The autophagy–lysosome pathway: a potential target in the chemical and gene therapeutic strategies for Parkinson’s disease

Abstract

Parkinson’s disease is a common neurodegenerative disease with movement disorders associated with the intracytoplasmic deposition of aggregate proteins such as α-synuclein in neurons. As one of the major intracellular degradation pathways, the autophagy-lysosome pathway plays an important role in eliminating these proteins. Accumulating evidence has shown that upregulation of the autophagy-lysosome pathway may contribute to the clearance of α-synuclein aggregates and protect against degeneration of dopaminergic neurons in Parkinson’s disease. Moreover, multiple genes associated with the pathogenesis of Parkinson’s disease are intimately linked to alterations in the autophagy-lysosome pathway. Thus, this pathway appears to be a promising therapeutic target for treatment of Parkinson’s disease. In this review, we briefly introduce the machinery of autophagy. Then, we provide a description of the effects of Parkinson’s disease–related genes on the autophagy-lysosome pathway. Finally, we highlight the potential chemical and genetic therapeutic strategies targeting the autophagy–lysosome pathway and their applications in Parkinson’s disease.

Introduction

Parkinson’s disease (PD) is a common neurodegenerative movement disorder clinically characterized by resting tremor, rigidity, bradykinesia, and ataxia, along with non-motor symptoms such as depression, constipation, and olfactory disorders (Lees et al., 2009). A typical pathological feature of PD is the formation of Lewy bodies (LBs) composed of abnormally aggregated α-synuclein (α-Syn) and the progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc).

Epidemiological studies suggest that the majority of PD are sporadic and 5%–10% cases are familial (de Lau and Breteler, 2006). The etiology of PD is still unclear, but it is believed to line with genetic factors, environmental toxins, and aging (Michel et al., 2016). To date, there are no effective treatments that can stop the progression or completely reverse PD. Recently, studies have found that targeting the etiology and pathogenesis of PD may contribute to improving or reversing its progression.

Autophagy is a major intracellular degradation system that delivers cytoplasmic materials such as abnormally aggregated or misfolded proteins and damaged organelles to the lysosome for degradation (Wang and Lu, 2023; Hernández-Cáceres et al., 2024; Nagayach and Wang, 2024). Autophagy is essential for maintaining neuronal homeostasis (Jia et al., 2024). Previous studies have shown that abnormal accumulation of autophagosomes was observed in the neurons of the SNpc from PD patients and animal models (Anglade et al., 1997; Dehay et al., 2010). Moreover, multiple genes associated with the pathogenesis of PD such as SNCA, LRRK2, PINK1, PARKIN, GBA, ATP13A2, VPS35, FBXO7, and DJ-1 are closely related to alterations in the autophagy/lysosomal pathways (Gan-Or et al., 2015). However, the precise mechanisms underlying the intriguing link between the autophagic-lysosomal pathway (ALP) deficiency and PD pathogenesis remain unclear. In this review, we briefly introduce the machinery of autophagy. Then, we provide a description of the effects of PD-related genes on ALP. Finally, we highlight the chemical and gene therapeutic strategies targeting ALP and their applications in PD.

Search Strategy

The electronic databases of Web of science, PubMed, and Google Scholar were systematically searched for published studies that investigated autophagy AND Parkinson’s disease. No date limits were applied. The search terms and keywords included as the following: (α-synuclein OR LRRK2 OR PINK1 OR VPS35 OR DJ-1 OR GBA OR ATP13A2 OR TMEM175) AND (autophagy OR lysosome), (autophagy activators OR autophagy inhibitors) AND Parkinson’s disease, (LRRK2 kinase inhibitors OR GCase OR c-Abl) AND (autophagy OR lysosome), (Natural compounds OR natural products) AND (autophagy OR lysosome), (Beclin1 OR TFEB OR LAMP2 OR ITPKB) AND (autophagy OR lysosome), (MicroRNAs OR Circular RNAs OR LncRNAs) AND (autophagy OR lysosome), (Nanotechnology OR Microbubbles OR Targeted protein degradation) AND (autophagy OR lysosome). The titles and abstract were screened first. The full-text of suitable papers only was further reviewed.

The Machinery of Autophagy

Autophagy is subdivided into three types: macroautophagy, chaperone-mediated autophagy (CMA), and microautophagy, according to the mechanism by which the cargoes are delivered to the lysosome (Yang and Klionsky, 2010).

Macroautophagy

Macroautophagy (hereafter referred to as autophagy) is the major pathway for the degradation of bulk cytoplasmic material. In the initial stage, the UN51-like Ser/Thr kinases (ULK) complex and the class III phosphatidylinositol 3-kinase (PI3K) complex are recruited to the phagophore assembly site (PAS) and form a crescent-shaped double membrane structure. During elongation, the phagophore enlarges and utilizes two ubiquitin-like conjugation systems for elongating the pre-autophagosomal structure, and elongation terminates with the formation of a complete bubble-like structure called the autophagosome. Autophagosomes subsequently fuse with either lysosomes (to form autolysosomes) or with endosomes (to form amphisomes), and cytoplasmic contents are degraded in autolysosomes.

A growing number of cytosolic cargoes including damaged organelles, aggregated proteins, and pathogens are cleared by selective autophagy (Vargas et al., 2023). In selective autophagy, the autophagy receptor proteins such as sequestesome (SQSTM)1/p62, neighbor of BRCA1 gene 1 (NBR1), and optineurin (OPTN) recognize ubiquitinated targets and then mediate the formation of selective autophagosomes via binding to small ubiquitin-like modifiers (UBLs)—Atg8/LC3/GABARAPs and ATG5 (Rogov et al., 2014). Finally, the ubiquitinated targets are degraded in the autophagosomes.

In particular, the selective degradation of damaged mitochondria via autophagy is called mitophagy, which is an important process for maintaining mitochondrial homeostasis. PTEN-induced putative kinase (PINK) 1/parkin–mediated pathway is the most important pathway in mitophagy (McWilliams and Muqit, 2017). PINK1 is a Ser/Thr kinase that monitors mitochondrial function. Under normal conditions, PINK1 is a Ser/Thr kinase that can be translocated to the mitochondrial inner membrane, where it is cleaved and inactivated by presenilin-associated rhomboid-like protein, and is then degraded via the N-terminal rule pathway (Yamano and Youle, 2013). When mitochondria are damaged, PINK1 cannot be translocated to the mitochondrial inner membrane and cleaved, resulting in its accumulation at the outer mitochondrial membrane (OMM). Then, PINK1 recruits and phosphorylates parkin, an E3 ubiquitin ligase (Narendra et al., 2010). Activated parkin induces the ubiquitination of mitochondrial membrane proteins and recruits autophagy receptors such as OPTN and nuclear dot protein 52 kDa (NDP52) to the damaged mitochondria (Wong and Holzbaur, 2014, 2015), followed by the formation of LC3-positive phagophores, which degrade the ubiquitinated mitochondria via lysosomes (Narendra et al., 2008; Harper et al., 2018).

Chaperone-mediated autophagy

Chaperone-mediated autophagy (CMA) is a type of selective autophagy characterized by degraded proteins containing the KFERQ motif recognized by the heat shock cognate 70 (HSC70) protein. HSC70 transfers the cargo proteins to the lysosome-associated membrane protein type 2A (LAMP2A), which serves as a CMA receptor (Cuervo and Dice, 1996). Subsequently, substrate unfolding triggers the assembly of LAMP2A into a multimeric protein complex, which mediates substrate translocation into the lysosome (Kaushik and Cuervo, 2012). During this step, HSP90 stabilizes the LAMP2A in the luminal side of the lysosomal membrane.

Microautophagy

Microautophagy is a biological process in which the cytoplasmic cargo is directly engulfed by lysosomes. Ten years ago, Sahu et al. (2011) first described that microautophagy occurs in late endosomes in mammals; it is now known as endosomal microautophagy (eMI). The cargo is then incorporated into luminal vesicles via membrane deformation internalization and degraded in late endosomes or until late endosomes fuse with the lysosomes. Although it is clear that microautophagy mediates the degradation of cytoplasmic cargo “in bulk” or in a selective manner, the mechanism and its functions in diseases largely remain unclear. Several studies have suggested that the membrane fusion systems (e.g., Rab and ESCRT I/III) in autophagy may contribute to microautophagy (Sahu et al., 2011; Wang et al., 2023).

Parkinson’s Disease–Related Genes and Their Effects on the Autophagic-Lysosomal Pathway

Numerous studies have shown that mutations in many high-risk genes of PD can lead to ALP dysfunction (Table 1), which in turn can result in abnormal protein aggregation and/or accumulation of damaged organelles, eventually causing neuronal death (Figure 1).

Table 1.

The role of Parkinson’s disease-related genes in the autophagy-lysosomal pathway

Genes	Forms of action	Mechanisms in ALP	References
SNCA	A53T and A30P mutants and oligomeric α-Syn; overexpression of α-Syn; LB-like α-Syn aggregates; α-Syn fibrils	Bind to the CMA receptor LAMP 2A and impair CMA activation; cause the loss of autophagosomes via Rab1a inhibition; lead to an attenuated fusion of autophagosomes with lysosomes via decreasing the expression of SNAP29; impair macroautophagy through decreasing the autophagosome clearance; affect lysosomal morphology and function and induce LMP mediated by TFEB	Cuervo et al., 2004; Winslow et al., 2010; Tanik et al., 2013; Dilsizoglu Senol et al., 2021
LRRK2	G2019S mutant of LRRK2; R1441C mutant of LRRK2; overexpression of wild-type LRRK2	Induce neurite degeneration with dependent on an increase in macroautophagy; impair autophagic homeostasis; Induce macroautophagy via the MAPK/ERK pathway; block the formation of the CMA transporter complex at the lysosomal membrane by inducing LAMP 2A and HSPA8/HSC70 proteins accumulation; enhanced mitophagy and dendrite shortening via disturbing intracellular calcium homeostasis; induce mitochondrial dysfunction through phosphorylating Drp1 at T595; reduce mitophagy rates via inhibiting the formation of mature autophagosomes; interact with OPTN through phosphorylation of Rab10 and then damage mitophagy	Plowey et al., 2008; Alegre-Abarrategui et al., 2009; Bravo-San Pedro et al., 2013; Orenstein et al., 2013; Cherra et al., 2013; Su and Qi, 2013; Korecka et al., 2019; Ho et al., 2020; Wauters et al., 2020
PINK1 and parkin	Nonsense (c.1366C>T; p.Q456X) or missense (c.509T>G; p.V170G) mutations in the PINK1	Reduce the expression of PPARGC1A and impair the recruitment of parkin to mitochondria and decrease the mitochondrial copy number; Impair the mitochondrial transport machinery by upregulation of Miro	Seibler et al., 2011; Liu et al., 2012
VPS35	VPS35 D620N mutant	Lead to defective autophagosome formation by impairing ATG9A trafficking to autophagosomes; affect the initiation of PINK1/Parkin-dependent mitophagy	Zavodszky et al., 2014; Ma et al., 2021
FBXO7	T22M, R378G and R498X mutations of FBXO7	Inhibit mitophagy by accelerating the accumulation of deleterious FBXO7 in mitochondria	Zhou et al., 2015
PARK7(DJ-1)	Knock down of DJ-1	Impair the autophagic flux and affect soluble α-Syn clearance by autophagy manipulation; suppress CMA pathway via inhibiting the upregulation of LAMP2A and decreasing the HSC70; lead to mitochondrial damage and accumulation of LC3 around mitochondria	Thomas et al., 2011; Nash et al., 2017; Xu et al., 2017
GBA	GBA L444P mutation; GBA knockout	Impair the ALR; increase mitochondrial content and mitochondrial oxidative stress, and impair autophagy; lead to abnormal accumulation of enlarged autophagic vesicles and increased release of insoluble α-Syn	Bae et al., 2015; Magalhaes et al., 2016; Li et al., 2019a
ATP13A2	L3292 and L6025 ATP13A2 mutations or knockdown of ATP13A2	Impair degradation of lysosomal substrates and increase the accumulation of autophagosome and α-Syn	Dehay et al., 2012; Usenovic et al., 2012; Tsunemi et al., 2019
TMEM175	p.M393T mutation or knockdown of TMEM175	Impair lysosomal degradation, lysosome-mediated clearance of autophagosome, and the function of mitochondrial respiratory chain; lead to lysosomal over-acidification, impaired proteolytic activity, and promoted α-Syn aggregation	Jinn et al., 2017, 2019; Hu et al., 2022

ALR: autophagy lysosomal reformation; ATP13A2: ATPase cation transporting 13A2; CMA: chaperone-mediated autophagy; FBXO7: F-box only protein 7; GBA: glycosylceramidase beta; LMP: lysosomal membrane permeabilization; LRRK2: leucine rich repeat kinase 2; OPTN: optineurin; PINK1: PTEN-induced kinase 1; PPARGC1A: PPARG coactivator 1 alpha; SNAP29: synaptosome associated protein 29; SNCA: synuclein alpha; TMEM175: transmembrane protein 175; VPS35: vacuolar protein sorting 35; α-Syn: α-synuclein.

Figure 1.

The role of ALP dysfunction in PD pathogenesis.

Macroautophagy, mitophagy, and CMA are involved in the pathogenesis of PD. Overexpression of α-Syn can cause loss of autophagosomes and prevent the fusion of autophagosomes with lysosomes and a decreased formation of autolysosomes. α-Syn fibrils can affect lysosomal morphology and function. Overexpression of the R1441C-LRRK2 mutation causes impaired autophagic homeostasis, as evidenced by accumulation of MVB and AVs. The LRRK2 G2019S mutation reduces mitophagy rates via inhibiting the formation of mature autophagosomes. Overexpression of wild-type LRRK2, G2019S, and R1441C mutants of LRRK2 can block the formation of the CMA transporter complex at the lysosomal membrane by inducing LAMP 2A and HSPA8/HSC70 proteins accumulation, and then inhibiting the CMA pathway. LRRK2 G2019S mutation also binds to and phosphorylates Bcl-2 at threonine 56, leading to p62-mediated mitochondrial degradation. The VPS35 D620N mutant impairs the autophagy protein ATG9A trafficking to autophagosomes, leading to defective autophagosome formation. VPS35 deficiency or mutation (D620N) reduces α-Syn degradation, thereby hindering the endosome-to-Golgi retrieval of LAMP 2A and accelerating its degradation. The VPS35 D620N mutant was found to be unable to accumulate PINK1 at the damaged mitochondrial surface and affect the initiation of PINK1/Parkin-dependent mitophagy. PD-linked FBXO7 mutants can recruit parkin into damaged mitochondria to facilitate its aggregation. PARK7 (DJ-1) knock down microglia exhibit an impaired autophagy-dependent degradation of p62 and LC3 proteins. DJ-1 deficiency suppressed the CMA pathway by inhibiting the upregulation of LAMP 2A and decreasing the level of HSC70, which leads to the aggregation of α-Syn. Mutation or deficiency of GBA impairs ALR through inhibition of mTOR activity. GBA knockout can increase abnormal accumulation of enlarged autophagic vesicles, accumulation of poly-ubiquitinated proteins which are lysosomal substrates, and release of insoluble α-Syn. ATP13A2 mutation or ATP13A2 knockdown can reduce lysosomal membrane stability and increase the abnormal accumulation of autophagosome and α-Syn. TMEM175 deficiency or p.M393T mutation can lead to impaired lysosomal degradation, lysosomal over-acidification, and impaired proteolytic activity. Created using Microsoft PowerPoint. ALR: Autophagy lysosomal reformation; ATP13A2: ATPase cation transporting 13A2; AVs: autophagic vacuoles; CMA: chaperone-mediated autophagy; FBXO7: F-box only protein 7; GBA: glycosylceramidase beta; LRRK2: leucine-rich repeat kinase 2; MVB: multivesicular bodies; PD: Parkinson’s disease; PINK1: PTEN-induced kinase 1; TMEM175: transmembrane protein 175; VPS35: vacuolar protein sorting 35; α-Syn: α-synuclein.

PD-related genes and defective autophagy

SNCA

SNCA is the first gene identified as being associated with PD. Mutations in SNCA (A53T, A30P, and E46K) or an increase of its copy numbers can lead to the development of familial PD (Polymeropoulos et al., 1997; Kruger et al., 1998; Zarranz et al., 2004). α-Syn is a protein encoded by the SNCA gene, which is made up of 140 amino acids with a molecular weight of 14 kDa. Previous studies have found that α-Syn is involved in synaptic vesicle release and regulates vesicle fusion through promoting the SNARE-complex assembly (Clayton and George, 1998; Burre et al., 2010). Nair et al. (2011) showed that the SNARE proteins are implicated in the formation of autophagosomes, which suggested that α-Syn may be involved in the process of autophagy.

In neuronal cells, α-Syn is mainly degraded through CMA and macroautophagy (Vogiatzi et al., 2008). α-Syn protein contains the KFERQ motif that can target it to the CMA pathway. Analysis in purified liver lysosomes confirmed that CMA may be the major pathway for wild-type α-Syn degradation (Cuervo et al., 2004). However, mutant and oligomeric α-Syn can tightly bind to the CMA receptor LAMP 2A and impair CMA activation (Cuervo et al., 2004). 3-MA, a specific inhibitor of autophagy inhibits autophagy at the sequestration stage. It was found that both endogenous and overexpressed α-Syn increased in PC12 cells and the ventral midbrain dopaminergic neurons treated with 3-MA, suggesting that α-Syn was mainly degraded by macroautophagy (Vogiatzi et al., 2008). Furthermore, they also found that wild-type α-Syn is degraded through the CMA pathway. In neuronal cell culture systems, there may be a crosstalk pathway between macroautophagy and CMA during α-Syn degradation, which suggests that the degradation of α-Syn by macroautophagy may be a compensatory mechanism for CMA dysfunction (Vogiatzi et al., 2008). Similarly, it has been shown that CMA regulates diquat (DQ)-induced α-Syn increase cooperatively with macroautophagy (Kim and Koh, 2021). Additionally, extracellular aggregated α-Syn can be internalized into cells via clathrin-dependent endocytosis and then enter the lysosome via the recycling endosomal pathway (Lee et al., 2008). Within the lysosome, α-Syn is degraded through many lysosomal proteases, including cathepsins D and cysteine cathepsins (cathepsins B and cathepsins L; Sevlever et al., 2008; Cullen et al., 2009; McGlinchey and Lee, 2015); these cysteine cathepsins likely contribute to the C-terminal truncation of α-Syn and production of toxic substances (McGlinchey et al., 2019).

Apart from being degraded by the ALP pathways, the accumulation of α-Syn possibly also interferes with the normal function of this system, thereby contributing to neurodegeneration (Bellomo et al., 2020). It has been reported that the mechanism by which wild-type α-Syn affects autophagy may be associated with protein expression levels or protein aggregation status (Ebrahimi-Fakhari et al., 2011). Overexpression of α-Syn causes loss of autophagosomes via Rab1a inhibition. Mechanically, it was demonstrated that neither Rab1a knockdown nor α-Syn overexpression results in mislocalization of the autophagy protein Atg9 and decreases the formation of omegasome (Winslow et al., 2010). A recent study found that α-Syn overexpression leads to decreases of SNAP29, a member of the SNARE complex, leading to attenuated fusion of autophagosomes with lysosomes and decreased formation of autolysosomes. Furthermore, autophagosomes can fuse with the plasma membrane (PM) to release vesicles into the extracellular space to compensate for the decreased autophagic flux (Tang et al., 2021). However, LB-like α-Syn aggregates cannot be effectively degraded by the ALP pathway and may impair macroautophagy through decreasing the autophagosome clearance, which may contribute to increased cell death in PD (Tanik et al., 2013). Dilsizoglu Senol et al. (2021) found that α-Syn fibrils can affect lysosomal morphology and function in neurons. Additionally, α-Syn fibrils also induce lysosomal membrane permeabilization mediated by transcription factor EB (TFEB), which increases the translocation of α-Syn fibrils to neighboring cells. Taken together, these data suggest that α-Syn overexpression and/or protein aggregates or its fibrillar forms may interfere with ALP at both early and late steps.

LRRK2

Autosomal-dominant mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are identified as the frequent genetic cause of PD (Zimprich et al., 2004). The G2019S and R1441C mutants of LRRK2 are the pathogenic variants in familial PD (Sosero and Gan-Or, 2023). Under normal conditions, LRRK2 is mainly localized in membrane microdomains, multivesicular bodies, and autophagic vesicles, involving multiple biological processes such as autophagy, vesicle trafficking, neurite outgrowth, and cytoskeleton maintenance (Rideout and Stefanis, 2014; Madureira et al., 2020). Accumulating evidence has shown that the process of ALP is altered in the LRRK2 mutants PD model, but it is still not conclusive whether LRRK2 plays a positive or negative role in the ALP (Bravo-San Pedro et al., 2012; Cogo et al., 2020). Several independent studies in animal models and cell lines with LRRK2 mutations have reported that PD-associated LRRK2 mutations can results in autophagic dysregulation. It has been shown that overexpression of the G2019S mutant of LRRK2 in cellular models induces neurite degeneration that is mechanistically dependent on an increase in macroautophagy (Plowey et al., 2008). In another study, overexpression of the R1441C-LRRK2 mutation in cells caused impaired autophagic homeostasis, as evidenced by accumulation of multivesicular bodies and large autophagic vacuoles (AVs), whereas LRRK2 silencing triggered an increase of autophagic activity (Alegre-Abarrategui et al., 2009). Additionally, elevated LC3-II levels were found in LRRK2 G2019S knock-in mice, but reduced MAP1LC3/LC3 activation and increased LC3-aggregates were found in aged Parkinsonian LRRK2 R1441G mutant knock-in mice, suggesting that LRRK2 with different mutation sites may have different effects on autophagy regulation (Yue et al., 2015; Liu et al., 2021). Moreover, the LRRK2 G2019S mutation induces autophagy via the mitogen activated protein kinase/extracellular signal regulated protein kinase (MAPK/ERK) pathway (Bravo-San Pedro et al., 2013), and fibroblasts from PD patients with the LRRK2 G2019S mutant are more sensitive to 1-methyl-4-phenylpyridinium ion (MPP⁺)-induced autophagy than controls without mutant in an mTOR-dependent manner, further emphasizing the synergistic roles of genetic and environmental factors in the pathogenesis of PD (Yakhine-Diop et al., 2014).

Additionally, wild-type LRRK2 protein bears eight putative CMA motifs that are recognized by HSC70 and can be selectively degraded by CMA. However, the LRRK2 G2019S mutant is poorly degraded by this pathway (Orenstein et al., 2013). Mechanically, brain autopsy studies on PD patients with LRRK2 mutations, LRRK2 transgenic mice, and human iPSC-derived dopaminergic neurons have shown that overexpression of wild-type LRRK2, and G2019S and R1441C mutants of LRRK2 can block the formation of the CMA transporter complex at the lysosomal membrane by inducing LAMP 2A and HSPA8/HSC70 protein accumulation, and then inhibiting the CMA pathway (Orenstein et al., 2013; Ho et al., 2020). Interestingly, LRRK2-induced CMA blockade can inhibit α-Syn degradation through the CMA pathway. Furthermore, accumulation of oligomeric α-Syn can be detected in the cortex and striatum of aged LRRK2 R1441G knock-in mice (Ho et al., 2020).

Several reports support that LRRK2 also plays an important role in mitophagy. Mitochondrial fission induced by LRRK2 G2019S mutation can be cleared by interacting with ULK1, a key regulator of autophagy (Zhu et al., 2013). Furthermore, LRRK2 also interacts and cooperates with the endogenous MKK4/7 and JIP3 to activate the JNK signaling pathway, suggesting that this pathway may be partially involved in LRRK2 G2019S-induced mitophagy (Zhu et al., 2013). The G2019S and R1441C mutants of LRRK2 in cortical neurons leads to enhanced mitophagy and dendrite shortening by disturbing the intracellular calcium homeostasis (Cherra et al., 2013). In addition, a study showed that LRRK2 G2019S mutation directly combined and phosphorylated dynamin-related protein 1 (Drp1) at threonine 595 and induced mitochondrial dysfunction. However, treatment with P110, a selective peptide inhibitor of fission Drp1, or expression of Drp1 T595A mutant reduces the mitochondrial impairment and neurite shortening (Su and Qi, 2013). The same group subsequently showed that LRRK2 G2019S mutation also binds to and phosphorylates Bcl-2 at threonine 56, leading to p62-mediated mitochondrial degradation (Su et al., 2015). Similarly, LRRK2 mutation impaired mitophagy by affecting DNM1L activation in LRRK2 R1441G mutant mouse embryonic fibroblasts (MEFs). Furthermore, impaired DNM1L-MAPK/ERK signaling that mediates mitochondrial fission and downstream mitophagic processes are found in mutant LRRK2 MEFs when stressed with cyanide-4-(trifluoromethoxy) phenylhydrazone (a uncoupler of mitochondrial oxidative phosphorylation; Liu et al., 2021). Additionally, in healthy subject (HS) and PD patient-derived fibroblast lines, overexpression of the LRRK2 G2019S mutation reduced mitophagy rates via inhibiting the formation of mature autophagosomes in fibroblasts compared to cells from controls (Korecka et al., 2019), suggesting that the LRRK2 protein plays an important role in the regulation of mitochondrial clearance by lysosomes. Another study showed that LRRK2 increases mitochondrial aggregation and decreases mitochondrial clearance by disturbing the interactions between parkin and Drp1 and their mitochondrial targets in PINK1/parkin-dependent mitophagy (Bonello et al., 2019). Recently, a study showed that G2019S and R1441C mutants of LRRK2 enhanced the phosphorylation of Rab10 at threonine 73, which interacts with OPTN and leads to accumulation of LRRK2 mutants on depolarized mitochondria, and then damages mitophagy. These defects can be rescued by knockdown of LRRK2 or inhibition of its kinase activity (Wauters et al., 2020).

There are accumulating data suggesting that LRRK2 likely participates in the regulation of the lysosomal system. LRRK2 could phosphorylate serval Rab proteins and interfere with their functions (Steger et al., 2017; Fujimoto et al., 2018; Madero-Perez et al., 2018; Pfeffer, 2023). Rab29 (also known as RAB7L1) is a candidate risk locus for PD. Upon lysosomal overload stress, Rab29 recruits LRRK2 to the lysosome, which in turn phosphorylates and stabilizes Rab8 and Rab10 to maintain lysosomal homeostasis (Eguchi et al., 2018; Komori et al., 2023). Additionally, the LRRK2 G2019S mutation can mediate α-Syn propagation by Rab35 phosphorylation, whereas administration of an LRRK2 kinase inhibitor can reduce the localization of α-Syn in Rab35-positive regions and enhance α-Syn clearance via the lysosomal degradation pathway (Bae et al., 2018).

Parkin and PINK1

Mutations in the PTEN-induced kinase 1 (PINK1) (and parkin genes are identified as the primary causes of autosomal recessive early-onset PD [EOPD]) (Pickrell and Youle, 2015). Both genes can work together to control the mitochondrial quality via mediating the autophagic degradation of mitochondria (mitophagy) (Li et al., 2023). During mitochondrial membrane depolarization, PINK1 accumulates at the OMM, where it phosphorylates serine 65 residues of ubiquitin (Ub) (pSer65Ub) (Swatek et al., 2019). Then, cytosolic parkin is recruited to the OMM and binds to pSer65Ub, leading to a series of conformational changes (Wauer et al., 2015). Notably, parkin remains partially autoinhibited after binding to pSer65Ub, and can be fully activated by PINK1 through phosphorylation of its Ub-like domain at serine 65 residues (Gladkova et al., 2018). Subsequently, phosphorylated Ub chains bind to the OMM and serve as a selective tag that was recognized by autophagy receptors such as OPTN, NDP52, and p62/SQSTM1 to mitochondria (Geisler et al., 2010; Wong and Holzbaur, 2014, 2015), followed by the selective degradation of damaged mitochondria through ALP (Heo et al., 2015; Lazarou et al., 2015; Wong and Holzbaur, 2015). It was found that pSer65Ub formation increased in the postmortem PD brain and decreased in patients with PINK1/parkin mutation, suggesting the relevance of the PINK1/parkin-mediated mitophagy in PD (Fiesel et al., 2015; Hou et al., 2018). The mechanism of PINK1/parkin-mediated mitophagy has been elegantly detailed in PD cellular or animal models. In iPSC-derived dopaminergic neurons from PD patients with PINK1 mutations, Seibler et al. (2011) showed that PINK1 mutant reduced the expression of PPARG coactivator 1 alpha (PPARGC1A), an important regulator of mitochondrial biogenesis, and impaired the recruitment of parkin to mitochondria and decreased the mitochondrial copy number. The turnover rates of mitochondrial proteins and mitochondrial respiratory chain subunits were found to be significantly decreased in Drosophila overexpressing PINK1 and parkin mutants according to the proteomics studies (Vincow et al., 2013). Additionally, PINK1 mutant impaired the mitochondrial transport machinery by upregulation of Miro protein level, whereas downregulation of Miro protein level could rescue PINK1 mutant phenotypes in Drosophila (Liu et al., 2012). In the striatum of parkin knockdown mice, parkin deletion resulted in reduced synaptic excitability, increased dopamine concentration in the extracellular space, and nigrostriatal deficits, yet the number of dopaminergic neurons in the substantia nigra was normal (Goldberg et al., 2003). PINK1/parkin-dependent mitophagy is essential for the clearance of damaged mitochondria, and its impaired function is closely associated with the pathogenesis of PD.

VPS35

The D620N mutation in the vacuolar protein sorting 35 (VPS35) gene has been identified as a pathogenic variant in late-onset familial PD (Luo et al., 2021). VPS35 protein is an integral component of the retromer complex that mediates the retrograde transport from late endosomes to the trans-Golgi network. VPS35 mutations or VPS35 knockdown may lead to the aberrant transport of proteases within the lysosome, which reduces the degradation of α-Syn (Follett et al., 2014; Miura et al., 2014). Tsika et al. (2014) found that exogenous expression of VPS35 D620N mutation significantly induces degeneration of dopaminergic neurons and axonal pathology in the substantia nigra of adult rats. Cells with the VPS35 D620N mutant reduced Wiskott-Aldrich syndrome protein and scar homolog (WASH) complex recruitment to endosomes and impaired the autophagy protein ATG9A trafficking to autophagosomes, leading to defective autophagosome formation (Zavodszky et al., 2014). VPS35 deficiency or mutation (D620N) in dopaminergic neurons reduces α-Syn degradation and promotes PD pathogenesis by hindering the endosome-to-Golgi retrieval of LAMP 2A (a receptor of CMA to mediate α-Syn degradation) and accelerating its degradation, suggesting that VPS35 is a critical regulator of LAMP 2A and α-Syn degradation in PD (Tang et al., 2015). Recently, the VPS35 D620N mutant was found unable to accumulate PINK1 at the damaged mitochondrial surface upon CCCP treatment, suggesting that the VPS35 mutation may affect the initiation of PINK1/Parkin-dependent mitophagy (Ma et al., 2021).

FBXO7

Mutations in the F-box only protein 7 (FBXO7) gene (locus PARK15) were identified as the cause of early-onset juvenile autosomal recessive PD with clinical features of Parkinsonian-Pyramidal syndrome (Tranchant et al., 2017; Wang et al., 2021b). FBXO7 is an adaptor protein in Skp-Cullin-F-box ubiquitin E3 ligase complex (SCF complex) and is involved in many kinds of biological processes, such as synapse neuroplasticity, cell proliferation, cell cycle, and mitochondrial and proteasome functions via interacting with target substrates (Zhong et al., 2023). Interestingly, wild-type FBXO7 has protective effects on neurons by promoting mitophagy, while FBXO7 mutations (T22M, R378G, and R498X) inhibit mitophagy by accelerating the accumulation of deleterious FBXO7 in mitochondria (Zhou et al., 2015). Mechanically, deficiency of FBXO7 is associated with the reduced level of cellular NAD⁺, which leads to impaired complex I activity in the mitochondrial electron transport chain, subsequently impairing mitochondrial function (Delgado-Camprubi et al., 2017). Both FBXO7 and parkin proteins have overlapping pathophysiologic mechanisms in autosomal recessive PD, which play important roles in mitophagy to clear damaged mitochondria. PD-linked FBXO7 mutants were reported to recruit parkin into damaged mitochondria and facilitate its aggregation. Furthermore, wild-type FBXO7 can rescue dopaminergic neuron degeneration induced by parkin deletion in Drosophila, suggesting that FBXO7 may be an independent upstream regulator of parkin (Zhou et al., 2016). Additionally, FBXO7 also regulates mitophagy by directly interacting with PINK1 and parkin (Kraus et al., 2023; Sanchez-Martinez et al., 2023).

PARK7

Mutations in the PARK7 gene encoding the protein DJ-1 can cause autosomal recessive early-onset PD. However, the oxidative damage of DJ-1 is involved in the development of late-onset sporadic PD (Chen et al., 2010). The effects of DJ-1 are anti-oxidative stress, transcriptional regulation, and maintenance of mitochondrial homeostasis, which play important roles in neuroprotection (Dolgacheva et al., 2019). DJ-1 has been reported to be involved in the autophagy regulatory mechanisms as an autophagy regulator. DJ-1 deficiency can impair the autophagic flux and affect soluble α-Syn clearance by autophagy manipulation in microglia (Nash et al., 2017). Overexpression of DJ-1 can enhance the expression of beclin-1 and LC3II and increase the ultrastructural signs of autophagy (Gao et al., 2012). Additionally, DJ-1 deficiency suppressed the CMA pathway via inhibiting the upregulation of LAMP 2A and decreasing the level of HSC70, which lead to the aggregation of α-Syn (Xu et al., 2017). Loss of DJ-1 leads to mitochondrial damage and accumulation of autophagic markers (LC3 puncta and lipidation) around the mitochondria in human dopaminergic cells (Thomas et al., 2011). Recently, Imberechts et al. found that DJ-1 is essential for PINK1/parkin-mediated mitophagy (Imberechts et al., 2022).

PD-genes related with lysosomal impairments

GBA

Heterozygous mutations of glycosylceramidase beta (GBA) gene encoding glucocerebrosidase (GCase) are associated with PD (Hopfner et al., 2020). GCase is a lysosomal enzyme that is synthesized in the endoplasmic reticulum (ER) and transported by the LIMP2. In the lysosomal compartment, GCase hydrolyses the simple glycosphingolipid-glucosylceramide (GlcCer) (Boer et al., 2020). It has been shown that there is a positive feedback loop regulation between GCase and α-Syn. Loss of function of GCase causes accumulation of α-Syn and neurotoxicity. GlcCer, a substrate of GCase, contributes to the aggregation of α-Syn by stabilizing soluble oligomeric intermediates. In turn, overexpression of α-Syn can inhibit the activity of GCase (Mazzulli et al., 2011; Bae et al., 2015). Additionally, mutation or deficiency of GBA has an influence on the activity of autophagy. Decreased levels of phopho-S6K, a marker of mTOR activity, and accumulation of Rab7 in GCase-deficient cells indicate the impairment of autophagy lysosomal reformation (ALR) (Magalhaes et al., 2016). Autophagic defects and impaired calcium homeostasis were found in iPSC-derived neurons from patients with GBA1-associated PD (Schondorf et al., 2014). Elevated mitochondrial content, mitochondrial oxidative stress, and defective autophagy have been found in the postmortem brain tissue of PD patients with heterozygous GBA L444P mutation, suggesting that GBA deficiency can also cause harm to the mitophagy pathways in PD (Li et al., 2019a). GBA plays a critical role in maintaining the function of normal lysosome and α-Syn homeostasis. Increased accumulation of poly-ubiquitinated proteins that are lysosomal substrates, abnormal accumulation of enlarged autophagic vesicles, and increased release of insoluble α-Syn have been reported in neuroblastoma cells with GBA knockout (Bae et al., 2015). In iPSCs-derived dopaminergic neurons from GBA-PD patients, Schondorf et al. (2014) found reduced GCase activity and increased accumulation of α-Syn. Additionally, GBA knockout or heterozygous mutation of GBA in mouse embryonic fibroblasts reduced the level of phopho-S6K, a phosphorylated substrate of mTOR that can be rescued by administration of recombinant GCase enzyme, suggesting the direct relationship between GBA and mTOR activity (Magalhaes et al., 2016).

ATP13A2

Mutations in the ATP13A2 gene are involved in autosomal-recessive PD (Ramirez et al., 2006). The ATP13A2 protein encoded by the ATP13A2 gene (locus PARK9) is a lysosomal type 5 P-ATPase and a lysosomal H⁺, K⁺-ATPase responsible for cation transport, which has been found to be decreased in dopaminergic neurons of PD patients and mostly accumulates within the LBs (Dehay et al., 2012; Fujii et al., 2023). Reduced lysosomal membrane stability, impaired degradation of lysosomal substrates, and accumulation of autophagosome and α-Syn have been reported in dopaminergic neurons with overexpression of ATP13A2 mutation or knockdown of ATP13A2 (Dehay et al., 2012; Usenovic et al., 2012; Tsunemi et al., 2019). In addition, depletion of ATP13A2 negatively regulates SYT11 (another PD-related gene) at the transcriptional and post-translational levels. Depletion of ATP13A2 leads to a reduction of SYT11 transcription that is mechanistically dependent on MYCBP2-induced ubiquitination of TSC2, which in turn leads to mTORC1 activation and a TFEB-mediated reduction of SYT11 transcription (Bento et al., 2016). It is interesting to note that ATP13A2 can recruit histone deacetylase 6 (HDAC6) to the lysosome to deacetylate cortactin, which in turn promotes autophagosome-lysosome fusion and degrades protein aggregates and damaged mitochondria (Wang et al., 2019).

TMEM175

Transmembrane protein 175 (TMEM175) is a K⁺ channel located in late endosomal and lysosomal membranes, which has been shown to regulate lysosomal pH stability and membrane potential, as well as lysosome-autophagosome fusion during autophagy (Cang et al., 2015). Mutation in the TMEM175 gene (p.M393T) is a common risk factor for PD (Jinn et al., 2019). TMEM175 deficiency or p.M393T mutation in neurons can impair lysosomal degradation, lysosome-mediated clearance of autophagosome, and the function of the mitochondrial respiratory chain, which in turn leads to an increased susceptibility of cells to exogenous α-Syn fibrils without affecting the endogenous level of α-Syn (Jinn et al., 2017, 2019). In an in vivo study, it was shown that TMEM175 deficiency leaded to lysosomal over-acidification, impaired proteolytic activity, and promoted α-Syn aggregation (Hu et al., 2022). In a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine plus probenecid (MPTP) mouse model of PD, knockout of TMEM175 alleviated motor impairment and reduced loss of dopaminergic neuron. Mechanically, activated TMEM175 increases the production of reactive oxygen species (ROS) through disruption of mitochondrial homeostasis, further activating TMEM175 and forms a positive feedback loop to promote cell apoptosis. This suggests that apoptosis is mediated by aberrant expression of TMEM175 in the pathogenesis of PD, while TMEM175 knockdown may be a potential molecular therapeutic target for PD (Qu et al., 2022).

Targeting the Autophagic-Lysosomal Pathway for potential therapy of Parkinson’s Disease

In recent years, it has been demonstrated that induction of autophagy by chemical or gene therapeutic strategies are beneficial in both cellular and animal models of PD, in which these therapeutic strategies can reduce intracellular aggregates and cell death (Polissidis et al., 2020).

The small-molecule regulators of ALP in PD

Various small-molecule compounds applied in PD models have been shown to promote the clearance of abnormal protein aggregates from neurons and/or protected neurons from apoptosis through the modulation of ALP (Table 2).

Table 2.

The small-molecule enhancers of ALP and their protective effects in PD

Molecule names	Mechanisms of action	Cells/animal models/patients	Clinical phase	Effects from PD models and/or clinical trials	References
Rapamycin	Increase lysosomal biogenesis and attenuate autophagosomes accumulation; Inhibit dephosphorylation of AKT by blocking the translation of RTP801	BE-M17 cells treated with MPP+; PC12 cell treated with 6-OHDA or MPP+, MPTP induced mouse model	N/A	Attenuate MPP+-induced cell death and MPTP-induced dopaminergic neurodegeneration in animals	Dehay et al., 2010; Malagelada et al., 2010
Temsirolimus	Enhance MTOCR1- mediated autophagy	MPTP induced mouse model	N/A	Ameliorate behavioral impairment, increase tyrosine hydroxylase and dopamine transporter expression, and reduce α-synuclein expression	Siracusa et al., 2018
DNL201	Inhibit LRRK2 kinase activity and enhance lysosomal function	LRRK2G2019S knockin mice; patients with PD	Phase I and phase Ib	Improve lysosome size and morphology and increase lysosomal protein degradation; well tolerated in clinical trials and have robust cerebrospinal fluid penetration and to significantly improve abnormal lysosomal biomarkers	Jennings et al., 2022
GSK3357679A	Inhibit LRRK2 kinase activity and enhance mitophagy	Primary MEF cultures established from LRRK2 WT, LRRK2 G2019S, and LRRK2 KO embryos	N/A	Rescue LRRK2 G2019S-associated mitophagy defects	Singh et al., 2021; Tasegian et al., 2021
PF-360	Inhibit LRRK2 kinase activity and enhance autophagy and lysosomal function	Rotenone induced mouse model; rats received a unilateral injection of AAV2-hSNCA	N/A	Prevent accumulation of pSer129-a-synuclein	Di Maio et al., 2018
BIIB122 (DNL151)	Inhibit LRRK2 kinase activity	Two randomized, double-blind, placebo-controlled studies in patients with mild to moderate PD and healthy participants	Phase I	Show well tolerated and no serious adverse events and reduce pS935-LRRK2 and pT73-Rab10 in peripheral blood and urine bis phosphate	Jennings et al., 2023
Ambroxol	Improve GCase activity by activating the CLEAR network	Fibroblasts from sporadic PD patients carried L444P or N370S heterozygous mutations in the GBA1 gene and healthy controls; α-Syn transgenic mice; fibroblasts from PD patients with homozygous E326K GBA mutation, α-Syn overexpressing SH-SY5Y cell; a single-center open-label noncontrolled clinical trial in patients with moderate PD	Phase I	Increase functional lysosomal mass; Decrease both α-Syn and phosphorylated α-Syn protein levels; Improve lysosomal biochemistry and oxidative stress, reduce α-Syn levels; Show safe and well tolerated; inhibit CSF GCase activity and increase total CSF α-Syn concentration and CSF GCase protein levels	McNeill et al., 2014; Ambrosi et al., 2015; Migdalska-Richards et al., 2016; Migdalska-Richards et al., 2017; Mullin et al., 2020
NCGC607	Restore GCase activity and protein levels	iPSC-derived dopaminergic neurons from PD patients	N/A	Reduce α-Syn levels and glycolipid storage	Aflaki et al., 2016
AT2101	Increase GCase trafficking and activity	Transgenic human wild-type α-Syn mice (Thy1-aSyn mice)	N/A	Improve motor and olfactory deficits, reduce α-Syn aggregates, abolish microglial inflammatory response in the substantia nigra	Richter et al., 2014
KYP-2047	Enhance macroautophagy	Transgenic homozygous a-Syn A30P mice	N/A	Enhance a-Syn clearance, increases striatal dopamine levels	Savolainen et al., 2014
Resveratrol	LC3 deacetylation and redistribution from the nucleus to the cytoplasm via activating SIRT1; upregulate autophagic flux via activating MEK/ERK pathway	MPTP induced mouse model; SH-SY5Y cells treated with rotenone	N/A	Enhance degradation of α-Syn; Protect against loss of dopaminergic neurons and motor impairment; Decrease ROS, apoptosis, mitochondrial membrane potential (Δψm) and mitochondria dynamics alteration, enhance both autophagic induction and autophagic flux	Guo et al., 2016; Lin et al., 2018
BL-918(33i)	Enhance the autophagic flux by triggering the ULK complex	SH-SY5Y treated with MPP+, MPTP-induced PD mouse model	N/A	Reverse MPP+-induced cell death, reduce loss of TH-positive neuron cells, attenuate the loss of DA and its metabolites and motor dysfunction	Ouyang et al., 2018
Nilotinib	Inhibit c-Abl kinase activity and enhance autophagy	Primary cortical neurons; A53T Tg mice; Patients with mild-to-moderate PD	Phase I; Phase II	Induce a-Syn degradation; Increase autophagic flux by inducing AMPK and ULK1 activation, and inhibiting mTORC1 activation; Has no significant clinical benefit	Mahul-Mellier et al., 2014; Karim et al., 2020; Pagan et al., 2020; Simuni et al., 2021
STI-571	Facilitate the nuclear translocation of TFEB via inhibiting c-Abl-GSK3b pathway; Inhibit phosphorylation of parkin by c-Abl	SN4741 cell and primary midbrain neurons treated with MPP+; SH-SY5Y cell treated with MPP+ and MPTP induced mouse model	N/A	Reverse ALP disfunction, protected against MPP+-induced neuronal cell death; Prevent the phosphorylation of parkin and maintain the catalytic activity of parkin	Ko et al., 2010; Ren et al., 2018
XCT 790	Induce mTOR-independent autophagy	MPTP induced PD mouse model	N/A	Alleviate dopaminergic neuronal loss and behavioral impairments, clear misfolded protein aggregates	Suresh et al., 2018
FCPR16	Enhance AMPK-dependent autophagy	SH-SY5Y cells treated with MPP+	N/A	Prevent production of ROS and the decline of Δψm, protect against MPP+-induced cell death	Zhong et al., 2019
Trehalose	Enhance autophagy	AAV a-Syn rat model; PC12 cells overexpressing A53T mutant α-Syn; Rotenone induced PD mouse model	N/A	Improve DA neuronal survival and inhibit α-Syn accumulation, attenuate behavioral impairment; Prompt degradation of A53T α-Syn; Ameliorate motor impairment and loss of nigral neurons, improve olfactory dysfunction and depressive-like behaviors and markedly reduce α-Syn and p62 deposition	Sarkar et al., 2007; Dehay et al., 2010; Lan et al., 2012; He et al., 2016; Moon et al., 2022; Casarejos et al., 2011
Curcumin	Enhance autophagy via downregulating mTOR/p70S6K signaling	SH-SY5Y cell overexpressing A53T α-Syn	N/A	Reduce the accumulation of A53T α-Syn	Jiang et al., 2013
Isorhynchophylline (IsoRhy)	Induce autophagy in a Beclin1-dependent manner	N2a cells overexpressing A53T and A30P a-Syn	N/A	Decrease the expression levels of wild-type and A53T a-Syn	Lu et al., 2012;
Corynoxine (Cory)	Induce autophagy through AKT/mTOR pathway	Inducible PC12/A53T-α-Syn	N/A	Promote the degradation of wild-type and A53T α-Syn	Chen et al., 2014
Glycyrrhizic acid	Enhance autophagy	SH-SY5Y cells treated with 6-hydroxydopamine (6-OHDA) and corticosterone (CORT)	N/A	Decrease the apoptosis, decrease the expressions of α-Syn and p-S1292-LRRK2 proteins	Yang et al., 2018
Piperlongumine	Enhance autophagy and phosphorylation of BCL2 at Ser70	Primary neurons and SK-N-SH cells treated with rotenone	N/A	Improve cell viability and enhance mitochondrial function, restore the balance between apoptosis and autophagy	Liu et al., 2018
Peiminine	Enhance the expression of PINK1/parkin and increase autophagy	SH-SY5Y cells treated with 6-OHDA, 6-OHDA induced Caenorhabditis elegans models	N/A	Reduce ROS production and DA neuron degeneration, improve the DA-mediated food-sensing behavior and prolonged their lifespan, diminish the accumulation of α-Syn	Hsu et al., 2021
Caffeic acid	Enhance autophagy via activating the JNK/Bcl-2 pathway	SH-SY5Y cells overexpressing A53T α-Syn, transgenic A53T α-Syn mice	N/A	Prompt A53T α-synuclein degradation, improve behavioral impairments, attenuate loss of dopaminergic neurons in SN	Zhang et al., 2019
Baicalein	Induce mitochondrial autophagy via inhibiting miR-30b and activating SIRT1/AMPK/mTOR pathway the NIX/BNIP3 pathway	6-OHDA induced rat model	N/A	Alleviate neuronal injury and partly recover mitochondrial dysfunction; Alleviate neurobehavioral defects, decrease the DA content in striatum, reduce apoptosis of neurons	Chen et al., 2021a, b; Kang et al., 2019
Caffeine	Enhance macroautophagy and CMA	A53T α-Syn fibrils induced mouse model	N/A	Blunt a cascade of pathological events including pSer129α-Syn-rich aggregates, apoptotic neuronal cell death, microglia, and astroglia reactivation	Yanan et al., 2018
Metformin	Enhance autophagy via activating AMPK; Inhibit mTOR activation and increase the JNK/c-jun pathway and FOXO3A;Enhance autophagy via activating AMPK	SH-SY5Y cells treated with MPP+, MPTP-induced mouse models; rotenone-induced rat models; PQ-induced mouse models	N/A	Improve motor impairment and increase dopamine level, prevent DA neuron degeneration and attenuate α-Syn accumulation; Prevents the depression-like behavior and motor deficits, as well as inhibits the increase of α-Syn; Ameliorated PQ-induced abnormal aggregation of α-Syn	Lu et al., 2016; Mendonca et al., 2022; Gopar-Cuevas et al., 2023
Rosuvastatin	Enhance macroautophagy and CMA	SH-SY5Y cells treated with rotenone	N/A	Reduce α-Syn expression and aggregation	Kang et al., 2017

ALP: Autophagic-lysosomal pathway; AMPK: AMP-activated protein kinase; c-Abl: nonreceptor tyrosine kinase Abelson; CSF: cerebrospinal fluid; DA: dopamine; mTOR: mammalian target of rapamycin; PD: Parkinson’s disease; ROS: reactive oxygen species; TH: tyrosine hydroxylase; ULK: UN51-like Ser/Thr kinases.

Small molecules or small-molecule inhibitors

Rapamycin and its analogs

Rapamycin, an inhibitor of mTOR, was identified as the first autophagy inducer (Sarkar et al., 2009). Rapamycin and its analog CCI-779 have been shown to reduce accumulation of α-Syn aggregates and protect neurons from death in both cellular and animal models of PD (Crews et al., 2010; Malagelada et al., 2010). Temsirolimus, a newly developed rapamycin analogue, has been shown to be protective against MPTP-induced neurotoxicity in animal models of PD. Temsirolimus ameliorates MPTP-induced behavioral impairment, increases tyrosine hydroxylase and dopamine transporter expression, and reduces α-Syn in the substantia nigra by enhancing mTOCR1-dependent autophagy (Siracusa et al., 2018). However, mTOR has diverse autophagy-independent functions, and its inhibition can lead to immunosuppression and impaired wound healing. Therefore, intra-neuronal targeted administration of mTOR inhibitors may be a better therapeutic strategy.

LRRK2 kinase inhibitors

Growing evidence in PD models suggests that lysosomal dysfunction is a common pathological feature of PD and suggests that therapeutic approaches targeting the improvement of PD-associated defects in lysosomal homeostasis may have a meaningful impact on PD progression (Wallings et al., 2019). The LRRK2 G2019S mutation results in enlarged lysosomes, diminishes the lysosomal degradation capacity, and leads to a reduction in lysosomal pH through an increase in its kinase activity (Henry et al., 2015). Therefore, LRRK2 kinase inhibitors could be useful for the treatment of PD. A recent preclinical and clinical study found that DNL201, which is a central nervous system (CNS)-penetrant, selective, ATP-competitive, small-molecule LRRK2 kinase inhibitor, improved lysosome size and morphology and increased lysosomal protein degradation in primary mouse astrocytes derived from LRRK2 G2019S knock-in mice by inhibiting LRRK2 kinase activity. In addition, administration of DNL201 partially restored the impaired lysosomal protein turnover in an aggressive cellular model of GBA-associated lysosomal dysfunction, suggesting that DNL201 treatment may improve lysosomal dysfunction not directly caused by LRRK2 mutants. Moreover, DNL201 was observed to be well tolerated in phase 1 and phase 1b clinical trials in patients with PD, to have robust cerebrospinal fluid penetration and significantly improve abnormal lysosomal biomarkers in vivo (Jennings et al., 2022). GSK3357679A is a potent and selective CNS penetrant LRRK2 kinase inhibitor, and recently showed that it can enhance mitophagy in dopaminergic neurons and microglia of normal genotype mouse brain and rescue LRRK2 G2019S-associated mitophagy defects (Singh et al., 2021; Tasegian et al., 2021). Di Maio et al. (2018) showed that wild-type LRKK2 kinase activity was enhanced in dopaminergic neurons of the substantia nigra region from patients with PD and in two different rat models of PD induced by adeno‐associated virus (AAV) 2-mediated -Syn overexpression and rotenone treatment. PF-360, another LRRK2 kinase inhibitor, prevents rotenone-induced activation of nigrostriatal LRRK2, which promotes pSer129-α-Syn autophagic degradation by improving lysosomal function and CMA, suggesting that PF-360 may be useful to treat patients with idiopathic PD who do not carry LRRK2 mutations (Di Maio et al., 2018). However, most of these studies are based on preclinical studies, which need further clinical validation of their therapeutic potential and side effects. Recently, in a randomized, double-blind, and placebo-controlled clinical study, BIIB122, a type I inhibitor of LRRK2, was well tolerated and showed no serious adverse events. Furthermore, dose-dependent median reductions of pS935-LRRK2 and pT73-Rab10 in peripheral blood and a decrease of urine bis (monoacylglycerol) phosphate, a lysosomal biomarker, were observed in both healthy volunteers and PD patients treated with BIIB122. These results suggest that BIIB122 can inhibit the activation of peripheral LRRK2 kinase and regulate its downstream lysosomal pathway (Jennings et al., 2023). Additionally, Christensen et al. (2017) and Wojewska and Kortholt (2021) have reviewed that some other LRRK2 kinase inhibitors such as MLi-2, GNE-0877, GNE-7915, GSK257821, HG-10-102-1, LRRK2-IN, and PF-064474 may have protective effects on PD, but there are currently no preclinical model studies of PD modeled by LRRK2 dysfunction.

Small molecule chaperone of GCase

In PD, mutations in GBA lead to reduction of GCase activity that subsequently cause severe lysosomal dysfunction and accumulation of α-Syn aggregates. Therefore, increasing GCase activity in the CNS may be a potential therapeutic strategy for PD (Abeliovich et al., 2021). Ambroxol is a small-molecule chaperone used for respiratory diseases, which has been proven to increase GCase activity and reduce expression of α-Syn in vitro and in vivo (Migdalska-Richards et al., 2016, 2017). Ambroxol can upregulate the mRNA and protein expression levels of GCase as well as its activity, and increase the expression of the GCase transporter LIMP2. Notably, ambroxol may block macroautophagy flux and drive cargo towards the secretory pathway (Magalhaes et al., 2018). In a single-center, open-label, noncontrolled clinical trial, the results after 186 days of ambroxol administration compared to baseline showed that ambroxol could significantly inhibit CSF GCase activity and increase blood leukocyte GCase activity, total CSF α-Syn concentration, and CSF GCase protein levels (Mullin et al., 2020). In addition, clinical studies of ambroxol in Parkinson’s disease dementia and dementia with LB are in phase IIa (Silveira et al., 2019; Chwiszczuk et al., 2023). Moreover, NCGC607, a small-molecule non-inhibitory chaperone of GCase, has been shown to increase GCase activity and protein levels and reduce α-Syn levels in iPSC-derived dopaminergic neurons (Aflaki et al., 2016). AT2101 (afegostat-tartrate, isofagomine), another pharmacological chaperone for GCase, has been shown to increase GCase trafficking and activity. Furthermore, it also abolishes microglial inflammatory response and reduces the number of α-Syn aggregates in the substantia nigra, thereby improving motor function and olfactory deficits of transgenic mice overexpressing human wild-type α-Syn under the Thy-1 promoter (Thy1-aSyn; Richter et al., 2014).

KYP-2047

Prolyl oligopeptidase (PREP), a serine protease that functionally hydrolyzes peptides of less than 30 amino acids, can accelerate the aggregation process of α-Syn (Brandt et al., 2008). KYP-2047, an inhibitor of PREP, can promote α-Syn aggregates through enhancement of macroautophagy. Additionally, KYP-2047 also increases striatal dopamine levels of α-Syn A30P transgenic mouse (Savolainen et al., 2014).

Resveratrol

Resveratrol (RV) is a polyphenolic compound extracted from grapes and red wine, which has been proven to protect against loss of dopaminergic neurons, decrease in dopamine levels, and motor impairment in MPTP-induced mouse models of PD. Mechanistically, treatment with RV activated SIRT1, which subsequently led to LC3 deacetylation and redistribution from the nucleus to the cytoplasm, enhanced the autophagic degradation of α-Syn, and this neuroprotective effect was antagonized by EX527, a specific inhibitor of SIRT1 (Guo et al., 2016). Additionally, in the rotenone-induced oxidative stress SH-SY5Y cellular model, RV administration exerted neuroprotective effects by improving rotenone-induced mitochondrial dynamics alteration and enhancing autophagic flux through upregulation of mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway (Lin et al., 2018).

BL-918

UNC-51-like kinase 1 (ULK1) is a Ser/Thr kinase and an initiator of autophagy. BL-918 (33i), a potent activator of ULK1 by structure-based drug design, effectively induces autophagy via the ULK1 complex activation and subsequently protects against MPTP-induced motor dysfunction and loss of dopaminergic neurons in a mouse model of PD (Ouyang et al., 2018).

c-Abl inhibitor

Studies in PD animal models and PD patients have shown that nonreceptor tyrosine kinase Abelson (c-Abl) was activated (Mahul-Mellier et al., 2014; Marin et al., 2022; Xiao and Tan, 2023). c-Abl can interact with α-Syn and phosphorylate its tyrosine residues at positions 39 and 125, thus preventing its degradation though autophagy and proteasome pathways (Mahul-Mellier et al., 2014). Nilotinib, a specific inhibitor of c-Abl kinase activity, can induce α-Syn degradation via enhancement of the autophagy and proteasome pathways (Mahul-Mellier et al., 2014). Karim et al. (2020) found that nilotinib or pifithrin-α, an inhibitor of p53, increases autophagic flux in neuronal cells by inducing AMP-activated kinase (AMPK) and ULK1 activation, and inhibiting mTORC1 activation, which suggests that c-Abl activation likely inhibits autophagy in a p53-dependent manner. However, two randomized clinical trials in patients with mild-to-moderate PD showed that nilotinib had no significant clinical benefit, possibly related to its drug properties of difficulty in crossing the blood-brain barrier (BBB; Pagan et al., 2020; Simuni et al., 2021). STI-571, another c-Abl inhibitor, has also been shown to provide neuroprotective effects via enhancing the function of ALP in PD. Mechanically, STI-571 promoted the nuclear translocation of TFEB via inhibition of the c-Abl-GSk3β pathway, and subsequently prevented MPP⁺-induced ALP defects and neuronal cell death (Ren et al., 2018). In addition, c-Abl leads to parkin inactivation through phosphorylating tyrosine 143 of parkin, thereby inducing the accumulation of aminoacyl-tRNA synthetase-interacting multifunctional protein type 2 and fuse-binding protein 1, the substrates of parkin. STI-571 can effectively prevent the phosphorylation of parkin and maintain the catalytic activity of parkin (Ko et al., 2010). Moreover, STI571 can protect against MPTP-induced oxidative stress by inhibiting phosphorylation of p38α by c-Abl, which can in turn protect against oxidative stress-induced PD (Wu et al., 2016). These results suggest that STI571 may be a neuroprotective molecule in the treatment of PD, but further clinical validation is needed.

XCT 790

An emerging study has shown that estrogen-related receptor α (ERRα) can keep autophagic flux by inhibiting autophagosome formation. XCT 790, one of the most selective inverse agonists of ERRα, cleared α-Syn aggregation by inducing the mTOR-independent autophagy, which exerted neuroprotective effects in the dopaminergic neurons, and ameliorated motor co-ordination deficits of a mouse model of PD (Suresh et al., 2018).

FCPR16

FCPR16, a novel phosphodiesterase 4 inhibitor, has protective effect against MPP⁺-induced toxicity and oxidative stress via enhancing AMPK-dependent autophagy (Zhong et al., 2019).

Natural small-molecule compounds

Natural plant compounds have been considered relatively safe, with fewer side effects, and are expected to become important agents for treatment of PD in the future. The effects of these natural small molecule compounds in the regulation of autophagy have received more attention.

Trehalose

Trehalose, a non-reducing disaccharide, which is a Food and Drug Administration (FDA)-approved compound has been proven to mediate transcriptional upregulation of a series of autophagy-related genes including LC3, Beclin1, SQSTM1, and Atg5 (Castillo et al., 2013). Existing literature supports the therapeutic effect of trehalose against neurodegeneration in PD mainly through the induction of mTOR-independent autophagic pathway. In animal and cellular models of PD, trehalose accelerated the clearance of α-Syn aggregates, enhanced dopamine neuronal survival, and improved the motor and non-motor deficits (He et al., 2016; Moon et al., 2022; Gopar-Cuevas et al., 2023). Additionally, in epoxomicin-treated NB69 cells, trehalose increased autophagosomes and autophagic markers in a dose- and time-dependent manner. Furthermore, trehalose alleviated intracellular aggregation of α-Syn via reducing activation of ERK and chaperone HSP-70, which in turn prevented epoxomicin-induced cell death (Casarejos et al., 2011). This result indicates that trehalose acts synergistically with other cellular machinery to tackle the proteotoxic load of aggregated proteins. However, it was found that in the absence of trehalose-specific transporter, cells were largely insensitive to trehalose (Kikawada et al., 2007). Therefore, whether trehalose and its derivatives can easily cross the BBB and play protective effects in the nervous system needs further investigation in more animal experiments.

Curcumin

Curcumin, a natural polyphenolic compound derived from turmeric, has low toxicity in normal cells (Ahsan et al., 1999). In A53T α-Syn overexpressing SH-SY5Y cells, curcumin could effectively reduce the accumulation of A53T α-Syn by downregulating mTOR/p70S6K signaling and restoring abnormal macroautophagy (Jiang et al., 2013). Compound E4, a curcumin derivative, promotes TFEB nuclear translocation mainly through AKT-mTORC1 inhibition, accompanied by enhanced autophagy and lysosomal biogenesis, which in turn promotes degradation of α-Syn and protects neuronal cells from MPP⁺-induced cytotoxicity (Wang et al., 2020). A recent study showed that a curcumin analogue-based nanoscavenger (NanoCA) was able to trigger both autophagy and calcium-dependent exosome secretion to clear α-Syn by stimulating nuclear translocation of TFEB. Moreover, brain-targeted administration of NanoCA promoted clearance of monomers, oligomers, and aggregates of α-Syn from the brain in an MPTP mouse model and significantly ameliorated behavioral deficits in PD mice (Liu et al., 2020).

Isorhynchophylline and corynoxine

Isorhynchophylline (IsoRhy) and corynoxine (Cory) are two major tetracyclic oxindole alkaloids isolated from Uncaria rhynchophylla (Miq.) Jacks (Gouteng in Chinese), which have been shown to promote clearance of wild-type, A53T, and A30P α-Syn mutants and α-Syn oligomers in neuronal cells through enhancement of ALP (Lu et al., 2012; Chen et al., 2014). Interestingly, IsoRhy-induced autophagy is dependent on the function of beclin1, but independent on the mTOR pathway; whereas, Cory enhances autophagy mainly through inhibition of the AKT/mTOR pathway.

Glycyrrhizic acid

Glycyrrhizic acid (GA) is a major bioactive component of Radix glycyrrhizae, which is used as an alternative medicine. In 6-OHDA- and corticosterone (CORT)-induced Parkinson’s disease dementia cell models, GA significantly inhibited 6-OHDA- and CORT-induced apoptosis through activation of autophagy and decreased the expression of α-Syn and p-S1292-LRRK2 proteins (Yang et al., 2018).

Piperlongumine

Piperlongumine is an alkaloid extracted from Piper longum L., which has anti-inflammatory and anti-cancer effects (Han et al., 2014). Liu et al. (2018) found that oral administration of piperlongumine for 4 weeks significantly attenuated rotenone-induced locomotor deficits and prevented the loss of dopaminergic neurons in the substantia nigra of mice. Furthermore, mechanistic studies revealed that piperlongumine inhibited apoptosis and induced autophagy through enhancement of Bcl2 (B cell leukemia/lymphoma 2) phosphorylation at Ser70, which in turn enhanced mitochondrial function and improved cell viability (Liu et al., 2018).

Peiminine

Peiminine extracted from Fritillaria thunbergii Miq, has significantly antioxidant and anti-neuroinflammatory effects. Mechanically, peiminine increased autophagy and ubiquitin-proteasome system via enhancement of the expression of PINK1/parkin, which subsequently decreased the accumulation of α-Syn (Hsu et al., 2021).

Caffeic acid

Caffeic acid (CA) is one of the active components of Radix Salviae Miltiorrhizae, which exhibits anti-inflammatory, antioxidant, and anti-cancer activity and has been shown to improve cognitive function (Wang et al., 2016b; Kassa et al., 2021; Bastidas et al., 2022; Kim et al., 2023). Zhang et al. (2019) reported that CA reduced expression of A53T mutant α-Syn through activation of the JNK/Bcl-2-mediated autophagic pathway. Furthermore, CA ameliorated motor deficits in A53T α-Syn transgenic mice and attenuated the loss of dopaminergic neurons in mice SNpc.

Baicalein

Baicalein is a natural compound that impacts the expressions of miRNAs that are regarded as a regulator for cell apoptosis and autophagy in cancer (Yan et al., 2018b; Deng et al., 2020). In a rat model of 6-OHDA-induced PD, administration of baicalein inhibited miR-30b-5p expression, which in turn activated mitochondrial autophagy via activating the SIRT1/AMPK/mTOR and NIX/BNIP3 pathways (Chen et al., 2021a, b). Additionally, in 6-OHDA-treated PC12 cells, baicalein reduced 6-OHDA-induced cell damage via down-regulation of miR-192-5p, and inhibition of the PI3K/AKT signaling pathway (Kang et al., 2019).

Caffeine

Evidence from both epidemiological and experimental studies showed that caffeine has a neuroprotective effect on PD and is a strong protective environmental factor (Kardani and Roy, 2015). In a mouse model of PD, chronic caffeine treatment selectively reversed α-Syn-induced macroautophagy defects and CMA, but did not affect normal striatal autophagic processes, which in turn reduced pSer129 α-Syn-rich aggregates and neuronal apoptosis, suggesting that caffeine exerts neuroprotective effects by targeting the autophagic pathway (Yanan et al., 2018).

Clinical drugs

Metformin

AMP-activated protein kinase (AMPK) is the upstream mechanistic target of mTORC1, and activated AMPK activates autophagy by inhibiting mTORC1 activity (Egan et al., 2011). Metformin, a typical oral hypoglycemic drug for type 2 diabetes, has been identified as an activator of AMPK, which can induce macroautophagy and mitophagy by prompting AMPK activation (Li et al., 2018; Chen et al., 2022; Ma et al., 2022). In MPTP-induced mouse model and MPP⁺-treated SH-SY5Y cells, metformin activated AMPK and subsequently enhanced autophagy to exert its neuroprotective effects (Lu et al., 2016). In addition to motor symptoms, depression is a major non-motor disorder symptom in PD patients. A recent study found that metformin significantly prevents the depression-like behavior and motor deficits, as well as inhibits the increase of α-Syn in rotenone-induced rat models. Mechanistically, metformin enhanced autophagy through inhibition of mTOR activation, increasing the JNK/c-jun signaling pathway and autophagy transcription factor FOXO3A (Mendonca et al., 2022). In paraquat (PQ)-induced mouse models, metformin enhanced autophagy by phosphorylating AMPK, which in turn ameliorated PQ-induced abnormal aggregation of α-Syn (Gopar-Cuevas et al., 2023).

Rosuvastatin

Treatment with rosuvastatin, a drug prescribed for reducing the levels of low-density lipoprotein (LDL), counteracted rotenone-induced neurotoxicity by increasing the expressions of upstream autophagy markers, including Beclin-1 and AMPK through an mTOR-independent manner in rotenone-induced SY5Y cell models (Kang et al., 2017).

Genetic approaches targeting for ALP in PD

Coding genes

Beclin1

Beclin 1 is a coiled-coil protein with a molecular weight of 60 kDa that plays an essential role in the initial steps of ALP (Furuya et al., 2005; Hill et al., 2019). In B103 neuronal cells overexpressing α-Syn, co-expression of beclin 1 alleviated autophagy deficits and reduced the accumulation of α-Syn, as well as ameliorated associated neuritic alterations. Furthermore, overexpression of beclin 1 in α-Syn transgenic mice significantly enhanced autophagy and lysosomal activity, improving the synaptic and dendritic pathology and decreasing α-Syn accumulation in the limbic system without any significant deleterious effects (Spencer et al., 2009). Similarly, in a PD rat model overexpressing α-Syn, overexpression of beclin-1 in the substantia nigra promoted sustained activation of the ALP pathway, reduced α-Syn oligomer formation, and resulted in increased protein expression in the majority dopaminergic neurons (Decressac et al., 2013).

TFEB

TFEB is a master regulator of ALP and its activation has been widely demonstrated to significantly ameliorate pathological changes in neurodegenerative diseases (Jiao et al., 2023). Under normal conditions, TFEB is inactive and mainly present in the cytoplasm. However, under stressful circumstances such as starvation or lysosomal dysfunction, TFEB translocates to the nucleus and promotes the transcription of genes associated with autophagy and lysosomes. The nuclear translocation of TFEB is mainly controlled by its phosphorylation status (Sardiello et al., 2009; Settembre et al., 2011). In postmortem human PD midbrains and the AAV‐α‐Syn rat model, TFEB has been reported to significantly reduce nuclear expression and co-localize with α-Syn and 14-3-3 in the cytoplasm (Decressac et al., 2013). In recent years, the neuroprotective effect of TFEB in PD has attracted more attention. TFEB overexpression can prompt the clearance of α-Syn aggregation in cells (Arotcarena et al., 2019). Interestingly, in the MPTP mouse model, overexpression of TFEB abrogates MPTP-induced neuronal atrophy and preserves neuronal integrity. Furthermore, TFEB overexpression can activate MAPK 1/3 and AKT pro-survival pathways and promote protein synthesis (Torra et al., 2018), suggesting that TFEB may counteract neurodegeneration by improving the dysfunction of ALP and other biological processes in PD.

LAMP2

Lysosome-associated membrane glycoprotein 2 (LAMP2) is a lysosomal membrane protein with a molecular weight of 45 kDa that contributes to the maintenance of lysosomal stability and the protection of the inner surface of the lysosomal membrane from hydrolytic enzymes (Fukuda, 1991; Grasland et al., 1998). Numerous studies have reported that LAMP2 concentrations in CSF reduced in patients with PD (Boman et al., 2016; Klaver et al., 2018). In the Drosophila brain, neutrally expressed LAMP2A markedly upregulated expression of Atg5 and increased formation of autophagosomes. Furthermore, LAMP2A overexpression reduces neuronal α-Syn accumulation and prevents α-Syn A30P mutation–induced locomotor defects, as well as prevents ROS accumulation and oxidative defects (Issa et al., 2018). Overexpression of LAMP2A in the rat substantia nigra through injection of recombinant AAV vectors can effectively increase the survival of dopaminergic neurons located in the substantia nigra and the axon terminals located in the striatum. In SH-SY5Y overexpressed wild-type a-Syn, overexpression of LAMP2A led to upregulation of CMA and selective protection against wild-type a-Syn neurotoxicity (Xilouri et al., 2013). These results suggested that overexpression of LAMP2A may be a novel therapeutic strategy in PD.

ITPKB

Inositol-1,4,5-trisphosphate (IP3) kinase B (ITPKB) is a ubiquitously expressed lipid kinase that phosphorylates IP3 leading to its inactivation and consequently inhibits IP3-mediated calcium release from the endoplasmic reticulum (ER) (Miller et al., 2015). It has been shown that ITPKB expression is increased in brain regions associated with PD pathogenesis such as the SNpc, striatum, and cerebral cortex (Zhang et al., 2016). Recently, a study has revealed that ITPKB overexpression promotes the degradation of phosphorylated, insoluble α-Syn by inhibiting calcium transport from the ER to the mitochondria, which in turn enhances the initiation of autophagy (Apicco et al., 2021). However, the regulatory mechanism by which ITPKB promotes α-Syn degradation is currently unknown.

Noncoding RNAs

Dysregulation of noncoding RNAs in PD can affect multiple pathways such as autophagy and apoptosis.

MiRNAs

MiRNAs are highly conserved, short, noncoding RNAs consisting of approximately 22 nucleotides, which regulate gene expression at the post-transcriptional level (Ranganathan and Sivasankar, 2014). Mechanistically, miRNAs bind specifically to the 3-untranslated region (UTR) of the target mRNA, thereby inhibiting mRNA translation or inducing mRNA degradation (Qadir et al., 2020). A systematic review showed that aberrant expression of various miRNAs plays a critical role in neurodegenerative diseases, including PD (Juzwik et al., 2019). MiRNA-124 is one of the brain-specific and highly expressed miRNAs whose expression is down-regulated in PD (Kanagaraj et al., 2014). MiR-124 overexpression in LPS-treated BV2 cells inhibits the secretion of pro-inflammatory cytokines in cells and promotes autophagy by suppressing the expression of p62 and p38 (Yao et al., 2019). Overexpression of miR-124 alleviated the accumulation of autophagosomes and lysosome depletion through inhibiting the expression of Bcl-2-interacting mediator of cell death (Bim), a BH3-only protein (Wang et al., 2016a). A previous study demonstrated that under normal conditions, Bim inhibits autophagosome formation through direct interaction with beclin1, whereas in response to stress such as starvation conditions, the interaction between Bim and beclin1 is disrupted, which ameliorates autophagy inhibition (Luo et al., 2012). Therefore, whether overexpression of miR-124 enhances autophagy by affecting the interaction between Bim and beclin1 in the PD model requires further investigation. Conversely, in an in vitro study, miR-124 overexpression was proven to significantly inhibit dopaminergic neuronal apoptosis and suppress autophagy by inhibiting the AMPK/mTOR pathway. Furthermore, miR-124 suppression significantly increased autophagy-associated protein expression, including beclin1 and the ratio of LC3 II/I compared with that in controls (Gong et al., 2016). Esteves et al. (2022) recently used extracellular vesicles derived from umbilical cord blood mononuclear cells as a biological vehicle to effectively deliver miR-124-3p to the brain. They found that miR-124-3p-loaded extracellular vesicles induced neuronal differentiation and protected N27 cells against 6-OHDA-induced toxicity in vitro. Moreover, the extracellular vesicles loaded with miR-124-3p were injected into the right lateral ventricles of mice and could prevent the loss of dopaminergic neurons in the substantia nigra and striatum and fully counteract motor deficits in PD mice. However, the formulation did not increase the number of newborn neurons in the lesioned striatum induced by 6-OHDA in mice (Esteves et al., 2022). MiR-7, which is expressed mainly in neurons, has been shown to bind directly to the 3′-UTR of α-Syn and down-regulate its mRNA and protein levels (Junn et al., 2009; Doxakis, 2010). Furthermore, in differentiated ReNcell VM cells, miR-7 also accelerated the clearance of α-Syn aggregates through enhancement of autophagy and promoted the degradation of pre-formed fibrils of -Syn transported from the extracellular space, further suggesting that miR-7 may be an important molecular target for the clearance of α-Syn in PD (Choi et al., 2018). In 10-month-old A53T α-Syn mice treated with chronic restraint stress (CRS), the expression of RTP801 was upregulated, which in turn triggered an autophagy obstacle, increased the accumulation of oligomeric α-Syn, and further aggravated ER stress. Moreover, miR-7 downregulation could inhibit RTP801 expression, reversing RTP801-induced autophagy impairment and α-Syn accumulation, thereby acting as a neuroprotective agent (Zhang et al., 2018). In α-Syn-overexpressed SH-SY5Y cells, miR-320a can decrease HSC70 expression at both protein and mRNA levels via specifically targeting the 3′ UTR of HSC 70, which would subsequently result in the intracellular accumulation of α-Syn (Li et al., 2014). The PTEN/AKT/mTOR/GSK3β signaling pathway plays important roles in autophagy. In MPP⁺-treated PC12 cells, increasing miR-199a expression inhibited autophagy and ameliorated MPP⁺-induced cell damage via inhibiting GSK3β expression, thereby activating the PTEN/AKT/mTOR signaling pathway (Ba et al., 2020). Additionally, many miRNAs also regulate mitophagy by targeting PINK1 or parkin. MiR-421 is highly expressed in MPTP-treated mouse models and in MPP⁺-treated SH-SY5Y cells, which inhibits mitophagy via directly targeting PINK1 and decreasing the expression of PINK1 and parkin (Dong et al., 2022b). Kim et al. (2016) showed that miR-27a and miR-27b can inhibit PINK1 expression by directly binding to the 3′-UTR of PINK1 mRNA and block the mitophagic influx. Moreover, reduction of ubiquitin phosphorylation, parkin translocation, and LC3-II accumulation were observed in damaged mitochondria, indicating a negative feedback regulation between PINK1-mediated mitophagy and miR-27a and miR-27b (Kim et al., 2016). Additionally, miR-106b 3p was downregulated in plasma extracellular vesicles (EVs) from PD patients compared to controls, suggesting that plasma EVs miR-106b may be a potential diagnostic biomarker for PD (Xie et al., 2022). Recently, it has been found that miR-106b shuttled by mesenchymal stem cells (MSCs)-derived extracellular EVs can increase the LC3II/I ratio, expression of Bcl-2 protein, and neuronal survival, and decrease Bax expression via inhibiting the expression of CDKN2B in MPTP-induced PD mouse models (Bai et al., 2021).

Circular RNAs

Circular RNAs (circRNAs) are peculiar noncoding RNAs that are known to be associated with various illnesses (Westholm et al., 2014). Recent reports implicated the participation of circRNAs in the pathogenesis of PD and considered them to be potential targets for PD therapy (Kong et al., 2021). Increased circSLC8A1 expression was observed in the substantia nigra of patients with PD and in cells exposed to the oxidative stress inducer—paraquat, which may produce oxidative stress via regulating miR-128 function and/or activity (Hanan et al., 2020). Recently, Zhang et al. (2023) demonstrated that expression of circular RNA homeodomain interacting protein kinase 3 (circHIPK3) was elevated in the serum and cerebral fluids of PD patients, LPS-treated BV2 cells, and conditioned SH-SY5Y medium. Furthermore, circHIPK3 could directly interact with and lower miR-124 expression, which subsequently promoted the activation of NLRP3 inflammasome through activation of the STAT3 signaling (Zhang et al., 2023). CircSAMD4A was found to be upregulated in PD animals and cellular models and promoted activation of AMPK/mTOR signaling pathway through the inhibition of miR29c3p expression, which in turn promoted MPTP or MPP⁺-induced apoptosis and autophagy (Wang et al., 2021a). CircDLGAP4 expression was decreased in PD mice and cellular models, whereas in vitro overexpression of circDLGAP4 can reduce mitochondrial damage, enhance autophagy, and attenuate MPP⁺-induced neurotoxicity. Mechanistically, circDLGAP4/miR-134-5p axis activates CREB signaling and increases the expression of its downstream target genes including BDNF, Bcl-2, and PGC-1a (Feng et al., 2020).

LncRNAs

Long non-coding RNAs (lncRNAs) are ncRNAs of more than 200 nucleotides in length that are involved in alternative splicing, transcriptional, and post-transcriptional regulation of genes; they also interact with miRNAs like a sponge, and whose dysfunction is closely associated with the development of many diseases (Peng et al., 2017). It has been confirmed that lncRNAs are involved in brain development and neuronal maintenance, and there is a growing interest in their role in PD (Riva et al., 2016). However, their role in the regulation of autophagy in PD is still unclear. Small nucleolar RNA host gene 1 (SNHG1) is a well-studied lncRNA whose expression is reportedly upregulated in PD cellular and animal models. Downregulation of SNHG1 can attenuate MPP⁺-induced cytotoxicity and decrease the autophagy levels by binding to and upregulating the miR-221/222 and inhibiting p27/mTOR pathway, which suggests that SNHG1 may be a therapeutic target for PD (Qian et al., 2019). Fan et al. (2020) found that the lncRNA brain-derived neurotrophic factor anti-sense (BDNF-AS) knockdown significantly promoted cell proliferation in MPP⁺-treated SH-SY5Y cell models, and inhibited apoptosis and autophagy by regulating miR-125b-5p in PD mouse models. In MPP⁺-treated SK-N-SH cells, the expression of lncRNA HOX transcript antisense RNA (HOTAIR) was upregulated, which promoted MPP⁺-induced neuronal injury via upregulating the expression of ATG10 (Zhao et al., 2020). Similarly, interfering lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) in mice effectively inhibits MPTP-induced autophagy and alleviates dopaminergic neuronal damage by targeting miR-107-5p or miR-374c-5p (Dong et al., 2021, 2022a). Additionally, NEAT1 can positively regulate the expression of PINK1 via inhibiting the degradation of PINK1 protein (Yan et al., 2018a). Polo-like kinase 2 (PLK2), a serine/threonine kinase upregulated in synucleinopathy-diseased brains, interacts with and phosphorylates the Ser129 site of α-Syn to enhance its selective clearance via the ALP degradation pathway (Oueslati et al., 2013). Furthermore, polyubiquitination of PLK2 by binding to the N-terminal region of α-Syn is the important mechanism for PLK2-mediated α-Syn degradation (Dahmene et al., 2017). Recently, it has been shown that lncRNA opa interacting protein five antisense RNA 1 (OIP5-AS1) can activate the PLK2-mediated autophagy by targeting miR-126 and reduce α-Syn aggregation and cell apoptosis in MPP⁺-treated SH-SY5Y cells (Song and Xie, 2021). Additionally, lncRNA homeobox DNA-binding antisense growth-associated long noncoding RNA (HAGLROS) was increased in MPTP-induced PD mouse models and MPP⁺-treated SH-SY5Y cells. Moreover, suppression of HAGLROS reduced autophagy and alleviated MPP⁺-induced cellular damage by activating the miR-100-mediated PI3K/AKT/mTOR pathway (Peng et al., 2019).

New strategies targeting α-synuclein degradation through ALP

Abnormal aggregation of α-Syn is an important etiologic factor leading to neuronal degeneration in PD. Targeting to promote the degradation of α-synuclein aggregates is a potentially effective option for the treatment of PD. In recent years, many emerging approaches such as nanoparticle technology, microbubbles combined with focused ultrasound (FUS), and autophagy-targeting chimera have been proven to promote the degradation of α-Syn aggregates through ALP.

Microbubbles and FUS

A large number of preclinical studies have revealed that some small molecules can protect against neuronal damage in PD through induction of autophagy. However, whether these small molecules can effectively cross the BBB and carry out their biological activities needs to be further investigated. Recently, it was found that FUS can mediate the opening of the BBB and improve the penetration of therapeutic drugs including chemotherapeutic agents, gene drugs, and nanoparticles, which have been widely used in drug delivery to the brain (Kamimura et al., 2019; Vince et al., 2021; Yang et al., 2021). Furthermore, in the presence of microbubbles (MBs), FUS can achieve local, transient, and reversible opening of the BBB (de Jong et al., 1992; Fan et al., 2015). However, naked MBs have a relatively low carrying efficiency, because it is unable to distinguish the target organ (Mayer et al., 2008). Triptolide (T10), a monomeric compound isolated from the traditional Chinese medicine Tripterygium wilfordii Hook F, has been shown to be an inducer of autophagy. Previous studies have shown that T10 can promote the degradation of α-Syn in vitro, but the efficiency of T10 in crossing the BBB is low in vivo (Zhou et al., 2012; Hu et al., 2017). Recently, Feng et al. (2022) developed a novel AHNAK-targeted microbubbles integrating T10 (T10-AHNAK-MBs) by using the film hydration method, which showed that FUS (T10-AHNAK-MBs-FUS) could significantly carry T10 into the substantia nigra by crossing the BBB, promote the clearance of various forms of α-Syn through ALP, and alleviate motor deficits of PD mouse models (Feng et al., 2022). However, in this study, they also found some limitations in the clearance of α-Syn aggregates by increasing doses of T10 and poor clearance of high molecular weight α-Syn aggregates by T10, suggesting that the function of T10-targeted autophagy for the treatment of PD needs further observation (Feng et al., 2022).

Nanotechnology

With the development of nanotechnology, nanomaterials are attracting increasing attention in the fields of drug delivery, diagnostics, and medical imaging (Chaturvedi et al., 2019; Pellico et al., 2021; Girija et al., 2022). Porous silica nanoparticles (SiO₂-NPs) have a wide range of applications in CNS research owing to their unique biological properties such as specific surface area and large porosity, controllable particle size, and good biocompatibility (Wang et al., 2015). Recently, it was shown that SiO₂-NPs promoted α-Syn aggregation and induced autophagy through inhibiting PI3K-Akt-mTOR signaling pathway in PC12 cells, which suggested that SiO₂-NPs negatively regulate the autophagy (Xie and Wu, 2016). Poly(lactic acid)-poly(ethylene glycol) (PLGA) acidic nanoparticles (aNP), a biodegradable synthetic material that has been approved by the U.S. Food and Drug Administration (FDA) for clinical therapeutic use, reportedly entered the lysosome and improved lysosomal pH and degradative function (Lu et al., 2009; Baltazar et al., 2012). Other studies showed that PLGA-aNP restored impaired lysosomal function by lowering lysosomal pH, enhanced α-syn degradation through ALP induction, and attenuated nigrostriatal dopaminergic neurodegeneration in LB-injected mice and MPTP-induced PD mouse model (Bourdenx et al., 2016; Arotcarena et al., 2022). These results indicate that PLGA-aNPs have a protective effect on lysosomal function. If PLGA-aNPs are used in combination with autophagy-inducing drugs, it may be more helpful for the degradation of macromolecular aggregates in neurodegenerative diseases, but this needs to be verified by more studies. HSA/Se nanoparticle (HSA/Se NPs), is a human serum albumin (HSA)-based selenium nanosystem characterized by low toxicity and high efficiency. Oral administration of HSA/Se NPs can cross the intestinal epithelial barrier (IEB) and BBB to enrich DA neurons and ameliorate behavioral deficits and DA neuron death in MPTP model mice. Mechanistically, HSA/Se NPs reduced MPTP-induced oxidative stress by activating the Keap1-Nrf2-SOD pathway (Xu et al., 2023). It has been shown that phosphorylation of p62 activated the Keap1-Nrf2 pathway during selective autophagy, which suggests that HSA/Se NPs may have a regulatory role in selective autophagy (Ichimura et al., 2013).

It has been shown that some autophagy agonists significantly ameliorate neuronal degeneration and motor deficits in PD model animals, but the delivery of these molecules to the brain remains a challenge. Currently, the main obstacle for the brain-targeted therapies is crossing the BBB, which can only be crossed by administering high doses of the drug to obtain effective concentrations in the brain (Silva Adaya et al., 2017). Recently, nanoparticle-mediated drug delivery therapies have gained attention for their superiority over conventional therapies (Khare et al., 2023). These delivery systems provide sustained release of the drug over time, which reduces the frequency of administration and the risk of dose dumping associated with other traditional controlled release systems (Jedinger et al., 2014; Saludas et al., 2018). In addition, the surface of NPs can make it easier for drugs to enter the brain by binding to specific ligands on the BBB cells (Re et al., 2012; Johnsen et al., 2017).

Although nanomaterials have better prospects for application, these nanomaterials may generate toxicity when interacting with biological systems. For example, an in vitro study showed that ambient ultrafine particles (UFP) and polystyrene (PS) nanospheres induced ROS production and toxic oxidative stress in phagocytic cell line (RAW 264.7) cells (Xia et al., 2006). A growing number of studies in recent years have found that SiO2-NPs exert some risk to the brain. For example, in SK-N-SH and neuro-2a cells, SiO2-NPs can induce amyloid-β (Aβ) plaque deposition and promote tau protein phosphorylation, which are the main pathological features leading to AD (Yang et al., 2014; Huang et al., 2015). In addition, an in vivo study found that SiO2-NPs can be transported to brain tissue via the olfactory bulb and deposited in the striatum, which can in turn trigger apoptosis of dopaminergic neurons (Wu et al., 2011). In a sense, the increase in autophagic vacuoles may be an adaptive cellular response for cells to degrade and remove potentially toxic nanomaterials, but it is also possible that nanomaterials may disrupt the autophagy pathway that is harmful. Thus, studies to assess the safety of nanomaterials is urgently required. Based on the above characteristics, nanomaterials could play an important function in PD therapy as a drug delivery system targeting ALP. However, the toxicity of those nanomaterials should also be paid close attention to.

Therapeutic strategies for TPD

Targeted protein degradation (TPD), a pathway that selectively targets pathogenic proteins and their aggregates for direct degradation, has emerged as a promising potential therapeutic modality (Zhao et al., 2022). Protein hydrolysis-targeted chimeras (PROTACs), a synthetic heterobifunctional molecule, is the TPD approach that has been received most attention (Qi et al., 2021). PROTACs binds to target proteins of interest (POI) and E3 ligase, respectively, which form a transient ternary complex and lead to polyubiquitination (polyUb) of the target POI and subsequent degradation via the ubiquitin-proteasome system (Sakamoto et al., 2001). Two PROTACs targeting the androgen receptor (ARV-110) and estrogen receptor (ARV-471) for the treatment of pancreatic cancer and breast cancer, respectively, have entered phase I clinical trials, respectively (Yin and Hu, 2020). However, the ubiquitin-proteasome system pathway has many limitations with respect to degradation of organelles, nonintracellular proteins, and aggregated proteins (Naito et al., 2019). More recently, many novel strategies for TPD mainly include the autophagy-targeting chimeras (AUTACs) inducing degradation via selective autophagy, mediated AUTOphagy-TArgeting Chimeras (AUTOTACs) directly combining with SQSTM1/p62, and the autophagy tethering compounds (ATTECs) directly degrading intracellular proteins and non-protein entities through the macroautophagy pathway (Li et al., 2019b; Takahashi et al., 2019; Fu et al., 2021; Ji et al., 2022). Moreover, the lysosome-targeting chimeras (LYTACs) technology, as an emerging TPD technology that can target extracellular and membrane-associated proteins, can degrade proteins through the endosomal-lysosomal pathway (Banik et al., 2020; Barghout, 2022). It has been demonstrated that ATTEC can degrade mHTT by interacting with the expanded polyQ stretch (Li et al., 2019b). LYTACs have been exploited to selectively degrade apolipoprotein E4 (ApoE), mHTT, and other membrane proteins (Banik et al., 2020; Jarosinska and Rudiger, 2021). Recently, Lee et al. (2023) reported that ATC161, a selective PD-AUTOTAC compound that target α-Syn aggregates, induces ALP degradation of α-syn aggregates in a SQSTM1/p62-dependent manner and alleviates the mitochondrial dysfunction in vitro and in vivo. Additionally, Fan et al. (2014) designed a protein-specific knockdown peptide containing a CMA-targeting motif that can target wild-type and A53T-mutant α-Syn for degradation in vitro and in vivo. Mei et al. (2023) designed a chimera for the targeted degradation of large proteins and/or organelles. The chimera is composed of ATG16L1 or LC3-binding peptide and a ligand for a target. Chimera is composed of a large number of standard ligands and ATG16L1- or LC3-binding peptides, which connect the target to phagophore-associated ATG16L or LC3, thereby inducing clearance of the target via selective autophagy (Mei et al., 2023). Taken together, these novel strategies provide exciting opportunities to degrade α-syn aggregates and other abnormal proteins in PD.

Limitations

Although the review provides a brief account and discussion of the chemical and gene therapeutic strategies targeting ALP in PD, some limitations need to be acknowledged. During the search progress, we only searched the literature published in English journals, but some relevant literatures published in Chinese journals such as studies on traditional Chinese medicine in autophagy were possibly missed. Moreover, in the progress that discusses the results of previous studies, this review only briefly describes the results and mechanisms of most preclinical studies and a small number of clinical application studies, and lacks specific and in-depth analysis of any specific aspect of the ALP pathway. Furthermore, in addition to the signaling pathways described in the text that regulate ALP, other pathways by which these small molecules or genes regulate ALP and whether they have effects on other biological pathways need to be further studied and analyzed in depth.

Concluding Remarks and Future Direction

Considerable data indicate that autophagy is compromised in PD, and there is extensive preclinical support for the use of autophagy inducers including small molecule drugs, traditional Chinese medicine, and overexpression of genes carried in AAV or nanoparticles in cellular and animal models of PD. However, one of the main difficulties in studying the autophagic process in the brain is currently to define autophagic flux. For example, studies have found that there are increased numbers of autophagosomes in the brain in mouse models or individuals, which does not mean an enhancement in autophagy. The increase in the number of autophagosomes may be due to increased autophagosome formation as a result of increased delivery of substrates to the lysosome, or may also be due to impaired lysosomal degradation or autophagosomes trafficking, which needs to be clarified in future research. In addition, there may be side effects when using a number of genetic manipulations of autophagy-related genes or chemical strategies to perturb or enhance autophagy; for example, beclin1 can indirectly regulate p53 levels (Liu et al., 2011). The PD pathological process is complex, and although we have induced some PD models with drugs, it cannot be faithfully modelled in animals. Therefore, a major future challenge is to develop multiple markers to infer autophagic fluxes in in vivo and postmortem samples, which may help to understand the specific mechanisms of autophagy in PD.

A large number of preclinical studies have demonstrated that many small molecule compounds targeting ALP exert neuroprotective effects in PD models, but there are several issues that need to be further validated in vivo before entering clinical applications. First, whether these small molecule drugs can cross the BBB, as well as their distribution and biological activities in the brain need to be observed in vitro. Second, the cell lines such as SH-SY5Y, N2a, and PC12 used in most of the studies are tumor cell lines, which may themselves have an effect on affection on the regulation of ALP, and the effects of these small molecule compounds need to be further verified in primary cultured neurons. Last, the effects of these small molecule compounds on polyamine synthesis and secretion in the substantia nigra and striatum need to be further investigated. In addition, overexpression of any genes regulating ALP by viruses can significantly ameliorate the neurological damage in PD models, which may be a promising therapeutic strategy in PD. However, whether the virus has pathological effects on brain tissues, as well as the uptake and metabolism of the virus in vivo need to be further observed in animals.

The local administration and/or drug delivery of micro- and nanomedicines is a very promising therapeutic option that can be applied in the clinic. Although several studies have explored the potential of nanomedicines in the treatment of PD, most of these studies have focused on animal and cellular levels, and the ability of those nanomedicines to effectively cross the BBB remains an important consideration in the development of nanotechnologies for the treatment of PD. Additionally, the ability of the formulation to deliver an effective therapeutic dose to a specific area and avoid unwanted build-up at other sites in the case of systemic administration needs to be further investigated in animal models and clinical trials of PD.

Funding Statement

Funding: This work was supported by the National Natural Science Foundation of China, No. 82101340 (to FJ).