The regulatory mechanism and therapeutic potential of transcription factor EB in neurodegenerative diseases

Abstract The autophagy‐lysosomal pathway (ALP) is involved in the degradation of protein aggregates and damaged organelles. Transcription factor EB (TFEB), a major regulator of ALP, has emerged as a leading factor in addressing neurodegenerative disease pathology, including Alzheimer's disease (AD), Parkinson's disease (PD), PolyQ diseases, and Amyotrophic lateral sclerosis (ALS). In this review, we delineate the regulation of TFEB expression and its functions in ALP. Dysfunctions of TFEB and its role in the pathogenesis of several neurodegenerative diseases are reviewed. We summarize the protective effects and molecular mechanisms of some TFEB‐targeted agonists in neurodegenerative diseases. We also offer our perspective on analyzing the pros and cons of these agonists in the treatment of neurodegenerative diseases from the perspective of drug development. More studies on the regulatory mechanisms of TFEB in other biological processes will aid our understanding of the application of TFEB‐targeted therapy in neurodegeneration.

located on chromosome 6p21.1 is composed of nine exons, with a postulated initiation ATG preceded by a perfect ribosomal binding sequence in exon 2. 20 This organization generates an mRNA transcript characterized by two non-coding and eight coding exons. Seven alternative TFEB transcripts containing distinct alternative 5′ exons, which contain the translational start site at exon 2, have been described with differential expression and different tissue distributions (Figure 2A). 20 TFEB protein has three essential regions similar to other Mif proteins, including DNA-binding regions, a helix-loop-helix (HLH) and leucine-zipper (Zip) regions. 21 The DNA-binding region is characterized by a HLH and a Zip domain flanked by an upstream basic region that is able to recognize an E-box sequence (CAYGTG) in the promoter of targeted genes. 22,23 In addition, the N-terminus of the TFEB protein contains a glutamine-rich domain encompassing the binding site of Rag C, while its C-terminus contains a proline-rich domain, the function of which has not been reported ( Figure 2B).
Lysosomes are key organelles of the cellular degradation and recycling processes, and they are required to maintain cellular homeostasis. 24 Lysosomes are involved in multiple essential cellular processes, including endocytosis, autophagy, and lysosomal exocytosis. 25 However, the regulation of lysosome biogenesis and function by cells has remained unanswered for a long time. In 2009, Sardiello, et al. first discovered that lysosomal biogenesis is transcriptionally regulated by a gene network called the coordinated lysosomal expression and regulation (CLEAR) element, a common 10-base E-box-like palindromic sequence, which is regulated by TFEB. 26 Furthermore, the authors also found that overexpression of TFEB upregulates the expression of genes related to lysosomal enzymes, lysosomal biosynthesis and functions. 26 TFEB-mediated activation of the CLEAR network regulates the lysosomal proteostasis by enhancing folding, trafficking and lysosomal activity of a severely destabilized glucocerebrosidase variant (L444P). 27 Subsequent work has shown that TFEB orchestrates the expression of genes involved in processes such as lysosomal biogenesis, lysosomal exocytosis, endocytosis, membrane repair, and autophagy by direct binding to the CLEAR motif at their promoters. 28 TFEB was identified as a main player in regulating autophagosome biogenesis and autophagosome-lysosome fusion by binding to the promoter regions of numerous autophagy genes. 11 In addition, TFEB enhances the degradation of long-lived proteins, the clearance of lipid droplets, and damaged mitochondria, 11,29,30 suggesting that it also plays an important role in modulating the biological process of lipophagy and mitophagy. In addition, TFEB has also been found to regulate lysosomal exocytosis by activating the lysosomal Ca2 + channel mucolipin 1 (MCOLN1). This promotes cellular clearance and increases the pool of lysosomes in the proximity of the plasma membrane (PM) to stimulate their fusion to PM. 31 In a number of disease conditions, the role of TFEB in the control of lysosomal biosynthesis, autophagy, and lysosomal exocytosis was primarily exploited to enhance cellular clearance. 32 This TFEBmediated effect has been widely demonstrated in several cellular and mouse models of human diseases which are characterized by an accumulation of undegraded substances(e.g. AD, [33][34][35] PD, [36][37][38][39]41 ) as well as lysosomal storage diseases (LSD), 27,31,42 among others.

| Transcriptional regulation of TFEB
TFEB expression is transcriptionally regulated by different factors (Table 1). Cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB), a key transcriptional activator that F I G U R E 1 Conservation analysis of TFEB gene. Graphical views showing multi-species comparisons of TFEB using UCSC genome browser. The conservation scores are indicated by the blue and red peaks. drives fasting responses, has been identified as a transcriptional activator of TFEB. In addition, CREB upregulates the expression of TFEB and other autophagy genes by recruiting its coactivator, the CREB regulated transcription coactivator 2 (CRTC2), in the liver of fasted mice. 43 TFEB was also identified as the downstream transcriptional target of a master activator of mitochondrial proliferation. The peroxisome proliferator-activated receptor γ (PPARγ) coactivator 1α (PGC-1α) binds to one of the promoters of TFEB, which is essential for TFEB-mediated mitophagy. 40,44 In turn, PGC-1α is a direct target of TFEB, which controls the starvation response by orchestrating PGC-1α-PPARα-mediated lipid catabolism. 30 Moreover, activated PPARα forms a complex with retinoid X receptor α (RXRα) and PGC-1α, which is recruited to the TFEB promoter and initiates transcriptional activation of TFEB in brain cells. 45 Scavenger receptor class B type I (SR-BI) is an integral membrane glycoprotein, which regulates autophagy, lysosome function, efferocytosis, cell survival, and inflammation. 46 Furthermore, SR-BI regulates both basal and inducible expression levels of TFEB by enhancing PPARα activation. 47 The positive feedback of TFEB on its own expression is also significantly enhanced during starvation. 30 Transcription factor cellular ets-2 gene (C-ETS2) has been identified as another transcriptional activator of TFEB, which mediates TFEB expression and further mediates upregulation of lysosomal genes under oxidative stress. 48 Forkhead box O1 (FoxO1), a transcription factor that controls mitochondrial function and morphology, interacts with Tfeb by directly binding its promoter, thereby regulating autophagy and mitochondrial uncoupling proteins (UCPs) expression in adipocytes. 49 Studies involving ChIP assays have revealed that signal transducer and activator of transcription 1 (STAT1), a downstream mediator of the nonreceptor kinase Janus kinase 2 (JAK2) signaling pathway, binds to the Tfeb promoter, which in turn regulates the TFEB promoter activity, expression, and nuclear localization. 50 Krüppel-like factor 2 (KLF2), a shear stress-responsive factor, upregulates Tfeb expression and promoter activity to exert antiinflammatory effects in endothelial cells. 51 Defective autophagy and endoplasmic reticulum (ER) stress contribute to a variety of diseases. Spliced X-box binding protein 1 (sXBP1), the key transcription factor that promotes adaptive unfolded protein response, occupies the −743 to −523 site of the Tfeb promoter and enhances TFEB transcription and autophagy in the context of obesity. 52 Interestingly, a novel splicing variant of TFEB has recently been cloned, which comprises of 281 amino acids and lacks the HLH and Zip motifs present in the full-length TFEB. One study found that the splicing variant of TFEB may act as a negative regulator of TFEB, and fine tune ALP activity during cellular stress. 53 SMAD family member 3 (SMAD3) directly binds to the 3′-UTR of Tfeb and inhibits its transcription, which triggers lysosome depletion in tubular epithelial cells in diabetic nephropathy. 54 Finally, it has been reported that histone deacetylase 2 (HDAC2) enhance c-MYC binding to Tfeb promoters to repress its expression. 55 In addition to the regulation of promoter activity, recent studies have found that noncoding RNAs (ncRNAs) are involved F I G U R E 2 Structure of human TFEB gene and protein. (A) Schematic of human TFEB gene. TFEB gene is composed of nine exons, and ATG start codon and TAG stop codon are located on exon 2 and exon 9, respectively. (B) Domain structure of human TFEB. The N-terminus of TFEB contains a glutamine-rich domain, and its C-terminus contains a proline-rich domain. The DNA-binding region is composed of a HLH and a leucine Zip domain. Relevant TFEB phosphorylation and acetylation sites and their regulatory role are indicated in different regions.
in regulating TFEB transcription. A recent study showed that microRNA (miRNA)-342-3p directly targets TFEB by inhibiting H 2 O 2 -induced autophagy, which contributes to exosomemediated heart repair. 56 Different studies have predicted and validated TFEB as a target miR-128-mediated regulation of autophagy-related gene transcription. 57 In addition, some TFEB co-activators, such as co-activator-associated arginine methyltransferase 1 (CARM1) 58 and Yes-associated protein (YAP), 59 can also increase autophagy and lysosomal function by enhancing TFEB transcriptional activity.

| Post-transcriptional regulation of TFEB
Under normal conditions, TFEB accumulates in the cytoplasm and is inactive. However, following conditions of stress, such as starvation or lysosomal dysfunction, TFEB rapidly translocates to the nucleus where it promotes transcription of its multiple target genes. 11,26 The phosphorylation and dephosphorylation of TFEB and its cytoplasmic-nucleus shuttling are regulated by multiple pathways ( Figures 2B and 3). TFEB activity and nuclear translocation are mainly controlled by its phosphorylation status. 11 and Ser211 respectively, resulting in cytoplasmic retention. 63,64 Mutations of either Ser142 or Ser211 into alanine (S142A, S211A, respectively) promote nuclear accumulation, similar to cells treated with the mTORC1 inhibitor Torin 1. Phosphorylation of Ser211 of TFEB promotes 14-3-3 proteins binding with its nuclear localization signal (NLS), thereby sequestering it in the cytoplasm. 63,64 In vitro kinase assays have shown that Ser122 is directly phosphorylated by mTORC1. 65 However, the S122A mutation does not affect TFEB nuclear localization by itself, but enhances the effects of the S211A mutation. Conversely, a S122D mutation is sufficient to block the effects of the S211A mutation on TFEB nuclear translocation.

Regulatory factors Treatment
Dephosphorylation at Ser122 is thought to be necessary for TFEB nuclear localization following mTORC1 inhibition. 65 The mechanisms by which Ser142 and Ser122 phosphorylation affect TFEB subcellular localization are still unclear.
In addition to mTORC1, other kinases are known to phosphorylate TFEB and modulate its localization and activity. Phosphorylation of TFEB Ser142 by the extracellular signal-regulated kinase 2 (ERK2) inhibits its nuclear localization and activity. Moreover, treatment with ERK inhibitors results in TFEB nuclear translocation. 11 However, the relationship between mTORC1-mediated and ERK2-mediated phosphorylation of TFEB Ser142 is still unclear. In osteoclasts, protein kinase C β (PKCβ) phosphorylates multiple serine residues (i.e., Ser461, Ser462, Ser466, and Ser468) located in the last 15 amino acids of TFEB, which is important for TFEB protein stability and activation but does not affect its subcellular localization. 66 TFEB can be phosphorylated by glycogen synthase kinase (GSK) 3β at Ser134 and Ser138 residues leading to its cytoplasmic retention. In contrast, GSK3β is inactivated by activated PKC and results in reduced phosphorylation and nuclear translocation of TFEB. 67 One hypothesis is that the effects of GSK3β on TFEB may not be truly independent from mTORC1. Interestingly, TFEB Ser138 phosphorylation by GSK3β is primed by its Ser142 phosphorylation, and phosphorylation of both sites but not either alone, activates a previously unrecognized nuclear export signal (NES) of TFEB. Furthermore, during glucose limitation, GSK3β is inactivated by AKT in response to mTORC2 signaling and inhibits TFEB nuclear export. 68 A recent study showed that TFEB is phosphorylated by AKT at residue Ser467 contributing to its cytosolic retention. TFEB S467A mutant shows an increased nuclear localization in normally fed conditions. 69 When amino acids are plentiful, TFEB is phosphorylated by mitogen-activated protein kinase 3 (MAP4K3) at residue Ser3. This phosphorylation modification is required for TFEB interaction with the mTORC1-RagGTPase-Ragulator complex and TFEB cytoplasmic retention, which also F I G U R E 3 Post-transcriptional regulation of TFEB. TFEB activity and nuclear translocation are regulated by its post-transcriptional modification status. (A) Upon inactivation (under normal conditions), TFEB is phosphorylated by multiple kinases, and phosphorylated TFEB interacts with 14-3-3 proteins, remaining sequestered in the cytosol. Moreover, TFEB is acetylated by the histone acetyltransferase in nucleus, suppressing its transcriptional activity. (B) Upon activation (under conditions of lysosomal stress or starvation or mTOR inhibition), TFEB is dephosphorylated, unmasking its nuclear localization signal and driving transcription of itself and other CLEAR network of target genes. In addition, TFEB is phosphorylated by multiple kinases increasing its protein stability and activation. TFEB is deacetylated by the histone deacetylase, enhancing transcriptional levels of TFEB.
precedes for mTORC1 phosphorylation at Ser211 of TFEB. 70 Another recent study showed that cyclin-dependent kinases4/6 (CDK4/6) interacts with and phosphorylates TFEB at residues Ser142 in the nucleus in Torin-1-treated cells, thereby inactivating them by promoting their shuttling to the cytoplasm. 71  in an mTORC1-independent manner. 80 Moreover, a recent study also found that TFEB activation is dependent on PP2A rather than calcineurin/PPP3 Ser211 dephosphorylation during inhibition of phos- Notably, growing evidence showed that the curcumin analogue, C1, a novel specific TFEB activator, promotes TFEB nuclear translocation in a phosphorylation-independent manner. 35,86,87 C1 directly binds to and activates TFEB, which promotes TFEB nuclear translocation by altering its structural conformation to expose nuclear localization signals. 86 Interestingly, together with our collaborators, we have revealed that proteasome impairment facilitates TFEB dephosphorylation and nuclear translocation, which significantly increases the expression of a number of downstream TFEB target genes. 88 In addition to phosphorylation and dephosphorylation modifica- This activates TFEB transcriptional activity and promotes its nuclear translocation. 90 In contrast, inhibition of the cytosolic deacetylase, HDAC6, enhances TFEB acetylation, which in turn increases TFEB nuclear localization in experimental kidney disease, but the deacetylation site on TFEB and the exact mechanisms behind this are still unclear. 91  Ubiquitination also plays an important role in TFEB activation.
One study showed that phosphorylation of Ser142 and Ser211 mediates TFEB-targeted degradation by the ubiquitin-proteasome system via binding to the E3 ubiquitin ligase, STIP1 homology and U-Box containing protein 1 (STUB1). This suggests that the activity and stability of TFEB in cells may be jointly regulated by phosphorylation and ubiquitination. 96 SUMOylation does not promote protein degradation like ubiquitination, but rather strengthens protein stability and affects protein transcription activity. It has been reported that TFEB contains the sumoylation consensus sequence ΨKXE, with SUMOylation of TFEB at a lysine site leading to its decreased transcriptional activity in vivo. 97

| IMPAIRED TFEB S I G NALING A S A CONTRIBUTOR TO THE PATHOG ENE S IS OF NEURODEG ENER ATIVE DIS E A S E S
Intracellular accumulation of aberrant protein inclusions is the primary molecular pathogenic event of neurodegenerative diseases.
Some protein aggregates associated with neurodegenerative diseases reportedly interact with TFEB. Indeed, studies of postmortem tissues from patients and animal models of neurodegenerative diseases have suggested that impaired TFEB signaling, including abnormal TFEB expression, changes in TFEB subcellular localization, and that abnormal expression of TFEB-targeting ALP genes may contribute to the pathogenesis of neurodegenerative diseases ( Figure 4).

| Alzheimer's disease
AD is the most common neurodegenerative diseases in the elderly and is clinically characterized by progressive cognitive impairment, often accompanied with psychobehavioural disturbances and language impairment in later stages. Extracellular amyloid beta (Aβ) plaques, intracellular neurofibrillary tangles (NFTs), and extensive neuronal degeneration are the three major pathological hallmarks of AD. 98 The production of Aβ, which is considered a crucial process in AD pathogenesis, is the proteolysis product of amyloidβ precursor protein (APP), which is overexpressed in AD. 99 Aβ is generated through a two-step processing of APP: cleavage of APP by β-site APP cleaving enzyme 1 (BACE1) to generate a β-C-terminal fragment (β-CTF), which upon cleavage by γ-secretase produces Aβ. In addition, cleavage of APP by α-secretases within the Aβ domain prevents Aβ production. 100,101 Moreover, hyperphosphorylated Tau protein is the main component of NFTs. 102 Multiple lines of evidence support that the ALP contributes to the removal of abnormally aggregated Aβ and tau proteins. [103][104][105] As a major regulator for ALP, TFEB plays an important role in the pathogenesis of AD. However, the role of TFEB currently showed in AD pathogenesis is controversial. An analysis in monocytes and lymphocytes from AD patients revealed a significant decrease in the expression of TFEB and its target lysosomal genes, suggesting its possible role in lysosomal deficits in AD. 57 A separate study later confirmed that the nuclear TFEB was striking reduced in brain tissue samples from AD patients. Furthermore, its reduction is positively correlated with AD pathology. 106 Similarly, TFEB nuclear transport was significantly impeded in an in vitro model of double presenilin knockout cells, indicating that TFEB cytosolic retention may contribute to AD pathogenesis. 107 Another study showed that Tfeb mRNA expression in microdissected CA1 neurons were unchanged in AD, whereas expression levels were significantly increased in AD hippocampal tissue. Notably, the study also demonstrated that TFEB nuclear translocation significantly increased in glial cells of AD hippocampal tissue, indicating an important role for TFEB in the glia of CA1 hippocampus. 108 An in vitro study using primary microglia showed that Aβ 1-42 induced reduction of the nuclear TFEB in a dose-dependent manner and significantly inhibited the expression of osteoporosis-associated transmembrane protein 1 (OSTM1), a vital molecule involved in lysosome acidification, resulting in lysosomal dysfunction. 109 In contrast, there was a significant increase in TFEB mRNA in AD patient-derived fibroblasts carrying the familial presenilin-1 (PS1) A246E mutation. 110 Another study of presenilin-1 conditional knock-out mice further revealed significant upregulation of a subset of CLEAR network genes related to lysosomal biogenesis, although failing to reveal a statistically significant difference in Tfeb mRNA expression. 111 Similarly, upregulation of TFEB target genes was also reported in 5xFAD mice, which contains five familial mutations related to AD. 112 In addition, computational modeling and DNA pull-down binding assays showed that APOE ɛ4, a key genetic risk factor for sporadic AD, may compete with TFEB for binding to CLEAR elements in the promoters of important genes involved in the ALP, including SQSTM1, MAP1LC3B, and LAMP2, thereby suggesting inhibition of the CLEAR network in APOE ɛ4/ɛ4 AD patients. 113 In agreement with this hypothesis, neuron-specific knockout of TFEB in the hippocampus of 2-month-old mice significantly increased the accumulation of total Aβ and paired helical filament (PHF) pTau in the brain, further suggesting that TFEB plays a key role in AD neuropathology. 107 Taken together, these studies clearly demonstrate the role of abnormal TFEB expression and activity, and subsequently impaired ALP, in the pathogenesis of AD.
However, upstream events affecting TFEB expression and nuclear translocation in AD remain unclear. in ALS. 135 Stage-dependent alterations in TFEB expression has been reported in SOD1 G39A transgenic mice. 136 In this study, TFEB was upregulated in the early stage of disease, but then reduced in the spinal cord at the middle and end stages of the disease. In vitro TFEB overexpression has been shown to increase cell survival and proliferation by inducing autophagy, although the effect of autophagy on SOD1-aggregation clearance has not been studied. 136 Furthermore, studies suggest that nuclear TFEB levels were reduced in ALS patient brain samples, suggesting decreased TFEB activity in ALS. 106 TFEB expression and nuclear translocation have been shown to increase in a C9orf72 knock-out mouse model. 137 A recent study in C. elegans found that deletion of C9orf72/ ALFA-1 leads to nuclear translocation of HLH-30/TFEB, subsequently leading to activation of lipolysis and premature lethality.

| TFEB A S A THER APEUTI C TARG E T FOR NEURODEG ENER ATIVE DIS E A S E S
Increasing evidence has shown that the accumulation of protein aggregates and ALP dysfunction are the major pathogenic mechanisms for neurodegenerative diseases. 141 As a master regulator of ALP pathways, TFEB has become an attractive target for alleviating ALP dysfunction in neurodegenerative diseases.

| Therapeutic effects of TFEB overexpression in neurodegenerative diseases
The beneficial effects mediated by TFEB have been demonstrated in multiple mouse and cell models of AD addressing Aβ and tau pathology. 33,142,143 With respect to Aβ, TFEB overexpression rescues the autophagic flux, accelerates Aβ 1-42 degradation by regulating the autophagy-lysosome pathway, and alleviates Aβ 1-42 -induced toxicity by reducing oxidative stress. 144 Similarly, intracranial stereotaxic injection of AAV-TFEB in hippocampal neurons of APP/PS1 mice leads to a reduction of APP, Aβ production, and amyloid plaque load by accelerating flux of the endosome-lysosome pathway. 142 Interestingly, nonneuronal cells in AD may also benefit from TFEB induction. In microglia, deacetylation of TFEB by SIRT1 stimulates fibrillar Aβ (fAβ) degradation by facilitating lysosomal biogenesis and reduces amyloid plaques in the brains of APP/PS1 mice. 92 In APP transgenic mice specifically overexpressing TFEB in hippocampal astrocytes, TFEB mainly localizes in the nuclei of astrocytes and enhances lysosome function, resulting in reduced Aβ levels and amyloid plaque load. These data suggest that TFEB can also facilitate Aβ clearance in astrocytes. 34 A recent study indicates that exogenous TFEB in both cells and the 3xTgAD mouse model strongly reduces C99 load by increasing the expression of cathepsins, key proteases involved in C99 degradation. This suggests that TFEB activation is an important strategy for preventing the accumulation of the early neurotoxic catabolite of AD. 143 A disintegrin and metalloproteinase 10 (ADAM10) has recently emerged as the major α-secretase responsible for APP processing, 145 which plays a crucial role in axon guidance and spine density regulation. 146,147 There is now definitive evidence that reduction of ADAM10 activity and impaired trafficking to the synapse of ADAM10 can cause AD, thereby suggesting that inadequate ADAM10 activity is likely the cause of AD. 148,149 A recent study showed that TFEB overexpression increased the expression of mature ADAM10 and its enzyme activity in both the cortex and hippocampus of flag-TFEB mice, resulting in increased generation of soluble-APPα (sAPPα). Furthermore, the TFEB-induced increase in mature ADAM10 protein levels is mediated via PPARα. 150 For tau pathology, TFEB overexpression in the brain of a rTg4510 mouse model of tauopathy dramatically reduces tau pathology, synaptic deficits, neurodegeneration and animal behavioral deficits. The proposed mechanism of this effect may be that TFEB induces ALP through upregulation of phosphatase and tensin homolog (PTEN), which is a lipid phosphatase that antagonizes PI3K-Akt-mTOR signaling. 33 In agreement with this conclusion, TFEB overexpression enhances uptake of extracellular tau protein, promotes lysosomal activity in primary astrocytes, reduces the pathology induced by hyperphosphorylated and misfolded tau protein, and significantly attenuates tau spreading in astrocytes in PS19 tauopathy mice. 151 Similarly, neuron-specific TFEB overexpression significantly reduces the expression of toxic p-tau and the number of lipofuscin puncta in the cortex and hippocampus of P301S tauopathy mice, and attenuates the learning and memory deficits in mice. 152 In a recent study, TFEB was found to regulate the secretion of truncated mutant tau lacking a microtubule-binding repeat (MTBR) by promoting lysosomal exocytosis. This study also showed an that this process is dependent on the lysosomal calcium channel, TRPML1. 153  Notably, TFEB overexpression has no effect on the expression and activity of mTORC2. Collectively, these findings suggest that TFEB may be a very promising target to counteract neurodegeneration by improving autophagy dysfunction and other biological processes in PD.
Studies from different groups have reported that overexpression of TFEB can reduce mHtt in both cellular and animal models of HD. 26,40,158

| Therapeutic effects of TFEB-targeted agonists in neurodegenerative diseases
A growing body of evidence has shown that some small molecules and natural products derived from traditional Chinese medicine (TCM) have therapeutic promise for neurodegenerative diseases.
These have been reported to activate TFEB through multiple mechanisms, including direct TFEB activation, mTORC1 inhibition, AKT inhibition, as well as Ca 2+ -dependence, among others. The TFEBactivating targets/pathways and the effects of these TCM-derived natural compounds and small molecules in neurodegenerative diseases models are summarized in Table 2.

Curcumin and its analogs
Curcumin, a natural polyphenol extracted from turmeric (Curcuma longa L.), has been reported to possess multiple pharmacological properties. 161 Curcumin has been shown to enhance autophagy by suppressing the PI3K-Akt-mTOR pathway and activating the ERK1/2 pathway. [162][163][164] Recently, a study found that curcumin promotes TFEB nuclear translocation by inhibiting GSK-3β activity and degrades aggregated APP and α-synuclein via the TFEB-autophagy/ lysosomal pathway in SH-SY5Y cells. 165 Due to its low bioavailability, several curcumin derivatives have been chemically synthesized to enhance its efficacy. A synthesized curcumin monocarbonyl derivative called C1(1,5-bis [2-methoxyphenyl]penta-1,4-dien-3-one) has been identified as a novel mTOR-independent activator of TFEB. 86 Compound C1 directly binds to the N terminus of TFEB and promotes it entry into the nucleus, without affecting TFEB phosphorylation or inhibiting upstreaming regulators of TFEB, including mTORC1 and MAPK1. 86 Compound C1 enhances autophagy and lysosomal activity by activating TFEB, and reducing APP, APP C-terminal fragments (CTFβ/α), β-amyloid peptides and tau aggregates in three AD animal models that represent β-APP pathology (5xFAD mice), tauopathy (P301S mice), and the APP/Tau combined pathology (3xTg-AD mice). In addition, compound C1 improves the motor and cognitive function in animal models of AD. 35 The protective effects of compound C1 have also been examined in PD cellular and animal models. In 6-hydroxydopamine (6-OHDA)/ascorbic acid (AA)-induced models of PD, compound C1 has been shown to enhance TFEB nuclear translocation and autophagy to exert neuroprotective effects.
Compound C1 significantly reduces oxidative stress-induced dopaminergic cell death and improves motor impairment. Moreover, these effects are prevented by silencing of TFEB. 38 Recently, the curcumin analog C1-based nanoscavenger (NanoCA), a self-assembled product from curcumin analog C1 molecules and polyethylene glycol, activates TFEB nuclear translocation in an mTOR-independent manner, and eventually promotes the autophagic degradation of αsynuclein. Furthermore, the brain-targeted NanoCA also promotes clearance of α-synuclein aggregates and improves the behavioral deficits of MPTP-intoxicated PD animal model, suggesting a promising approach for PD intervention. 166 Curcumin analog E4, another curcumin derivative, potently activates TFEB via AKT-mTORC1 inhibition. Compound E4 enhances autophagy flux and lysosomal biogenesis, promotes α-synuclein degradation, and protects against MPP + -induced cytotoxicity in neuronal cells. 167 Trehalose Trehalose, a disaccharide homologous to sucrose, has recently been shown to have potential utility in neuroprotection by reducing the aggregation of misfolded proteins and promoting the clearance of abnormal protein aggregates by autophagy induction. 168,169 Trehalose enhances the degradation of polyQ-AR (which is associated with SBMA), TDP-43, and SOD1 (which are associated with ALS) via the TFEB pathway in an mTOR-independent manner, while silencing TFEB disturbs the pro-degradation activity of trehalose. 170,171 Similarly, trehalose is able to activate TFEB, as indicated by its nuclear translocation upon treatment, and attenuates MPP + -induced cell death. 37 In addition, trehalose also activates TFEB in an indirect manner. Trehalose reportedly induces TFEB nuclear translocation and upregulate TFEB target genes, in a manner that is mediated by PPP3CB downregulation. 170  Pseudoginsenoside F11 Promote TFEB nuclear translocation via mTOR inhibition AD Primary rat microglial cells treat with oligomeric Aβ Increase the degradation of oligomeric Aβ in microglia [190] Fisetin Promote TFEB nuclear translocation via mTOR inhibition

AD T4 cells treated with doxycycline
Promote degradation of phosphorylated tau [192] Paeoniflorin Increase the transactivation of TFEB, increase TFEB expression via the upregulation of NF-YA, and promote TFEB nuclear translocation SBMA NSC34 cells overexpressing mutant (97Q) AR, AR-97Q mice Enhance both the UPS and autophagy systems, mitigate the behavioral and pathological impairments [193] Compound

Mechanism of TFEB activation
Disease Cell/animal models

References
Chlorogenic acid Promote TFEB nuclear translocation via mTOR inhibition AD Aβ25-35-exposed SH-SY5Y cells, APP/PS1 mice Alleviate neuron damage and cognitive impairment [194] Qingyangshen Increase expression of TFEB and PPARα AD HT-22 cells overexpressing APP and Tau, 3xTg AD mice Reduce expression of APP and phospho-Tau, improve learning and spatial memory behavior [198] Celastrol Induce dephosphorylation of TFEB (S142 and S211) via mTORC1 inhibition AD P301S Tau and 3xTg mice Reduce phosphorylated Tau aggregates, attenuate cognitive deficits, enhance autophagy and lysosomal biogenesis [199] Small molecule inhibitor and clinical drugs Rapamycin and its analogs CCI-779 Induce dephosphorylation of TFEB via mTORC1 inhibition; restore PGC1α- Reduce 6-OHDA-and PQ-induced dopaminergic cell death [210] Dynasore Repress the lysosomal localization of mTOR and block the activity of mTORC1, which in turn enhance the nuclear translocation of TFEB

HD HEK 293 cells overexpressing Nhtt60Q
Promote the clearance of protein aggregates formed by mutant polyQ-Htt protein [211] Aspirin Induce the activation of PPARα and stimulated the transcription of Tfeb via PPARα AD 5xFAD mice, primary mouse astroglia Decrease amyloid plaque pathology [212,213] Ibudilast Enhance TFEB nuclear translocation by inhibiting the mTORC1 activity ALS HEK 293 overexpressing TDP-43 or SOD1 G93A , NSC-34 cells Enhance the clearance of TDP-43 and SOD1 protein aggregates [214] TA B L E 2 (Continued) (Continues) multisystemic phenotype associated with neurodegeneration. 173 Mucopolysaccharidosis is an LSD characterized by altered metabolism of glycosaminoglycans (GAGs). 174 Genistein, a natural isoflavone, has been shown to reduce lysosomal GAGs storage. 175 Genistein has also been shown to facilitate the nuclear translocation and target gene expression of TFEB, and promote lysosomal synthesis. This suggests that genistein not only involved in the synthesis and degradation of GAGs but also enhances lysosomal function via TFEB. 176 Niemann-Pick disease Type C (NPC) is a rare neurodegenerative disease caused by mutations in NPC1 or NPC2 gene which results in an accumulation of cholesterol in lysosomes. Additionally, NPC is also considered to be an autosomal recessive LSD. 177 Loss of function in NPC1 or NPC2 proteins can causes lysosomal dysfunction and autophagy defects. 178 A recent study has shown that genistein promotes TFEB translocation to the nucleus and induces autophagy and lysosomal exocytosis, leading to a reduction of cholesterol accumulation in NPC1 patient fibroblasts. 179 Therefore, genistein may promote the development of novel therapeutic strategies to treat some LSDs.

Oleuropein aglycone
Oleuropein aglycone, the major phenolic component found in olive oil, has been shown to fight against neurodegeneration by autophagy activation. 180 Oleuropein aglycone triggers autophagy in cultured neuroblastoma cells through activation of Ca 2+ -calmodulindependent kinase kinase β (CaMKKβ)-AMPK axis and mTOR inhibition. 181 In transgenic TgCRND8 mice, which overexpressed the Swedish and Indiana mutations in human APP, dietary supplementation of oleuropein aglycone (50 mg/kg of diet) significantly reduced β-amyloid levels and plaque deposits and improved the cognitive performance in TgCRND8 mice. 182 However, the authors did not investigate the effect of oleuropein aglycone on TFEB, which may be a potential target for autophagy enhancement.

Ouabain
Ouabain, a cardiac glycoside, is a potent inhibitor of the sodium/ potassium pump (Na + /K + -ATPase). 183 Ouabain has been reported to inhibit cell growth by blocking the AKT/mTOR signaling pathway. 184 Ouabain increases the TFEB dephosphorylation and promotes its nuclear translocation, thereby inducing the expression of ALPrelated genes targeted by TFEB. Ouabain also reduces phospho-

Cinnamic acid
Cinnamic acid, a naturally occurring plant-based product, activates the nuclear hormone receptor PPARα to transcriptionally upregulate TFEB and induce lysosomal biogenesis in mouse primary neurons.
Moreover, cinnamic acid treatment in 5xFAD mice remarkably reduce cerebral Aβ plaque burden and improve memory deficit via PPARα. 187

Pseudoginsenoside-F11
Pseudoginsenoside-F11(PF11), an ocotillol-type saponin derived from leaves of Panax pseudoginseng subsp. himalaicus HARA, has been shown to be protective against AD both in vivo and in vitro. 188

Rapamycin and its analogs
Rapamycin, a food and drug administration (FDA)-approved drug, is an inhibitor that interferes with the mTOR signaling pathway, which may be a TFEB agonist. 200 A number of studies have reported the protective effects of rapamycin and its analogs CCI-779 in various models of neurodegenerative diseases. 201 In an animal model of PD induced by intracerebral injection of AAV α-synuclein, peripheral administration of CCI-779 efficiently triggered a proautophagic response in the brain via inhibiting mTORC1 activity and increasing the nuclear translocation of TFEB. In addition, CCI-779 significantly improves motor impairment of PD animals, increases survival of nigral dopamine neurons, maintains the striatal dopamine content, and reduces the accumulation of toxic oligomeric α-synuclein. 36 In a Parkin Q311X mutant mouse model, treatment with rapamycin independently restores PGC1α-TFEB signaling in a manner not requiring parkin E3 ligase activity and abrogates subsequent mitochondrial impairment and age-related neurodegenerative effects. 122 Rapamycin has also been proven to effectively reduce Aβ and tau pathology leading to improve cognitive function in several different mouse models of AD. [202][203][204] However, it is not clear whether rapamycin and its analogs exert neuroprotective effects by regulating TFEB or ALP in these models. Furthermore, more studies are required to fully elucidate this mechanism.

Cyclodextrin
The FDA-approved excipient, 2-hydroxypropylβ-cyclodextrin (HPβCD), is used to improve the stability and bioavailability of drugs and is widely used as a drug delivery vehicle. HPβCD administration results in activation of TFEB and enhances the cellular autophagic clearance capacity in cells derived from a patient with LSD. 205 In addition, pharmacological activation of TFEB by HPβCD also enhances autophagic clearance of α-synuclein aggregates in human neuroglioma cells. 39

F-SLOH
F-SLOH, a versatile theranostic agent, can selectively bind to Aβ oligomers with strong fluorescence enhancement and also efficiently crosses the blood-brain barrier (BBB). 218 Recently, F-SLOH has been found to significantly reduce the levels of APP, Aβ oligomers, and hyperphosphorylated Tau aggregates in the brains of 5xFAD and 3xTg-AD mice. In addition, F-SLOH also significantly ameliorates synaptic deficits and cognitive impairment in AD mouse models. Mechanistic studies have shown that F-SLOH promote autophagy and lysosomal biogenesis via TFEB dephosphorylation and nuclear accumulation through MAPK1/ERK2 inhibition and PP2A activation. 219 In addition to some of the small-molecule compounds described above, a recent study showed that electroacupuncture activates TFEB by inhibiting the AKT-MAPK1-mTORC1 pathway and significantly reduces APP and Aβ load and improves cognitive deficits in 5xFAD mice. 220 Several studies have shown that the natural small-molecule compounds Mucuna pruriens, Ursolic Acid and Chlorogenic Acid have significant neuroprotective effects on MPTP-induced dopaminergic neuron damage through their antioxidant and anti-inflammatory effects in an MPTP-induced Parkinsonian mouse model, but the mechanism of action is unclear. [221][222][223] Nrf2 is a critical transcription factor that neutralizes ROS to restore the redox balance of cells, which can be degraded by the Kelch-like ECH-associated protein 1 (Keap1), an adapter protein of the Cul3-ubiquitin E3 ligase complex. 224 In PD and AD cellular models, the small molecules, myriocin and fisetin, or stimulation of PINK1-dependent mitophagy are found to be neuroprotective through Nrf2 activation, but the exact mechanism has not been elucidated. 121,192,225 Moreover, p62 has also been shown to bind to aggregates of ubiquitylated proteins to increase its affinity for Keap1 when phosphorylated at Ser351 in a redox-independent manner. This results in Keap1 degradation and leaves Nrf2 to translocate in the nucleus to regulate antioxidant and detoxifying genes. 226 Interestingly, an in vitro study found that TFEB overexpression stabilizes and activates Nrf2 through repression of the Nrf2-specifc E3 ubiquitin ligase, DDB1 and Cullin4 associated factor 11 (DCAF11). This results in phosphorylation of p62 at Ser349, which further disrupts the binding of Keap1 and Nrf2, thus leading to sustained Nrf2 activation through a positive feedback loop. 227 This event suggests that TFEB-mediated positive feedback regulation of p62-Nrf2 may be a key mechanism linking oxidative stress and autophagy. Furthermore, in PD the above small molecules may mechanistically mitigate oxidative stressinduced neuronal damage by enhancing the TFEB-mediated positive feedback regulation of p62-Nrf2. However, further investigations are required in cellular and animal models.

| CON CLUDING REMARK S
The crucial role of TFEB and its mediated ALP in the clearance of abnormal aggregates has been supported in several preclinical models of neurodegenerative diseases. Several studies have shown that genetic and pharmacological modulation of TFEB are increasingly important strategies for the prevention and treatment of neurodegenerative diseases. However, there are still many uncertainties about whether the small molecules can be used as drug targets for the treatment of neurodegenerative diseases. First, most studies that reported that specific small molecules indirectly activate TFEB by regulating upstream kinases, including mTORC1, PKC, and AKT.
However, these kinases also participate in the regulation of several other important cellular biological processes. [228][229][230] Therefore, whether activation of TFEB by inhibiting or activating these key regulators affects other normal cellular functions needs to be considered and further examined in neurodegenerative diseases models.
Secondly, TFEB is also activated by various stressors such as nutrient deficiency, oxidative stress, ER stress, and lysosomal stress, which have been considered as considered risk factors for neurodegenerative diseases. It was necessary to determine whether these investigated small molecules activate TFEB by triggering the cellular stress.
Furthermore, it remains unclear whether these small-molecule TFEB activators can cross the BBB and directly act on brain neurons or glial cells to exert drug effects, which should be carefully determined in animal models. In addition, although acute induction of intracellular protein aggregates degradation can ameliorate some symptoms of disease in several animal models, any long-term effects of such treatments have not yet been evaluated.
Moreover, as an important intracellular clearance molecule, the level and duration of TFEB activation needs to be tightly regulated.

CO N FLI C T O F I NTE R E S T
The authors declare that there are no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analyzed in this study.