances the acetylation of TFEB at sites K91, K103, K116, and K430	Mice with insulin resistance-induced cognitive deficit; R6/2-HD mice	Improves cognitive functions; reduces SDS-insoluble aggregate load	Mielcarek et al., 2011; Sharma and Taliyan, 2016; Zhang et al., 2018
Siramesine		Inhibits mTORC1	N.A.	N.A.	Zhitomirsky et al., 2018
Sulforaphane		Increases intracellular ROS, then stimulates Ca2+ release	PS1V97L-AD mice	Inhibits the generation of Aβ; alleviates the cognitive deficits	Hou et al., 2018; Li et al., 2021
Sunitinib		Inhibits mTORC1	AD2576APPSwe-AD mice; 3xTg-AD mice	Reduces Aβ; improves cognitive function	Grammas et al., 2014; Zhitomirsky et al., 2018
Torin-1		Effectively blocks phosphorylation of mTOR (both mTORC1 and mTORC2)	PD mice (C57BL/6J female mice were injected with 6-OHDA in striatum)	Alleviates loss of dopaminergic neurons	Thoreen et al., 2009; Zhuang et al., 2020
Trehalose		Increases transient lysosomal membrane permeability, and releases Ca2+ from the lysosomes to activate the phosphatase PPP3/calcineurin	APP23-AD mice; R6/2-HD mice	Reduces Aβ generation and plaque formation; decreases polyglutamine aggregates improves motor function	Tanaka et al., 2004; Rusmini et al., 2019; Liu et al., 2020
Trichostatin A (TSA)		Enhances the acetylation of TFEB at sites K116, K236, K237, and K431, and decreases TFEB ubiquitination at site K347	APP/PS1-AD mice	Reduces Aβ plaques; decreases Aβ40 and Aβ42; improves learning, memory, and cognitive functions	Li et al., 2022
Tripterine		Inhibits mTORC1, thus inducing TFEB dephosphorylation at sites S142 and S211	P301S Tau-AD mice; 3xTg-AD mice	Attenuates Tau pathology and cognitive deficits	Yang et al., 2022

6-OHDA: 6-Hydroxydopamine; AD: Alzheimer’s disease; AMPK: AMP-activated protein kinase; APP: amyloid precursor protein; Aβ: amyloid beta; BDNF: brain-derived neurotrophic factor; HD: Huntington’s disease; MPTP: 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine; mTOR: mammalian target of rapamycin; N.A.: not available; PD: Parkinson’s disease; PKC: protein kinase C; ROS: reactive oxygen species; SDS: sodium dodecyl sulfate; TFEB: transcription factor EB.

LYECs mainly enhance lysosome-dependent cargo degradation, while ATTEC, AUTAC, LYTAC, AUTOTAC, GlueTAC, and AbTAC technologies are used to enhance cargo recognition. In the future, if LYECs are combined with ATTEC, AUTAC, LYTAC, AUTOTAC, GlueTAC, or AbTAC strategies, they could have synergetic effects for the enhancement of both cargo recognition and cargo degradation by autolysosome-dependent systems. Drug cocktail therapy targeting protein aggregates will be a potential approach to enhance the degradation efficiency of autolysosomes and ameliorate neurodegenerative diseases (Figure 2).

Figure 2.

A schematic illustration showing how a combination of LYECs with ATTEC, AUTAC, LYTAC, AUTOTAC, GlueTAC, or AbTAC can be used as a potential therapeutic approach to ameliorate neurodegenerative diseases.

AbTAC: Antibody-based PROTAC; ATTEC: AuTophagosome-TEthering Compound; AUTAC: Autophagy-TArgeting Chimera; AUTOTAC: AUTOphagy-TArgeting Chimera; CPP: cell-penetrating peptide; GlueTAC: nanobody-based PROTAC; LSS: lysosome-sorting sequence; LYECs: LYsosome-Enhancing Compounds; LYTAC: LYsosome-TArgeting Chimera; M6P: mannose 6-phosphate; POI: protein of interest. Created with Adobe Illustrator CC 2018.

Additional file: Open peer review report 1 ^{(73.6KB, pdf)} .