Emerging views of OPTN (optineurin) function in the autophagic process associated with disease

ABSTRACT

Macroautophagy/autophagy is a highly conserved process in eukaryotic cells. It plays a critical role in cellular homeostasis by delivering cytoplasmic cargos to lysosomes for selective degradation. OPTN (optineurin), a well-recognized autophagy receptor, has received considerable attention due to its multiple roles in the autophagic process. OPTN is associated with many human disorders that are closely related to autophagy, such as rheumatoid arthritis, osteoporosis, and nephropathy. Here, we review the function of OPTN as an autophagy receptor at different stages of autophagy, focusing on cargo recognition, autophagosome formation, autophagosome maturation, and lysosomal quality control. OPTN tends to be protective in most autophagy associated diseases, though the molecular mechanism of OPTN regulation in these diseases is not well understood. A comprehensive review of the function of OPTN in autophagy provides valuable insight into the pathogenesis of human diseases related to OPTN and facilitates the discovery of potential key regulators and novel therapeutic targets for disease intervention in patients with autophagic diseases.

Abbreviations: ATG: autophagy-related; APAP: acetaminophen; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CC: coiled-coil; HACE1: HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1; MYO6: myosin VI; IKBKG/NEMO: inhibitor of nuclear factor kappa B kinase regulatory subunit gamma; IKK: IκB kinase; LIR: LC3-interacting region; LZ: leucine zipper; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NFKB/NF-κB: nuclear factor kappa B subunit; OPTN: optineurin; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RTECs: renal tubular epithelial cells; SQSTM1/p62: sequestosome 1; TBK1: TANK binding kinase 1; TOM1: target of myb1 membrane trafficking protein; UBD: ubiquitin-binding domain; ULK1: unc-51 like autophagy activating kinase 1; WIPI2: WD repeat domain, phosphoinositide interacting 2; ZF: zinc finger.

Introduction

The lysosomal degradation pathway of autophagy is a highly conserved pathway in eukaryotic cells and plays an essential role in cellular, tissue, and organismal homeostasis by selectively removing dysfunctional organelles, intracellular bacteria, and aggregated proteins [1]. In general, cellular stress induces the formation of a cup-shaped structure called a phagophore that can mature into an autophagosome with the cooperation of autophagy-related (ATG) proteins and autophagy receptors. These autophagosomes and lysosomes fuse to form autolysosomes to ultimately degrade cargos [2]. The autophagic process is modulated sequentially and linked to numerous fundamental physiological functions [3].

OPTN (optineurin) is a conserved protein that can be found in humans, rhesus monkeys, rats, pigs, and many other species [4–6]. Generally speaking, OPTN plays roles in vesicular trafficking, NFKB/NF-κB (nuclear factor kappa B subunit) signaling and autophagy. In specific, OPTN has been identified as an autophagy receptor that connects the ubiquitinated autophagy substrates with MAP1LC3/LC3 (microtubule associated protein 1 light chain 3)-positive phagophore membranes [7]. Moreover, increasing evidence has shown that OPTN is also an autophagy inducer that initiates the autophagic process [8,9]. Studies have suggested that OPTN’s participation in autophagic initiation can start as early as the process of autophagosomal membrane formation [10,11]. These novel findings highlight the role of OPTN as a potential autophagy receptor with multiple functions during the autophagic process, rather than merely a common autophagy receptor that functions at a single stage of autophagy [12].

Mutations in the human OPTN gene have been found in many familial diseases, such as OPTN^E478G in amyotrophic lateral sclerosis (ALS) and OPTN^E50K in glaucoma [13–15]. OPTN^E478G mutation occurs in its autophagy-associated ubiquitin-binding domain (UBD) [7,16], indicating the close connection between OPTN-induced autophagy and the disease of ALS. Moreover, low OPTN expression was detected in a subset of patients with Crohn disease [6]. These results indicate that OPTN may be involved in many biological processes and act as a key disease-driving gene. Given this new function of OPTN as an autophagy receptor, it may have implications for OPTN-associated diseases with novel autophagic mechanisms.

In this review, we first summarize the current knowledge of the protein structure and cellular function of OPTN, with an update of new OPTN interaction partners identified to act during autophagosome formation. Then, we give a comprehensive review of OPTN participation in the autophagic processes, including its role in cargo recognition, autophagosome formation, autophagosome maturation, lysosomal quality control, and autophagic degradation. We further discuss OPTN-induced autophagic mechanisms in various diseases, such as neurodegenerative diseases, inflammatory diseases, cancer, and nephropathy. Overall, an understanding of the autophagic functions of OPTN may have implications in the landscape and intervention of OPTN-associated human disorders.

The protein structure and cellular function of OPTN

The human OPTN protein is a 74-kDa scaffold protein comprised of 577 amino acids, and the mouse Optn gene codes for a 584–amino acid protein (67 kDa), which is 78% identical to human OPTN [4]. OPTN is a highly conserved protein that is expressed in many tissues including brain, liver, and heart and plays an important role in normal physiology and disease pathogenesis [4,17].

OPTN contains several structural domains, including two coiled-coil (CC) domains, a leucine zipper (LZ) domain, an LC3-interacting region (LIR), a ubiquitin-binding domain (UBD), and a zinc finger (ZF) domain. In OPTN, the LIR domain is an LC3-II binding site, and the UBD is the ubiquitinated cargo binding site. Both the LIR and UBD are critical regulatory elements in OPTN-associated autophagy. OPTN binds with LC3-II-conjugated-autophagic membranes via this LIR. Then, OPTN-bound-ubiquitinated cargos are enclosed by autophagic membranes and form autophagosomes for degradation in autolysosomes. Moreover, the LZ domain is also showing an autophagic function in interacting with ATG9A [10], which is the sole multi-spanning membrane protein among ATG proteins that is essential for autophagosome formation [18–20] (Figure 1).

Figure 1.

Schematic representation of OPTN functional domains and OPTN mutations. TBK1-binding domain (amino acids [aa] 1–127), RAB8-interacting region ([aa] 1–209), LC3-interacting region (LIR; [aa] 169–181), coiled-coil (CC) domains ([aa] 230–445), leucine zipper (LZ) domain and ATG9A-interacting region ([aa] 143–164), HACE1-interacting region ([aa] 411–456), MYO6-interacting region ([aa] 417–512), ubiquitin-binding domain (UBD; [aa] 454–514) and zinc finger (ZF; [aa] 556–577). OPTN can be phosphorylated on S177, S473, and S513 by TBK1 and ubiquitinated on Lys193 by HACE1.

Many biological functions of OPTN have been identified to date. First, OPTN has been found to colocalize with the Golgi apparatus and plays a vital role in the maintenance of Golgi integrity [14,21]. Second, OPTN shows strong homology with IKBKG/NEMO (inhibitor of nuclear factor kappa B kinase regulatory subunit gamma), a regulator of IKK/IκB kinase complexes [22]. To activate NFKB, IKBKG is recruited to the RIPs (poly-ubiquitin chains of receptor-interacting proteins) to form a functional complex with IKK [23,24]. As a homolog of IKBKG, OPTN competes for binding to the poly-ubiquitin chains of RIPs (poly-ubiquitin chains of receptor-interacting proteins), disrupts the formation of a functional IKK complex and negatively regulates NFKB signaling [22]. Third, an in vitro study identified the specific interaction between OPTN and LC3 using yeast two-hybrid and affinity-isolation assays [7]. This study provides a foundation for subsequent studies of OPTN in autophagy, which has received increasing attention in recent years.

OPTN can interact with a large number of proteins, and some of those interactions have been shown to be disrupted by mutations (Figure 1). For instance, OPTN can interact with ATG9A, LC3, MYO6 (myosin VI), TBK1 (TANK binding kinase 1) and the ubiquitin chains of cargos to execute its functions at multiple steps in autophagy [10,21,25,26]. Specifically, OPTN can be phosphorylated by TBK1 on serines 177 (S177), S473, and S513 [27,28]. OPTN^E50K, a glaucoma-associated mutation, can abnormally enhance the interaction between OPTN and TBK1, leading to a blockage in autophagy [29]. The ALS-associated mutations OPTN^Q398E and OPTN^E478G disrupt the interaction between OPTN and MYO6, and OPTN^E478G also loses the function of its UBD and thus fails to interact with the ubiquitinated cargos [7,16].

OPTN is a key regulator of autophagy

OPTN has been proposed to contribute to selective autophagy of depolarized mitochondria (mitophagy), protein aggregates (aggrephagy), and intracellular bacterial pathogens (xenophagy) through ubiquitin‐signaling. However, there have been no systematic and comprehensive reviews the authors are aware of highlighting the vital role of OPTN in autophagy fully. In this section we review the different stages of the autophagic process in which OPTN is actively involved, including cargo recognition, autophagosome formation, autophagosome maturation, lysosomal quality control, and autophagic degradation (Figure 2).

Figure 2.

Schematic of OPTN’s roles in the autophagic process. In this figure, we take mitophagy as an example, and the cargo is mitochondrion. In response to stress, PINK1 is stabilized on the mitochondrial outer membrane (MOM), promoting the phosphorylation of PRKN on mitochondria. OPTN is recruited to the mitochondria immediately after PRKN recruitment and associates with ubiquitin chains (Ubs), which can be disrupted by mutation of OPTN^E478G. The OPTN-TBK1 interaction can be abnormally enhanced by OPTN^E50K, leading to the blockage of autophagy. Induction of autophagy results in the recruitment of ATG proteins to autophagy initiation sites, where OPTN can promote ULK1 translocation to the phagophore. This function can be disrupted by OPTN^Q398E mutation. The ULK1 complex drives the downstream activation of PtdIns3K to trigger nucleation of the phagophore. Then, OPTN directs the lipidation of LC3 at the phagophore by recruiting ATG12–ATG5-ATG16L1, which can be disrupted by the OPTN^E478G mutant. Also, OPTN can interact with ATG9A vesicles to seed local autophagosomal membrane formation. After this, the ubiquitinated-cargo-connected OPTN links cargo to autophagosomal membranes via binding to LC3. The autophagosomal membrane seals and gives rise to a double-layered vesicle called the autophagosome. OPTN directly interacts with MYO6, which can be disrupted by OPTN^E478G and OPTN^Q398E mutation. OPTN and MYO6 bind to TOM1, delivering endosomal membranes to autophagosomes, which is the ultimate maturation step for autophagosomes and is required for autophagosome-lysosome fusion. Finally, the autophagosome fuses with the lysosome and becomes the autolysosome for degrading and recycling cellular contents.

The role of OPTN in cargo recognition

It has become clear that specific cargos induce the initiation of autophagy in selective autophagy. Autophagy receptors recognize the ubiquitinated cargos and recruit scaffold proteins to these cargos [30–32]. As an autophagy receptor, OPTN acts to recognize ubiquitinated cargos, which forms the molecular basis for selective autophagy [33]. In selective autophagy, OPTN has to be recruited around these cargos and then recognizes such ubiquitinated cargo.

OPTN can be recruited to different kinds of cargos in different manners. In mitophagy, OPTN can be stabilized by PRKN (parkin RBR E3 ubiquitin protein ligase) on the surface of damaged mitochondria [16]. In the presence of PRKN in HeLa cells, OPTN was shown to be recruited to mitochondria right after PKRN translocation, and this recruitment was stabilized by the UBD binding with ubiquitinated mitochondria. In the absence of PRKN, OPTN puncta can only localize to damaged mitochondria transiently [16]. In addition to PRKN, PINK1 (PTEN induced kinase 1) can dynamically recruit OPTN to damaged mitochondria to activate mitophagy directly [34]. PINK1 works as a mitochondrial protein kinase that protects cells from stress-induced mitochondrial dysfunction [35]. While it has been shown that OPTN recruitment requires PINK1 kinase activity, but how PINK1 recruits OPTN remains unclear [34]. Together, these two studies highlight that PINK1 facilitates OPTN recruitment, and PRKN promotes OPTN stability in damaged mitochondria to activate mitophagy.

Like mitophagy, in xenophagy, intracellular bacterial pathogens are also targeted by cargo receptors via a ubiquitin-mediated pathway. At least four cargo receptors, namely CALCOCO2/NDP52 (calcium binding and coiled-coil domain 2), SQSTM1/p62 (sequestosome 1), NBR1 (NBR1 autophagy cargo receptor) and OPTN have been shown to bind ubiquitin chains on intracellular bacteria and direct ubiquitin-coated intracellular bacteria to autophagosomes [12,33,36,37]. Many E3 ubiquitin ligases, such as RNF166 (ring finger protein 166), LRSAM1 (leucine rich repeat and sterile alpha motif containing 1), PRKN and SMURF1 (SMAD specific E3 ubiquitin protein ligase 1), can localize to the surface of bacteria and ubiquitinate substrates [38–40]. However, how OPTN is recruited to ubiquitinated bacteria (cargos) to trigger autophagy is still an unclear process. Future studies of specific bacterial ubiquitinated substrates and their respective E3 enzymes may bring new insights into the molecular mechanism driving this process.

After being recruited, OPTN binds to ubiquitinated cargos. Interestingly, OPTN does not bind to linkage types of ubiquitin chains randomly. Rather, it has a binding preference for linear polyubiquitin chains and K63 chains rather than K48 chains or monoubiquitin modified substrates [29]. Crystal structure analysis revealed a complex of the OPTN UBD and linear diubiquitin, and the phosphorylation of OPTN plays a critical role in complex formation [41]. Furthermore, phosphorylation at S473 and dual phosphorylation of S473 and S513 in the UBD domain by TBK1 have been shown to enhance the binding of OPTN to multiple ubiquitin chains [27]. Moreover, using GST affinity-isolation assays, a study showed recently that phosphorylation of ubiquitin at S65 catalyzed by PINK1 prevents interaction with OPTN. However, OPTN UBD phosphorylation by TBK1 can partially rescue this loss of binding [27]. While many cargo-recognition studies have been done, how OPTN or other autophagy receptors selectively recognize the ubiquitin-modified cargos and how OPTN recognizes these cargos efficiently are still far from clear.

The role of OPTN in autophagosome formation

To form an autophagosome, OPTN binds to a ubiquitin-decorated cargo and links the ubiquitinated cargo to autophagosomal membranes via binding to LC3. Increasing evidence suggests that OPTN is actively participating in autophagosome formation steps, including the initiation of a phagophore, the biogenesis of a phagophore, and interaction with LC3 [42,43].

A variety of membrane sources and proteins are required for phagophore nucleation, and much evidence suggests that OPTN is involved in this process. At the early stage of PINK1-PRKN mitophagy, OPTN promotes the translocation of the ULK1 (unc-51 like autophagy activating kinase 1) complex to autophagy initiation sites. ULK1 is the first autophagy-specific complex recruited to autophagy initiation sites during autophagosome formation [34]. The ULK1 complex can drive the activation of the phosphatidylinositol 3-kinase (PtdIns3K) complex, which serves as the second kinase complex recruited to form an autophagosome [44–46]. The PIK3C3 (phosphatidylinositol 3-kinase catalytic subunit type 3) first generates a simple phosphoinositide, PtdIns3P, and then recruits the WIPI1 (WD repeat domain, phosphoinositide interacting 1)-WIPI2 (WD repeat domain, phosphoinositide interacting 2) effector to translocate to PtdIns(3,4,5)P₃-enriched sites. Furthermore, WIPI1-WIPI2 binds to the ATG16L1 from the ATG12–ATG5-ATG16L1 complex, and then conjugates Atg8-family proteins to phagophore membranes [47,48]. In addition to ULK1 complex recruitment, OPTN can also form a complex with the WIPI2 and ATG12–ATG5 conjugate, facilitating the recruitment of the ATG12–ATG5-ATG16L1 complex to WIPI2-included phagophores [49]. The ATG12–ATG5-ATG16L1 complex is believed to act in the covalent modification of LC3 to the phosphatidylethanolamine (PE) in the phagophore membrane, suggesting that OPTN may also participate in the process of LC3 lipidation [50].

Interestingly, a recent study using a Fluoppi assay revealed that OPTN could interact with ATG9A vesicles to initiate local autophagosomal membrane formation, which is crucial for PRKN-mediated mitophagy [10]. ATG9, a sole transmembrane protein, provides a lipid/membrane source during the early steps of autophagosome formation [51]. However, it is enigmatic that ATG9-containing vesicles do not supply the bulk of the autophagosomal membrane in this process. Recently, Sawa-Makarska et al. recapitulated autophagosome formation’s initial steps using autophagy factors purified from yeast. This revealed that ATG9 vesicles serve as nucleators to establish membrane contact sites with a donor compartment such as the endoplasmic reticulum (ER) during the de novo formation of autophagosomes [11]. OPTN recruits ATG9 vesicles by interacting with ATG9A and cargos to initiate local autophagosomal membrane formation, indicating that OPTN is a very upstream regulator of autophagosomal membrane formation.

Generally speaking, OPTN is known to bind to Atg8-family proteins via its LIR domain, and this binding can be enhanced by the Ser177 phosphorylation of OPTN in the LIR domain by TBK1 [7]. The LIR motif within autophagy receptors is the main feature of selective autophagy models, in which autophagy receptors, such as SQSTM1, mediate the combination of Atg8-family protein associated membranes with the autophagic cargos. A previous study demonstrated that OPTN could recruit LC3 via its LIR domain and promote the lipidation of LC3 to the phagophore [50]. Unexpectedly, though OPTN^F178A mutation led to loss of LC3 binding and impaired mitophagy, this mutant could still recruit all Atg8-family proteins, suggesting that the LIR domain of OPTN might be dispensable for LC3 recruitment in mitophagy [52]. This refreshing study showed that OPTN with a UBD mutation (OPTN^D474N) could still be recruited to depolarized mitochondria by Atg8-family protein-positive phagophores via the LIR domain, which could amplify the rate of autophagosome biogenesis after the initiation of autophagosome biogenesis [52].

In summary, during the autophagosome formation, OPTN recruits the ULK1 complex to initiate phagophore biogenesis and then directs LC3 lipidation by recruiting the ATG12–ATG5-ATG16L1 complex to phagophores [34,49]. Subsequently, OPTN recruits ATG9 vesicles to assist local autophagosomal membrane formation and promotes autophagosomal membrane recruitment to cargos by interacting with LC3 [10,53]. Autophagosome is generally formed by the interaction between autophagy receptors and LC3, which is a common feature of autophagy receptors such as OPTN, SQSTM1, and CALCOCO2 [2]. However, the recruitment of the ULK1 complex, the ATG12–ATG5-ATG16L1 complex and ATG9 vesicles is a unique function of OPTN, suggesting that it acts as a multifunctional autophagy receptor in autophagosome formation.

The role of OPTN in autophagosome maturation

OPTN plays a vital role in the maturation of autophagosomes through its interactions with MYO6 and LC3 family members. The fusion of autophagosome with lysosome is a crucial step in autophagy [54]. MYO6 is the only known unconventional myosin motor that moves in the opposite direction along actin filaments [55]. Recent studies have also clarified the importance of MYO6 in autophagy and mitochondrial homeostasis. Defects in MYO6 cause impeded autophagy and the accumulation of mitophagosomes [56]. Moreover, MYO6 can form actin cages to ensure the sequestration of damaged mitochondria [57]. Additionally, OPTN directly interacts with MYO6, binding with TOM1 (target of myb1 membrane trafficking protein) to deliver endosomal membranes to autophagosomes, which is required for autophagosome-lysosome fusion [58]. MYO6 or TOM1 dysfunction can reduce the autophagosomal delivery of endocytic cargo and block maturation from autophagosomes to autolysosomes [25,59,60]. Consistently, a similar molecular process can also be found during the maturation of S. typhimurium-containing autophagosomes [61]. Via a non-conventional LIR domain, CALCOCO2/NDP52 and OPTN can promote autophagosome maturation by interacting with several Atg8 orthologs, such as LC3A/B and GABARAPL2 (GABA type A receptor associated protein like 2), but not via an interaction with LC3C [61]. Another autophagy receptor, TAX1BP1 (Tax1 binding protein 1), also mediates the maturation of autophagosome by binding to MYO6, suggesting that OPTN is not the only autophagy receptor involved in the maturation of autophagosome [62]. However, whether OPTN is highly conserved in the maturation of autophagosomes is worth further attention.

The role of OPTN in lysosomal quality control

The lysosome is the central organelle for cellular degradation and recycling in eukaryotic cells. Autophagosomes fuse with lysosomes containing degrading enzymes in autophagy, leading to autophagosome content digestion. Though the lysosome’s functions have often been overlooked, they play an essential role in ensuring autophagy proceeds without a hitch. A recent study employed correlative light-electron microscopy (CLEM) in alpha synuclein-stimulated microglial and found that instead of SQSTM1 and CALCOCO2, OPTN was recruited to ubiquitinated lysosomes in cases of lysosomal quality impairment. In this study, OPTN restored the lysosomal quality control by lysophagy, a kind of selective autophagy mediating the degradation of lysosomes [63]. Autophagy induction is frequently associated with the upregulation of hydrolase synthesis and lysosome biogenesis [64]. When lysosomes are unstable or their functions are impaired, autophagy inhibitors tend to lessen the stress of autophagy on dysfunctional lysosomes by reducing autophagic cargo delivery to lysosomes rather than reducing an excessively aggressive autophagic process [64]. Generally, when lysosomal function is impaired, cells cannot tolerate robust autophagy induction, and autophagic flux is consequently inhibited [64]. Collectively, OPTN contributes to successful lysosomal quality control, which guarantees autophagic flux.

The role of OPTN as a substrate of autophagy

It has been established that OPTN can be degraded by the autophagic process and the ubiquitin-proteasome system. HACE1 has been identified as the E3 ubiquitin ligase responsible for the ubiquitylation of OPTN by yeast two-hybrid [28]. Moreover, endogenous OPTN can be stabilized by the lysosomal inhibitor bafilomycin A₁, but not the proteasome inhibitor bortezomib [28]. Although another study demonstrated that the ubiquitin-proteasome system contributes to the major degradation of exogenous OPTN, OPTN can also be stabilized by bafilomycin A₁ [65]. Lysosomes participate in the autophagosome-lysosome fusion in autophagy as well as the endosomal system. The results of OPTN accumulation by bafilomycin A₁ illustrates autophagy might be a system for OPTN degradation. Further investigations should be performed to define the degradation mechanisms regulating OPTN.

OPTN mediates autophagic dysfunction in diseases

Autophagy dysfunction is associated with many pathological conditions, such as cancer, neurodegenerative diseases, and microbial infection, making autophagy a promising target for disease intervention. Particularly, OPTN-mediated autophagy-dysfunction is closely related to a variety of diseases (Table 1). Understanding the autophagic mechanisms of OPTN dysfunction in diseases may ultimately pave the road for novel disease intervention methods.

Table 1.

The different types of OPTN related diseases and autophagy related mechanisms

Type of disease	Diseases	Mechanism	Reference
Neurodegenerative diseases	Glaucoma	OPTNE50K disrupts the interaction with RAB8 and induces oxidative stress; OPTNE50K enhances the interaction with TBK1 but reduces autolysosomes; OPTNE50K disrupts the oligomeric state of OPTN	[29,82,133,134]
	ALS	OPTNE478G disrupts the ubiquitin-binding function of OPTN and affects PRKN-mediated mitophagy	[16]
	PD	Increased OPTN expression and colocalized puncta in PD-relevant brain regions with specific impairments of autophagy	[135]
	HD	Impaired autophagic clearance of aggregates colocalized with OPTN	[96,136–138]
	NIID	Unclear, abnormal accumulation of OPTN and its binding partner MYO6 in intranuclear inclusions of NIID	[87,136]
Inflammatory diseases	CD	Clearance of intracellular bacteria by OPTN mediated xenophagy	[95,96]
	PDB	There are several PDB genes (including OPTN) with functions related to the autophagic system	[106]
	RA	OPTN is upregulated while the related autophagy mechanism is unclear	[110]
	OP	optn deficiency caused inhibition of selectively degradation of FABP3 by autophagy	[120]
	ALF	OPTN is upregulated accompanied with autophagy activation after APAP treatment	[122]
Cancer	Lung cancer	HACE1-OPTN axis mediated autophagy	[28]
	Pancreatic cancer	OPTN involved chaperone-mediated autophagy (CMA)	[124]
Nephropathy	DN	OPTN mediated mitophagy	[128,129]
	AKI	OPTN mediated mitophagy	[130]

AKI: acute kidney injury; ALF: acute liver failure; ALS: amyotrophic lateral sclerosis; CD: Crohn disease; DN: diabetic nephropathy; HD: Huntington disease; NIID, neuronal intranuclear inclusion disease; OP: osteoporosis; PD: Parkinson disease; PDB: Paget disease of bone; RA: rheumatoid arthritis.

OPTN-mediated autophagy alleviates neurodegenerative diseases

The most prevalent pathological feature of neurodegenerative diseases is the aggregation of proteins and dysfunctional mitochondria, which are both primarily degraded by autophagy. Many autophagy-associated genetic mutants have been found in neurodegenerative diseases, such as the OPTN^E50K mutant in glaucoma and the OPTN^E478G mutant in ALS. Abnormal protein aggregates, along with compromised autophagy, are found to co-occur in these neurodegenerative diseases. However, the mechanism of OPTN-mutant-related pathology in neurodegenerative diseases is still unclear. Herein, we provide a summary of OPTN-mediated autophagic mechanisms in these diseases.

Glaucoma

Glaucoma is the second leading cause of blindness in the world [14]. The name of “optineurin” was designated when the disease was first identified to correspond to one of the genes encoding the glaucoma form of the “optic neuropathy inducing” protein [14]. Mutations in the coding region of OPTN are associated with 16.7% of families with hereditary primary open-angle glaucoma [14]. The OPTN^E50K mutant has been frequently and widely studied in glaucoma. Transgenic mice with the OPTN^E50K mutation present a glaucoma phenotype, including a loss of retinal ganglion cells (RGCs) and the thinning of various cell layers of the retina and gliosis [66], which further confirms the association between OPTN^E50K and glaucoma. Moreover, OPTN expression is significantly increased in human anterior segment organ cultures treated with glaucoma-related stimuli such as elevated-intraocular pressure (IOP), TNF (tumor necrosis factor) and dexamethasone [67], also indicating OPTN’s involvement in the occurrence of glaucoma.

Many studies have tried to elucidate the mechanism of OPTN^E50K induced glaucoma. It has been reported that the E50K mutation can abnormally enhance OPTN’s interaction with TBK1 while disrupting its interaction with RAB8, leading to compromised autophagy and increased oxidative stress [68–71]. In different studies, there have been some disputes of OPTN^E50K-mediated autophagy in glaucoma. Shim et al. presented the idea that OPTN^E50K can induce mitophagy in RGCs. Increased mitophagosome number, upregulated LC3-II expression, and LC3 puncta were observed in the glial lamina axons of OPTN^E50K−tg mice [72,73]. In this case, the accumulation of mitophagosomes and LC3 may have been caused by either autophagic activation or blockage of downstream steps in autophagy [74]. Therefore, further investigations are needed to determine if OPTN^E50K can induce mitophagy. In another study, Madhavi et al. used an mCherry-GFP-LC3B plasmid to monitor autophagy. Once the mCherry-GFP-LC3B plasmid entered into autolysosomes, only red fluorescence was detected while the GFP fluorescence was quenched in the acidic autolysosomes. In amino acid starvation conditions, OPTN^E50K retinal cells showed fewer autolysosomes indicated by reduced red fluorescence, suggesting the inhibition of autophagic flux by OPTN^E50K [75]. In addition, OPTN^E50K colocalized more often with autophagosomes than wild-type OPTN. The autophagy activator rapamycin treatment significantly inhibited OPTN^E50K induced retinal cell death, indicating that OPTN^E50K disrupted autophagy in retinal cells [75].

It is worth mentioning that OPTN^E50K mutant retinal cells show abnormal insolubility, and present small dot-like deposits in the outer plexiform and inner nuclear layers. Studies have suggested that the insoluble aggregation in OPTN^E50K mutant retinal cells may be related to an enhanced TBK1 interaction with OPTN^E50K. As shown in this study, abnormal insolubility could be resolved by BX795, a specific TBK1 inhibitor [29,68]. Though the mechanism of OPTN^E50K mediated autophagy in glaucoma is elusive, we think that an enhanced interaction with TBK1 may reduce the binding affinity of OPTN to its other interaction partners. In addition, as a delayed glaucoma diagnosis may lead to more severe symptoms without effective treatment [76], it is worth testing if TBK1 inhibitors could act as a potential medication for OPTN^E50K-related glaucoma.

Amyotrophic lateral sclerosis

ALS is a progressive neurodegenerative disease characterized by the degeneration of motor neurons in the primary motor cortex, brain stem, and spinal cord [77]. Many genes have been reported to be associated with familial ALS/fALS, including SOD1, TBK1, SQSTM1, and OPTN [78–80]. Several types of OPTN mutations occur in ALS patients, including a homozygous deletion of exon 5, a homozygous Q398X nonsense mutation, a heterozygous E478G missense mutation, and a novel Q454E missense mutation. Both E478G and Q454E are located in the UBD of OPTN [13,81]. OPTN^E478G and OPTN^Q454E mutations result in impaired mitophagy after carbonyl cyanide 3-chlorophenylhydrazone/CCCP treatment in HeLa cells [82]. Moreover, optn^−/- mice exhibit accumulated mitochondria in the hypoglossal nerve axons, which further implicates the involvement of OPTN-mediated autophagy in ALS development [83].

The OPTN^E478G mutation is located in the UBD region, which abolishes OPTN’s binding to ATG12–ATG5 conjugates, ubiquitin, and MYO6 and leads to severe disruption in several stages of autophagy [7,49]. OPTN^E478G mutation impairs the ability of OPTN to form autophagosomes probably because it disrupts complex formation between OPTN and ATG12–ATG5 conjugates, which further undermines the recruitment of the ATG12–ATG5-ATG16L1 complex to WIPI2-positive structures and leads to defective autophagy [49]. In addition, OPTN^E478G mutation disrupts ubiquitin binding and thus impairs autophagosomal membrane recruitment [7]. In HeLa cells, OPTN^E478G consistently reduces the capacity of LC3 recruitment to mitochondria and slows autophagosome formation [16]. It is worth noting that OPTN^E478G can also cause a defect in linear chain ubiquitin-binding capacity, resulting in the dysregulation of IKK-mediated NFKB activation and the consequent aggravation of the pathogenesis of ALS [84]. In addition, the interaction between OPTN and MYO6 is an essential process for facilitating autophagosome maturation in autophagy [25]. A potential mechanism could be that OPTN^E478G mutants disrupt the association of OPTN with MYO6 [16]. Therefore, OPTN^E478G mutation causes less colocalization with MYO6, which inhibits autophagosome-lysosome fusion and results in autophagy impairment. In summary, although how OPTN^E478G causes defective autophagy is becoming clearer, the connection between ALS pathogenesis and defective autophagy caused by OPTN^E478G is still not resolved.

Other neurodegenerative diseases

In addition to the ubiquitin-dependent mechanism, OPTN can also recognize and actively participate in the degradation of HTT exon 1 Q103 aggregation in Huntington disease (HD) and SOD1^G93C aggregation in ALS through its C-terminal CC domain in a ubiquitin-independent manner, suggesting a diverse role of OPTN in autophagic clearance in different neurodegenerative diseases [85]. Moreover, OPTN is frequently observed in the protein inclusions of neurons and glia in Parkinson disease (PD) and neuronal intranuclear inclusion disease (NIID) [86–88]. We theorize that this OPTN positive protein inclusion accumulation may be mainly caused by defective autophagy, leading to neurodegeneration. Moreover, several investigations have shown that OPTN deficiency leads to progressive demyelination in the CNS (central nervous system), indicating a connection between OPTN and demyelination diseases [83,89]. Nevertheless, it is not clear whether OPTN-associated autophagy is involved in demyelination, and further studies are required in this field to resolve this important question.

Interestingly, SQSTM1 also acts as an autophagy receptor to remove the abnormal protein aggregates and destroy organelles to protect form neuron death in several neurodegenerative diseases such as PD, AD, HD, and ALS [90]. Except for working as an autophagy receptor like SQSTM1, OPTN with specific mutations can also cause defective autophagosome nucleation and autophagosome maturation in ALS [25,49], indicating the multiple functions of OPTN as an autophagy receptor during disease pathology.

OPTN activates autophagy to reduce inflammation

A growing body of evidence suggests that autophagy plays a protective role in inflammatory diseases. In addition, several inflammatory diseases have been reported to be closely related to OPTN. Given that OPTN has a direct role in inflammation, its autophagic functions in inflammation are often underestimated. Here, we review current research on the association of OPTN with inflammatory diseases, aiming to disclose the autophagic mechanism of OPTN in these diseases.

Crohn disease

Crohn disease (CD) is characterized by chronic inflammation in the gut, with aberrant immune responses to gut microbiota during disease pathogenesis [91]. Patients with CD may experience an abnormal intestinal bacteria overgrowth, and bacterial infection control is critical in the management of CD. On the molecular level, xenophagy is a type of selective autophagy that specifically targets intracellular pathogens [92]. Many genetic studies have demonstrated that mutation of autophagy-associated genes can increase susceptibility to CD, which reveals the critical role of autophagy in intestinal homeostasis [93]. Susceptibility loci for this disease include a coding variant in ATG16L1 (ATG16L1^T300A) and polymorphisms in IRGM (immunity related GTPase M), both of which are autophagy-related genes, implicating a role for xenophagy in intestinal homeostasis and disease [94]. Interestingly, in macrophages, OPTN expression was shown to decrease in a subpopulation of CD patients revealed by a microarray study. Additionally, macrophages from both optn-deficient mice and OPTN^low patients showed lower levels of TNF and IL6 (interleukin 6) after HkEc (heat-killed Escherichia coli) stimulation than control cells [6]. Moreover, optn-deficient mice were shown to be more susceptible to Citrobacter colitis, E. coli peritonitis, and Salmonella infection [95]. Also, optn-knockdown zebrafish infected with Salmonella experience higher mortality risk [96]. In intestinal epithelial cells, impaired autophagy always results in more susceptibility to intestinal inflammation [97]. To restrict bacterial proliferation, OPTN, which mediates xenophagy, competes with IKBKG, and triggers local NFKB activation, allowing for ubiquitin-coated bacteria binding [98]. However, a deficiency in OPTN would not affect NFKB activation after treatment with TNF, lipopolysaccharides or polyinosinic-polycytidylic acid/poly(I:C) [99]. Together, these data indicate that OPTN-mediated xenophagy is essential for restricting bacterial proliferation in the pathogenesis of CD.

Paget disease of bone

Paget disease of bone (PDB) is a common bone disorder characterized by disorganized bone remodeling with unknown pathogenesis [100]. The most likely cause of PDB is chronic paramyxovirus infection in genetically susceptible people [101]. Genome-wide association studies (GWAS) reveal a locus situated on chromosome 10p13, the locus of OPTN, which showed significant association with familial PDB. Therefore, the risk haplotype can be associated with rare allele(s) within OPTN that markedly increases susceptibility to PDB [102,103]. Consistently, it has been reported that 100% of aged optn^−/- mice present the clinical features of human PDB patients, including polyostotic osteolytic lesions, mixed-phase lesions, and elevated serum ALP (alkaline phosphatase) levels [104]. Mechanistically, optn-deficiency enhances osteoclast differentiation, which significantly decreases the production of type I interferon (IFN) and type I IFN signaling through IFNAR/IFNα/βR (interferon alpha and beta receptor), and meanwhile increases NFKB activity through a CYLD-dependent pathway [104,105]. Although current mechanistic studies mainly focused on the OPTN modulation of NFKB and IFN signaling [105], OPTN mediated autophagy may also be involved in the progression of this disease. In addition to OPTN, PDB is also related to several other genes in the autophagic system, including SQSTM1, VCP (valosin containing protein), and ATG16L1, which suggests a strong connection between autophagy and PDB [106–108]. For instance, a SQSTM1 mutant in PDB has been recently reported to induce autophagy blockage [109], suggesting autophagy’s potential role in PDB progression. Future studies focusing on OPTN or SQSTM1 in PDB will provide further insight into the relationship between autophagy and PDB.

Rheumatoid Arthritis

OPTN has also been associated with rheumatoid arthritis (RA), a chronic erosive polyarthritis characterized by systemic inflammatory autoimmunity. OPTN was determined to functionally downregulate TNFSF11 (TNF superfamily member 11), a protein primarily responsible for the development of bone erosions in RA patients [110]. OPTN knockdown in rheumatoid arthritis synovial fibroblasts results in increased osteoclast formation when co-cultured with monocytes from RA patients [110]. Additionally, cytokines derived from immune responses, such as TNF and IFNG/IFN-γ, have been shown to increase OPTN expression in a mimic of inflammatory conditions to develop RA joint [111,112]. The finding above provides further evidence that OPTN may play a protective role in RA pathogenesis. Given that autophagy degradation of polyubiquitinated proteins is one of the fundamental mechanisms for maintaining rheumatoid arthritis synovial fibroblast homeostasis [113], autophagy tends to negatively regulate the innate immune signaling pathway by degrading the cytoplasmic signaling complexes in RA. The current opinion is that OPTN inhibits NFKB-induced TNFSF11 (TNF superfamily member 11) expression [22,114]; however, whether OPTN-mediated autophagy affects RA progression should also be examined.

Osteoporosis

Osteoporosis is an inflammatory disease characterized by osteoporotic bone loss during aging [115]. In the bones of aged mice, osteopenia is associated with lower autophagic activity, which can be alleviated by autophagy activation [116–118]. Recent studies have shown that OPTN plays dual roles in osteogenesis. OPTN inhibits inflammation-related STAT1 (signal transducer and activator of transcription 1) signaling to promote osteoblast differentiation [119]. OPTN deficient mice display impaired autophagic activity, decreased bone mass, and reduced bone rigidity [120]. Mechanistically, optn deficiency in mesenchymal stem cells impairs autophagy and inhibits selective recognition and degradation of adipogenesis and osteogenesis-related FABP3 (fatty acid binding protein 3), leading to decreased osteogenesis, increased adipogenesis, and a phenotype with elevated senescence [120]. Though autophagy is known to prevent cellular senescence during aging, the molecular mechanism remains elusive, especially the effect of OPTN-regulated autophagy on senescence phenotypes.

Other inflammatory diseases

Autophagy is closely related to hepatic inflammatory diseases, including drug-induced liver injury, virus-induced liver injury, nonalcoholic fatty liver disease, and liver fibrosis. In most liver diseases, autophagy fights to keep cells alive in the presence of “life-threatening” stimuli [121]. Specifically, increased OPTN expression in mice livers injured by acetaminophen (APAP) overdose were found to be accompanied by increased autophagic flux, indicating that OPTN mediated autophagy may also play a role in the progression of acute liver injury [122]. Another study shows that OPTN downregulates TCR (T Cell Receptor)-induced NFKB activation and TNF production to regulate T cell activation during autoimmune diseases [123], revealing a new biological function of OPTN in inflammatory diseases.

Taken together, it is clear that OPTN functions in these inflammatory diseases. Mechanistically, on the one hand, OPTN directly modulates inflammatory signaling pathways independent of autophagy [105]. On the other hand, OPTN-mediated autophagy can remove intracellular bacteria to regulate inflammation indirectly [98]. However, it is still confusing whether OPTN regulates other inflammatory processes such as inflammatory cytokine secretion in an autophagy-dependent manner. It is worth noting that, similar to OPTN, SQSTM1 also plays a role in PDB, RA, and APAP-induced acute liver injury, indicating a general role of autophagy receptors in inflammatory diseases.

OPTN-mediated autophagy in cancer progression

OPTN is regarded as a versatile factor in the progression of cancer in general. HACE1 can ubiquitinate OPTN on Lys193 and then form an autophagic complex with SQSTM1. This is a critical step in HACE1-activated autophagy to accelerate the total cellular autophagic flux and facilitate tumor suppression, suggesting that OPTN is a tumor suppressor in lung cancer [28]. The Cancer Genome Atlas (TCGA) database analysis showed high expression of the OPTN gene across several tumor types, especially in pancreatic and renal cancers [124]. Furthermore, OPTN knockdown had a limited effect on the proliferation of different human pancreatic cancer cell lines, but it could significantly increase cell migration and reduce colony formation. Although the authors of this study discussed the role of OPTN knockdown induced autophagy inhibition in increased cell migration and apoptosis, the precise mechanism is still obfuscated [124].

It has been reported that sustained SQSTM1 expression resulting from autophagy defects promotes tumor formation [125]. SQSTM1 overexpression in clear cell renal cell carcinoma/ccRCC lines was reported to promote resistance to oxidative stress and increased tumor formation [126]. These data suggest an oncogenic role of SQSTM1 during tumorigenesis. Unlike SQSTM1, OPTN suppresses tumor growth by activating autophagy in lung cancer [28], while optn knockdown induces apoptosis in pancreatic cancer cells [124], suggesting dual roles of OPTN in different tumors. In summary, in contrast to SQSTM1, OPTN-mediated autophagy has different effects on tumorigenesis.

OPTN enhances mitophagy to protect nephropathy

Diabetic nephropathy (DN) is one of the most severe and frequent chronic complications of type 1 diabetes [127]. Recent studies have revealed that OPTN presents anti-senescent and protective effects in diabetic nephropathy through enhancing mitophagy [128]. Renal OPTN expression has been shown negatively correlate with tubulointerstitial injury scores in clinical specimens [128]. Moreover, OPTN expression is negatively correlated with serum creatinine levels and positively correlated with eGFR (estimated glomerular filtration rates), suggesting a close relationship between OPTN and DN progression. Interestingly, the expression of OPTN in human renal tubular epithelial cells (RTECs) is always negatively correlated with the senescence marker CDKN2A/p16 (cyclin dependent kinase inhibitor 2A). OPTN in mouse RTECs enhances mitophagosome formation, leading to the downregulation of cellular senescence markers, such as CDKN2A, CDKN1A/p21 (cyclin dependent kinase inhibitor 1A), SA-GLB1/β-gal (senescence-associated galactosidase beta 1), and senescence-associated heterochromatin foci [128,129]. Though OPTN-induced mitophagy can negatively regulate RTEC’s senescence, it is not clear if cellular senescence or OPTN-mediated mitophagy is a critical factor of DN. Therefore, more definitive studies are needed. Similarly, OPTN-mediated mitophagy may also be protective in AKI (acute kidney injury) induced by sepsis, one of the major types of AKI with extremely high morbidity and mortality rates [130]. Although the protective role of OPTN-mediated mitophagy in kidney injury has been well established, further study is needed to decipher how OPTN enhances mitophagy to protect the kidney from injury.

Perspectives

Except for an autophagy receptor, OPTN regulates autophagosome nucleation, autophagosome maturation, lysosomal quality control, and autophagic degradation, making OPTN a multifunctional receptor in the autophagic process. OPTN^E478G mutant-related autophagic flux disruption cannot be restored by activating other autophagy receptors, indicating the essential role of OPTN in the autophagic process [34]. Moreover, OPTN is linked to various diseases through its autophagic roles. The pathogenesis of disorders related to OPTN mutation or dysfunction, unfortunately, is not yet fully understood. Although the autophagic mechanisms mediated by OPTN are very well clarified in ALS, Crohn disease, and lung cancer, the studies in other diseases are relatively superficial and only show correlations between OPTN and these diseases.

Interestingly, several different mutations of OPTN with autophagic function loss are associated with different diseases, such as OPTN^E478G in ALS and OPTN^E50K in glaucoma [13,66]. We think this is because of the heterogenicity of OPTN’s function in different kinds of cells or tissues. Therefore, the choice of specific target cells is quite important in any etiological and mechanistic studies of different diseases.

Collectively, OPTN usually plays a protective role in diseases via OPTN-mediated autophagy, such as in lung cancer, neurodegenerative diseases, osteoporosis, and kidney injury. Coincidently, these diseases all appear with low autophagic activity, which might be associated with either mutation or low expression of OPTN [131,132]. Unexpectedly, our unpublished data have shown that OPTN may act as a negative regulator in experimental autoimmune encephalomyelitis pathology via an autophagy-independent mechanism. Thus, OPTN may play distinctive roles in different diseases, depending on its primary molecular function in each respective condition.

In summary, the multiple roles of OPTN in the autophagy process are of great significance, and new tools are required to monitor autophagy flux to better clarify the precise function of OPTN. Given the close relationship of OPTN and autophagy-related diseases, OPTN can be a key regulator and a potential novel target for disease intervention, and will require further studies in the future.

Funding Statement

This work was supported by the National Natural Science Foundation of China [82073857]; Natural Science Foundation of Zhejiang Province [LR21H310001].

Disclosure statement

The authors declare no competing interests.