Canonical and Noncanonical Autophagy Pathways in Microglia


Autophagy is a vital cellular degradation process, second to the ubiquitin-proteasome system. It transports unwanted or harmful materials, such as pathogens, damaged organelles, and aggregated proteins, from the cell’s interior to the lysosome, where they are broken down. This standard process of autophagy helps maintain balance within cells and supports the immune system. Research has shown the importance of autophagy due to its links to various human diseases like cancer, immune diseases, and neurodegenerative disorders. Interestingly, some elements of the autophagy system are involved in other processes that don’t involve this typical degradation – termed “noncanonical autophagy.” In the context of neurodegenerative diseases, while neurons have been the primary focus, microglia (brain’s immune cells) are now gaining attention. As microglia play a crucial role in brain health, understanding autophagy’s role in these cells is essential. This article aims to provide an overview of the core and recent findings about both traditional and non-traditional autophagy roles in microglia.

Referred articles;1. Julg J, Strohm L, Behrends C. Canonical and Noncanonical Autophagy Pathways in Microglia. Mol Cell Biol. Feb 23 2021;41(3):e0038920. doi:10.1128/MCB.00389-20


Cellular homeostasis in all eukaryotic cells relies on preserved systems for controlled degradation of proteins, organelles, and metabolites. The term “autophagy,” derived from Greek and meaning “self-eating,” refers to an intracellular recycling mechanism wherein cytoplasmic contents are directed towards lysosomal degradation. Depending on the mode of delivery to the lysosome, autophagy can be categorized into three distinct processes:

  1. Microautophagy involves the internalization of small cytoplasmic sections via inward-budding vesicles originating from the lysosomal membrane.
  2. Chaperone-mediated autophagy sees cytoplasmic proteins specifically identified by chaperones and subsequently integrated into the lysosome in a transporter-reliant fashion.
  3. Macroautophagy (often just referred to as autophagy) is marked by the enclosure of cytoplasmic components within double-membrane formations known as autophagosomes, which eventually merge with lysosomes (Fig. 1)..

Regardless of the method of transportation, all materials targeted by autophagy are ultimately broken down by enzymes within the lysosome.

Julg J, Strohm L, Behrends C. Canonical and Noncanonical Autophagy Pathways in Microglia. Mol Cell Biol. Feb 23 2021;41(3):e0038920. doi:10.1128/MCB.00389-20

The Process of Autophagy: Advanced Insights

In the complex process of autophagy, autophagy-related (ATG) proteins play a pivotal role in shaping the autophagosome. This formation is orchestrated through a series of phosphorylation and ubiquitin-like events, as depicted in Fig. 1.

  1. Initiation: The autophagy process is kickstarted by assembling the ULK1 complex, which comprises subunits like ULK1, ATG13, ATG101, and RB1CC1. A key activation signal is the dephosphorylation of ATG13.
  2. Activation and Recruitment: Post-activation, ULK1 works to phosphorylate and then bring in the ATG9 transmembrane protein and the PI3KC3 complex I. This collective action sets the stage for autophagosome creation.
  3. PI3KC3 Complex: This complex, essential for mammalian cells, has core components such as hVps34, hVps15, BECN1, and ATG14. It also collaborates with other factors like AMBRA1 and NRBF2 .
  4. Omegasome Formation: PI3KC3 leads to the creation of PI3P, which then triggers the inception of the omegasome. This membrane structure is closely tied to the endoplasmic reticulum (ER) and paves the way for the phagophore or isolation membrane, which is integral in the autophagy process.
  5. Recruitment: With increased PI3P, proteins WIPI2 and DFCP1 get attached to the omegasome. Later, WIPI2 facilitates the attachment of hATG8 proteins to phosphatidylethanolamine (PE), securing them to the emerging phagophore.
  6. hATG8 Protein Family: This family includes seven members, classified into two subfamilies. The first is linked with microtubule-associated proteins (LC-3A, LC3-B, LC-3B2, and LC3C), while the second is associated with GABARAP proteins.
  7. Ubiquitin-Like Machinery: This involves ATG7 (similar to E1 activating enzyme), ATG3 (akin to E2 conjugating enzyme), and the ATG12-ATG5-ATG16 complex, which acts like an E3 ligase.
  8. Role of hATG8 Proteins: Once bound to the phagophore membrane, these proteins stay within the autophagosome. They undergo degradation alongside the engulfed cargo. Meanwhile, hATG8-PE complexes outside the autophagosome’s membrane are eventually detached by the ATG4 hydrolase family.
  9. Importance of LC3 and GABARAP: Their conjugation dynamics are vital for the autophagosome’s formation, impacting membrane tethering and fusion. Furthermore, LC3 has been identified to aid in moving autophagosomes to lysosomes. It does this by partnering with Rab7 and FYCO1, optimizing vesicle transport along the microtubules .

Interestingly, the structured arrangement of ATG proteins isn’t strictly necessary for autophagy—the process of degrading the cell’s internal contents. For instance, there’s a type of autophagy, termed BECN1-independent autophagy, that can be initiated under certain conditions like the presence of resveratrol. This particular mechanism isn’t affected by inhibiting components of the PI3KC3 complex such as BECN1 or hVps34. Over time, researchers identified other similar pathways bypassing proteins like ATG3, ATG5, ATG7, and even ULK1 and ULK2.

Autophagy isn’t a static process; while cells maintain a baseline autophagy level, they can enhance this pathway when required. For example, during nutrient scarcity, cells amplify autophagy, turning internal components into essential resources like proteins, sugars, and fats. The cell’s nutrient conditions significantly dictate this process. Two primary energy-detecting proteins—mTORC1 and AMPK—play pivotal roles here:

  1. mTORC1: Acts as an inhibitor for autophagy. When nutrients are abundant, mTORC1 senses amino acids, growth factors, and metabolic stress, which leads it to suppress autophagy by targeting the ULK1 complex.
  2. AMPK: Comes into play when the cell detects a drop in its energy reserves, especially ATP. Once activated, AMPK promotes autophagy. It does this by stimulating the ULK1 complex in two ways: by directly targeting ULK1 and by inhibiting mTOR activity.

Essentially, when cells face starvation, they ramp up autophagic activity, transcending its basic levels. This is a cell’s survival instinct—it’s a relatively indiscriminate mechanism designed to source nutrients and ensure survival during tough times.

Selective Autophagy: A Targeted Approach to Cellular Maintenance

While the broader autophagy process can be non-specific, there exists a more tailored version called selective autophagy. This sophisticated system specifically targets and degrades cellular components, such as protein clumps, malfunctioning organelles, or invasive pathogens, ensuring the cell remains in optimal health.

How does selective autophagy distinguish between what to preserve and what to discard? The answer lies in specialized receptors that pinpoint and latch onto designated cellular materials. These receptors are equipped with unique functional domains that allow them to identify their targets, and one of the most prevalent signals prompting this recognition is polyubiquitination. Polyubiquitination acts as a ‘tag’, highlighting specific substrates for degradation.

However, a pertinent question remains: how do different polyubiquitin chains give these receptors their specificity? While many studies have observed lysine-63 ubiquitination as the primary marker for autophagic degradation, it’s not the sole type. Experiments using autophagy-deficient mice revealed the accumulation of various polyubiquitin chains, hinting that it might not be the structure of these chains that matters. Instead, it seems that the way substrates group together, their oligomerization, dictates which receptor will act upon them, rather than the specific polyubiquitin pattern they exhibit.

Deciphering Autophagy Receptors and Targeted Elimination

Autophagy receptors work in tandem with the hATG8 protein family, allowing for a precise engulfment of the chosen cargo by the autophagosome. The collaboration hinges on the LC3 interaction region (LIR), a distinct sequence present in autophagy receptors and various hATG8-associated proteins.

A select group of autophagy receptors, encompassing entities like SQSTM1 (or p62), NBR1, OPTN, NDP52, TAX1BP1, and TOLLIP, are endowed with both the ubiquitin-binding domain (UBD) and the LIR (37). Intriguingly, alterations in some of these receptors have been linked to neurodegenerative conditions, underscoring the pivotal role of targeted autophagy in safeguarding neuronal health.

Microglia: The CNS Custodians

In the central nervous system, comprising the brain and spinal cord, microglia serve as vigilant caretakers, overseeing tissue upkeep and discarding cellular detritus (39). Recognizing microglia’s foundational role in the CNS, this piece illuminates the latest research on their autophagic responses to inflammation and varied degradation pathways. Beyond the traditional, internal-focused autophagy, innovative external-focused pathways are also coming to the fore. We aim to elucidate the molecular mechanisms underpinning both autophagy avenues within microglia.

Canonical and Noncanonical Autophagy Pathways in Microglia


Microglia: The Guardians of the CNS

Microglia are the primary immune defenders of the central nervous system (CNS). These sentinel cells make up a significant portion of the CNS’s cellular population. Tracing their lineage, these cells emerge early in development from embryonic yolk sac-derived macrophages and continue to play multifaceted roles in brain functionality throughout life.

Acting as stewards of tissue equilibrium, microglia release both inflammatory and neurotrophic factors and are renowned for their phagocytic abilities. Phagocytosis, the act of engulfing and digesting extracellular particles, is a hallmark of their function. Within a healthy brain, microglial processes constantly adjust, extending and retracting to monitor their surroundings for any signs of abnormalities. This adaptability is due to their sensitivity to a range of neurotransmitters, immune receptors, and ion channels.

In response to disturbances, microglia can morphologically and functionally adapt based on environmental cues and migrate to injury sites. Such adaptations might involve adopting an amoeboid shape, altering receptor expressions, or adjusting their phagocytic capabilities. When faced with potential threats, microglia can recognize them using a variety of receptors, including Toll-like receptors (TLRs) and triggering receptor expressed on myeloid cells 2 (TREM2).

After identifying a threat, the microglial cell engulfs the target, initiating a process known as ingestion. This leads to the formation of a phagosome, an internal membrane vesicle containing the ingested particle. This phagosome then merges with lysosomes for degradation. This phagocytic process is vital for removing various harmful entities. Additionally, microglia can release a spectrum of both pro-inflammatory and anti-inflammatory factors. Notably, emerging research suggests that autophagy, a cellular self-digestion process, plays a pivotal role in microglia’s immune functions.


The canonical autophagy process is characterized by a sequential assembly of the autophagic machinery. Although most cells do not rely exclusively on specific components, the induction of autophagy frequently involves the integration of the ULK1 kinase complex, the incorporation of the PI3KC3 complex, ATG9, PI3P effector proteins, and the attachment of hATG8s to PE on the developing phagophore. Through this mechanism, cellular materials from the cytosol are captured and directed to lysosomes for degradation. It remains uncertain if these components are essential for microglial autophagy. Within the CNS, the focus of autophagic research has primarily been on neurons, leaving a gap in our understanding of microglia’s role in neuronal immune responses in the brain.

Microglial Autophagy and Intracellular Aggregates

Protein aggregate accumulation is a key feature of neurodegenerative diseases. While these aggregates are mostly present in neurons, they are notably less common in microglia. Recent findings suggest that microglia can clear extracellular aggregates that enter their cytosol through autophagy.

One such process, termed “synucleinphagy,” involves the degradation of neuron-released α-synuclein by microglial selective autophagy. α-synuclein is implicated in vesicular trafficking and is notorious for its association with Parkinson’s disease, where it forms neuronal inclusions named Lewy bodies. When excessive human α-synuclein is present in transgenic mice, microglia become activated and consume extracellular α-synuclein deposits. These interactions boost levels of p62, an autophagy receptor, through the action of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). This increased p62 is believed to collaborate with internalized α-synuclein, facilitating its isolation in autophagosomes. It’s assumed that p62 identifies ubiquitinated α-synuclein. However, the exact way microglia intake α-synuclein remains a mystery. Contrary to traditional TLR4 signaling processes, α-synuclein binding doesn’t instigate TLR4 endocytosis, suggesting other mechanisms at play. One possibility is that α-synuclein might pass through the microglial cell membrane, influenced by lipid rafts.

In another context, Cho and team discovered that autophagy assists in clearing extracellular β-amyloid (Aβ) fibrils. This autophagic initiation is believed to be controlled by AMPK and serine/threonine-protein kinase 11 (STK11) pathways. It’s postulated that OPTN, an autophagy receptor, lures the autophagy machinery to cytosolic Aβ by linking to LC3B. The exact mechanism by which OPTN targets Aβ aggregates remains uncertain, though it’s known that OPTN can associate with protein aggregates in multiple ways, including ways independent of ubiquitin, leading to their autophagic removal. While extracellular Aβ uptake is possible via receptor-mediated phagocytosis, Aβ could also permeate through the phagosomal membrane and join with OPTN for autophagic degradation.

Delving deeper into the molecular intricacies between Aβ internalization and microglial autophagy is essential. Notably, TREM2 has the capability to detect Aβ via lipoproteins, facilitating microglial phagocytosis of Aβ. Absence of TREM2 has significant implications for mTOR signaling, resulting in heightened autophagy activation.

Microglial Autophagy in Pathogen Defense

Autophagy acts as a cellular shield, regulating the immune response by seizing intruding pathogens from the cytosol and channeling them towards lysosomal destruction. This specialized autophagic pathway is known as xenophagy. In macrophages, detection of pathogen-associated molecular patterns and damage-associated molecular patterns initiates phagocytosis, which leads to the elimination of these pathogens through autophagy. Just as with the selective degradation of other cytoplasmic entities, xenophagy employs core autophagic mechanisms, autophagosome creation, and ubiquitination as a beacon for degradation. Key autophagy receptors like p62, NDP52, and OPTN can specifically target these ubiquitinated pathogens or the compromised pathogen-holding phagosomes by anchoring them to LC3. Moreover, NDP52 has a unique interaction with galectin-8, a cytosolic lectin and sentinel receptor, which can identify exposed β-galactosides on damaged phagosomes. This intricate interplay serves as another route to single out and direct invading bacteria for autophagic breakdown.

However, the battle isn’t one-sided. As microorganisms use host cell invasion for replication, various bacterial pathogens have evolved tactics to sidestep xenophagy. For example, during the invasion of epithelial MDCK cells by the Gram-negative bacterium Shigella flexneri, its virulence factor, VirG, binds to ATG5 to induce autophagy. Yet, by releasing another bacterial agent, IscB, Shigella flexneri can cloak VirG’s presence. IscB and VirG have a competitive relationship, ultimately shielding Shigella flexneri from autophagic detection. Other pathogens, like Legionella pneumophila, sabotage lysosomal degradation by targeting key components of the xenophagy process, ensuring their proliferation and persistence. For instance, during its invasion, Legionella employs an effector called RavZ, a protease, to sever the connection of LC3 to PE. This action renders the LC3 protein ineffective, thwarting autophagy within the host cell. Additionally, certain virulence factors in Salmonella enterica interfere with critical signaling pathways, hampering autophagy initiation.

Despite these bacterial strategies, the role of microglial autophagy as a defense mechanism remains somewhat mysterious. Given that several bacterial invaders can breach the blood-brain barrier and infiltrate CNS cells, it’s plausible that microglial xenophagy acts as a crucial protective measure for the nervous system.

Microglial Autophagy and its Role in Proinflammatory Responses

Microglia play a pivotal role in orchestrating proinflammatory responses post brain injury, primarily through the secretion of cytokines. Studies indicate that autophagy proteins can influence microglial inflammation, displaying either stimulatory or inhibitory effects. Central to this proinflammatory signaling is the inflammasome: a complex intracellular protein structure comprising a sensory component for ligand recognition, an adaptor for caspase binding, and caspases for the subsequent processing of cytokines.

Within this inflammasome framework, the sensor known as NACHT, LRR, and PVD domain-containing protein 3 (NLRP3) becomes activated in the presence of Aβ. This leads to the cascading events of the adaptor component, apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization, and the intrinsic cleavage of caspase-1 (CASP1). The net result is the secretion of the cytokine interleukin-1β (IL-1β) and a heightened proinflammatory response to Aβ plaques. Research highlights that impaired autophagy exacerbates the activation of the NLRP3 inflammasome. For instance, microglia exposed to Aβ exhibit amplified CASP1 cleavage and elevated IL-1β release when certain autophagy proteins like LC3B and ATG7 are reduced. Notably, chronic and excessive inflammatory responses can be toxic to neurons, potentially causing cellular harm. There’s an intriguing perspective that heightened autophagic activity might mitigate Aβ-induced inflammation, thus favoring cell survival.

The exact role and participation of specific autophagy proteins in microglial inflammation regulation remain somewhat enigmatic. Houtman’s team identified an innovative function of microglial autophagy in controlling the NLRP3 inflammasome via BECN1. BECN1-deficient microglia, as opposed to their wild-type counterparts, showcased amplified IL-1β secretion and heightened NLRP3 protein concentrations. Additionally, there’s a belief that NDP52 might orchestrate the recruitment of NLRP3 for autophagosomal degradation, given that reducing NDP52 has been linked to increased IL-1β levels. Parallel research in macrophages has highlighted the role of p62 in aligning ubiquitinated inflammasomes with autophagosomes. Drawing from this, it’s postulated that microglia might modulate inflammasome activity through the autophagosomal degradation of NLRP3.

Another intriguing insight comes from Saitoh’s team, revealing an ATG16L1-dependent regulation of inflammation in macrophages following endotoxin exposure. Given ATG16L1’s central role in autophagosomal membrane lipidation, its absence results in macrophages displaying heightened IL-1β expression. The direct implications of this observation on microglial function are yet to be fully understood. However, the accumulating evidence undeniably points to autophagy’s significance in modulating microglial proinflammatory activities.

Finally, considering the dynamic morphological adjustments microglia undergo during confrontations with harmful agents, it’s imperative they maintain high energy levels to preserve their functionality. Thus, beyond its role in regulating inflammatory responses and removing external aggregates, autophagy also serves as a continuous energy-generating process. This ensures microglia are consistently replenished with vital nutrients and maintains intracellular protein and organelle integrity.

Canonical and Noncanonical Autophagy Pathways in Microglia


In the realm of noncanonical autophagy, components of the autophagy system are utilized for roles that go beyond the lysosomal delivery of cytoplasmic entities. A pioneering insight into these pathways in microglia was presented by Lucin and his team. Their research highlighted the importance of BECN1 in the phagocytic uptake and degradation of Aβ. When ex vivo studies were conducted using BECN1-deleted microglia from BV2 cells introduced to APP transgenic mouse brain slices, there was a noticeable reduction in Aβ phagocytosis. Even more telling, microglia isolated from postmortem human brains afflicted with Alzheimer’s disease (AD) displayed diminished levels of BECN1. This hints at a potential correlation between the autophagic modulation and phagocytic uptake of Aβ deposits.

Furthering the understanding of noncanonical autophagy, recent revelations spotlight two pathways crucial in myeloid cells: LC3-associated phagocytosis (LAP) and LC3-associated endocytosis (LANDO). In both LAP and LANDO processes, autophagy machinery components come into play to attach LC3 to the membranes of phagosomes and endosomes, respectively. Given their significant role in myeloid cells, a deeper exploration of these two pathways is warranted.

LC3-Associated Phagocytosis and Its Role in Microglia

LC3-associated phagocytosis (LAP) utilizes elements from the canonical autophagy process to link LC3 to the phagosome. Various membrane receptors initiate this, especially in macrophages. These receptors range from TLRs, which detect pathogen-associated patterns, to immunoglobulin receptors and T-cell immunoglobulin mucin receptor 4, which recognize opsonized pathogens and dead cell signals, respectively. Upon binding to the receptor, the extracellular target is engulfed.

Upon complete cargo engulfment and phagosome sealing, LAP effectors are recruited. Yet, the specific mechanism of this recruitment remains elusive. Central to LAP is the PI3KC3 complex II, distinct from its counterpart in canonical autophagy. Notably, the Rubicon protein plays a dual role in PI3P generation and the stabilization of the NADPH oxidase 2 (NOX2) complex at the phagosome. This NOX2 activity and its resulting reactive oxygen species are vital for LC3 lipidation, but the mechanism is yet to be fully understood.

Differences in LC3 lipidation between LAP and canonical autophagy are prominent. For instance, LC3 lipidation’s location varies, with LAP occurring on the single-membrane phagosome’s outer leaflet, leading to the formation of a LAPosome. Despite differences, a common LC3 function in both pathways might be to promote the fusion with lysosomes, facilitating engulfed material degradation.

LAP’s importance is underscored in its role against bacterial and fungal infections. Studies have shown the crucial nature of LAP in efferocytosis and in regulating proinflammatory cytokines. Additionally, LAP plays a part in antigen presentation through the major histocompatibility complex (MHC) class II mechanism, emphasizing LC3’s contribution to enhanced antigen presentation.

While most LAP studies have been on macrophages, microglia, with their similar features, have also been under investigation. Recent findings confirmed LAP’s role in microglial uptake of specific agents. However, the precise targets of microglial LAP remain an area of ongoing research.