Neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD) are progressive in nature and often consist of both inherited as well as sporadic forms (DiFiglia et al., 1997; Baba et al., 1998; Näslund et al., 2000; Meredith et al., 2002; Le and Appel, 2004; Li et al., 2004; Orr et al., 2008; Genuis and Kelln, 2015). Within the context of an increased aging population, as is the case today, the incidence of sporadic AD and PD cases has been seen to be much greater than those of the genetic variants (Alzheimer’s Association, 2019, 2022). The loss of cognitive function is an overlapping feature of each of these diseases. Additionally, each disease is histologically characterized according to the dysfunction of a particular protein that leads to the formation of inclusion bodies that consist primarily of these proteins within regions of the brain that correlate with the symptoms of the disease in question. For example, AD is characterized by the formation of senile plaques that present with β-amyloid deposition in the cerebral cortex (Braak and Braak, 1991), PD by the formation of Lewy bodies containing α-synuclein in the neurons of the substantia nigra (Spillantini et al., 1998) and HD by intranuclear inclusions containing of mutant huntingtin protein (Yu et al., 2003). Prior to the manifestation of a molecular pathology and symptoms these proteins form a necessary part of normal brain physiology, however, there is an increased production of their misfolded forms, which are aggregate-prone, leading to the eventual formation of inclusion bodies (Chiti and Dobson, 2009; Pechmann et al., 2009). Misfolded proteins have been shown to be a fact of life but are quickly cleared before they are produced to such an extent that they outnumber the presence of the native protein forms (Swart et al., 2014; Sweeney et al., 2017). This clearance is maintained by degradative systems such as the ubiquitin proteasomal system (UPS) and macroautophagy (Bence et al., 2001; Komatsu et al., 2006; Tanaka and Matsuda, 2014). However, it is known that the UPS becomes overwhelmed and ineffective when there is an overproduction of aggregated proteins, as is the case in neurodegenerative diseases, leading to a greater dependence of the system upon autophagy as a means of clearance (Figure 1). Although the components are well known in isolation, the interplay between protein cargo and its solubility state, mitochondrial health and lysosomal dysfunction appears central to the development of neurodegenerative diseases. In this review we will discuss the mechanisms in place for protein quality control; particularly macroautophagy, as well as the cellular dysfunction that impacts mitochondria and lysosomes which appear to be at the heart of neurodegenerative disease pathogenesis.

Proteostasis, that is the maintenance of protein homeostasis by controlling the abundance, conformation, binding interactions, and distribution of proteins by means of synthesis, degradation or modification is an integral part of normal metabolic functioning of a cell (Balch et al., 2008). Indeed, due to the important roles that proteins play in cellular processes such as signaling, the immune response, and structure, changes to proteostasis may result in cellular dysfunction and detrimental effects such as ER stress and autophagy dysfunction (Chen et al., 2001; Mcnaught et al., 2002; Chiti and Dobson, 2006; Hara et al., 2006). Therefore, there is a clear need for this balance to be carefully maintained for the sake of cell survival. Two degradative pathways exist and are used by the cell to ensure that proteostasis is maintained.

The first degradative pathway is the ubiquitin-proteasomal system (UPS) which is critical for the rapid degradation of short-lived, monomeric proteins (Kraft et al., 2010). The ubiquitin tagging system component is responsible for covalently binding cytoplasmic entities with ubiquitin, which acts as the recognition motif for the 26S subunit of the proteasome. Thus, the ubiquitination system provides the UPS with a high degree of specificity. It is however possible for the proteasome to become overwhelmed by substrates present (Bence et al., 2001; Mcnaught et al., 2002; Rideout et al., 2004; Pandey et al., 2007; Wang et al., 2020). This occurs when protein targets express irregular structures, resulting in misfolded conformations which are hydrophobic and aggregate-prone. As these proteins aggregate, they from larger clusters and are unable to pass through the proteasome (Johnston et al., 1998). In this instance, the proteostatic balance has been disrupted and the potential for the formation of insoluble structures such as aggresomes or inclusion bodies has been increased. This disruption leads to a proteostatic shift in which the system becomes more reliant upon autophagy as the means to degrade proteins.

Autophagy is the second key degradative pathway operating in cells. Three types of autophagy exist, namely microautophagy, macroautophagy and chaperone-mediated autophagy. Although each of these are well distinguished from one another, they share the function of delivering cytoplasmic components to lysosomes which contain acidic hydrolases; thereby aiding in degradation of these recruited components. Macroautophagy (hereafter autophagy) is the most well studied of its variants and morphologically distinct from the other forms due to the use of vesicular structures known as autophagosomes to shuttle cytoplasmic components to lysosomes, thereby facilitating degradation (Jahreiss et al., 2008; du Toit et al., 2018a). Autophagy has been shown to display pro-survival functions in cells following metabolic perturbation and cellular stress conditions (Mizushima et al., 2004, 2008; Ravikumar et al., 2010; Sumpter and Levine, 2010). Its degradative capabilities have been shown to extend to many cytoplasmic components, including proteins, mitochondria, peroxisomes and even micro-organisms.

In the past, autophagy was described as a non-selective form of degrading cytoplasmic components for the purpose of generating amino acids to be used in ATP production. Although this is true given nutrient-poor conditions (Sahani et al., 2014), it is now known that autophagy also controls selective cargo degradation given imbalanced homeostasis; including disruption in mitochondrial, lysosomal and ferritin homeostasis (Galluzzi et al., 2017). This selective targeting of cytoplasmic components as cargo is accomplished by proteins that act as receptors of ubiquitinated elements, effectively enabling the distinction between different types of disruptive components and targeting them to the autophagosome for subsequent degradation by lysosomal hydrolases (see section 6). Should the synthesis of misfolded proteins and aggregated proteins outweigh the synthesis and presence of fully functional proteins, proteostasis will be disrupted (Figure 1). Misfolded proteins have been shown to interact with other components of the cell such as mitochondria and the endoplasmic reticulum (ER), disrupting their function and further contributing to disrupted conditions (Lasagna-Reeves et al., 2011; Fang et al., 2019; Sharoar et al., 2019; Evans and Holzbaur, 2020). It becomes crucial therefore for the presence of these proteins to be minimized to preserve the viability of the cell.

A basal level of autophagy is constantly present within the cell and serves a “housekeeping” function; eliminating old or damaged cellular components that would otherwise disrupt homeostasis (Mizushima et al., 2004; Alers et al., 2012; Sarkar, 2013). However, in the event of a disruption in cellular homeostasis due to, for example, starvation or increased misfolded protein production, the rate of clearance through autophagy will need to increase (Yang and Klionsky, 2010; Kim et al., 2011).

Autophagy can be characterized as a step-by-step process (Figure 2) which begins with the initial synthesis of the phagophore which then matures into an autophagosome; the double-membrane vesicle that characterizes the pathway (Loos et al., 2014). Once it has sequestered cargo, the autophagosome is transported to and fuses with a lysosome, resulting in an autolysosome, which ensures the degradation of cargo. The term ‘autophagy flux’—the rate of protein degradation through the autophagy pathway—can be used to quantitatively describe the overall degradative activity and capacity of the pathway (Klionsky et al., 2016). Given conditions such as starvation or increased misfolded protein load, it becomes necessary for the autophagy flux to be increased. Indeed, given such conditions it has been observed that the cellular system will increase the number of autophagosomes thereby effectively increasing the autophagosome pool size available for cargo sequestration and delivery (Boland et al., 2008; du Toit et al., 2018b; de Wet et al., 2021).

As the autophagy pathway involves the participation of many molecular role players, including autophagosomes, lysosomes and cargo, it becomes necessary to discern which of these are being used to measure and define autophagy flux. The term “autophagosome flux” has been proposed and is defined as the rate of flow along the vesicular pathway. The techniques used to measure it infers a distinction between the vesicular—that is, the molecular machinery that make up the autophagosome—and the cargo flux (Loos et al., 2014). This is of importance as not all proteins are degraded at the same rate nor does autophagy occur at the same rate in every tissue type. Indeed, whilst there may be indications of high autophagic activity—such as large autophagosome pool sizes— the inherent degradation rate of proteins may differ according to different tissue types or even brain regions (Mizushima et al., 2004; Lumkwana et al., 2017). Additionally, we have previously shown that an increase in autophagosome pool size does not necessarily infer an equal or proportional increase in the cargo receptor abundance and availability (de Wet et al., 2021). Hence, a distinction between the cargo clearance and autophagosome flux as separate entities may be necessary. To measure autophagosome flux one must consider the total number of autophagosomes within the cell at a time point and contrast this with the number of autolysosomes at the same time, in the absence and presence of an autophagosome/lysosome fusion inhibitor or lysosomal deacidifying agent, such Bafilomycin A1 or chloroquine (Li et al., 2013; du Toit et al., 2018a). In doing so, it becomes possible to measure the change in the number of autophagosomes, lysosomes and autolysosomes, respectively, that are present in the cell within a given time frame and thereby reveal their relationship in contributing toward the degradative potential of the cell. For this reason, autophagosome, lysosome and autolysosome pool sizes should be measured simultaneously to better understand the autophagosome flux, while measuring both the autophagosome and cargo flux may enable greater insight into the clearance capabilities of the pathway.

Indeed, the step-by-step nature of the autophagy pathway allows a greater number of parameters to be assessed to describe the degradative activity of the system. The following sub-sections will be used to discuss the control and stages of autophagy flux as separate entities such that it is clearer what factors may influence the degradative capacity of autophagy (Figure 2).

The molecular events necessary following the signaling event that led to the generation of a mature autophagosome that contains sequestered cytoplasmic cargo and is functionally able to interact with a lysosome for degradation, is highly complex and comprises of multiple steps (Figure 4). This section will address the autophagosome machinery, that is, the core proteins recruited to the phagophore membrane, leading to the formation of a mature autophagosome. Autophagosome biosynthesis is marked by the initial translocation of the ULK complex to the ER, the site of autophagosome initiation (Koyama-Honda et al., 2013). Once present on the ER membrane, the ULK complex recruits the PI3K complex III (PI3KC3) containing Atg14L (Russell et al., 2013). Once recruited to the ER membrane, PI3KC3 phosphorylates phosphatidylinositol (PtdIns)—a lipid present on the membranes of many organelles, including the ER—to produce PI(3)P (phosphatidylinositol-3-phosphate). In parallel with this event, there is recruitment of proteins that specifically bind to PI(3)P, such as DFCP1 (double FYVE domain-containing protein 1) and WIPI (WD-repeat-interacting phosphoinositide protein). The exact role of these proteins remains unclear; however, it seems clear that their recruitment precedes that of the autophagy-related proteins (Axe et al., 2008; Koyama-Honda et al., 2013; Amaya et al., 2015). Additionally, these proteins contain a FYVE domain, which enables them to bind with the membrane of the ER and possibly the Golgi apparatus and therefore the vesicle derivatives of each. As such, PI(3)P-binding is considered to form a scaffolding matrix on the phagophore that facilitates the recruitment of autophagy-related (Atg) proteins to the phagophore after the nucleation event.

Before an autophagosome can sequester and deliver cargo, it requires modification from two ubiquitin-like conjugation systems to facilitate its elongation (Figure 4). First, Atg12 is conjugated to Atg5, which occurs due to a reaction with Atg7 and then with Atg10. Next, Atg16L is non-covalently conjugated to the Atg12-Atg5 complex, resulting in the formation of the Atg12-5-16 L complex (Mizushima et al., 2003; Tanida et al., 2004). The complex is then targeted toward phosphatidylethanolamine (PE), another membrane lipid located on the pre-autophagosome membrane (Mizushima et al., 1999).

The second conjugation system involves the transfer of microtubule-associated protein 1 light chain 3 (LC3). This process begins with the cleavage of pro-LC3 by Atg4 resulting in LC3-I. LC3-I is subsequently conjugated with Atg7 and then with Atg3. The interaction between Atg12 and Atg5 enables Atg12 to sequester Atg3, removing it and bringing LC3-I into proximity with PE. The reaction between LC3-I and PE results in the lipid-bound LC3-II (Fujita et al., 2008b). The presence of LC3-II marks the end of autophagosomal elongation and autophagosomes are now prepared for cargo sequestration (section 6). Once cargo has been sequestered, Atg proteins dissociate from the autophagosome membrane. This dissociation is thought to enable the closure of the autophagosome (Cebollero et al., 2012). LC3-II remains as part of the autophagosome membrane for the duration of its lifetime and is used as a marker of mature autophagosomes when assessing autophagosome abundance or autophagosome puncta counts (Pankiv et al., 2007; Boland et al., 2008; Jahreiss et al., 2008; du Toit et al., 2018a). Additionally, LC3-II is useful in enabling interaction between autophagosomes and microtubules, acting as an anchor point for molecular motors; proteins that drive the movement of the autophagosome (Fass et al., 2006; Jahreiss et al., 2008; Pankiv et al., 2010). Microtubules play an important role in the maturation of autophagosomes as well as their delivery, docking and fusion with lysosomes (Shen and Mizushima, 2014). The role of microtubule-dependent delivery in the autophagy process will be further discussed in section 7.

After lysosomal-dependent degradation, amino acids are released into the cytosol where they can be used for aerobic respiration by mitochondria (Scherz-Shouval and Elazar, 2007; Rabinowitz and White, 2010). LC3-II is delipidated to LC3-I by Atg4, thereby replenishing the cytoplasmic pool for future autophagy events (Fujita et al., 2008a; Kaizuka et al., 2016). The proteins involved in ensuring functional autophagosome generation and providing autophagosomes with the ability to successfully sequester cargo and interact with microtubules to enable their delivery to lysosomes demonstrate a high level of complexity. The molecular machinery necessary for autophagosome synthesis is of key interest and studies are ongoing to elucidate the precise role of many of these protein during autophagy enhancement and cargo sequestration.

Once elongated, the autophagosome is ready to receive cargo that is to be delivered to the lysosome for degradation. This step has received major attention as the autophagosome may either sequester cytoplasmic components as cargo in a non-selective, bulk manner (Mizushima, 2007), or in a highly selective manner (Kirkin et al., 2009; Zhang and Ney, 2009; Deosaran et al., 2013). The understanding of the specificity of autophagy has created a need amongst researchers to differentiate between several subtypes of autophagy according to the specific cargo targets in the context of metabolic or homeostatic perturbation (Galluzzi et al., 2017). The exact mechanism by which cargo recognition is regulated is not clear, however ubiquitinated cargo appears to be an overlapping feature (Kim et al., 2008) which is exploited by proteins bearing a ubiquitin binding area (UBA) domain as well as a LC3-interacting region (LIR). In this way, these receptor proteins, sometimes termed adaptors, are able to bind ubiquitinated components as well as LC3-II, thereby linking cargo with autophagosome machinery for effective cargo sequestration (Pankiv et al., 2007; Fujita et al., 2008a; Figure 2).

One such receptor protein is p62/SQSTM1 (hereafter p62). Due to its various functional domains, p62 has been shown to play an important role in many physiological processes such as cellular signaling pathways, inclusion body formation and tumorigenesis (Moscat and Diaz-Meco, 2009; Lim et al., 2015). In the context of cargo degradation, p62 has been shown to be present in autophagy progression and is therefore useful as a marker of autophagy activity (Ravikumar et al., 2005). Additionally, p62 has been shown to bear a Phox and Bem1 (PB1) domain which enables it to oligomerize with other p62 receptors. In so doing, p62 can self-aggregate along with its cargo to further enhance degradation (Kirkin et al., 2009). Although p62 appears to be involved in many forms of autophagy, its importance lies in its function to serve as a receptor that is primarily associated with aggrephagy; the autophagy-dependent degradation of protein aggregates (Kopito, 2000; Bjørkøy et al., 2005; Caccamo et al., 2017). This role is vital in the context of neurodegenerative diseases in which harmful misfolded proteins are generated and may lead to the formation of inclusion bodies if left unchecked (Komatsu et al., 2007; Salminen et al., 2012; Sarkar et al., 2014).

Another receptor protein, neighbor of BRCA1 gene 1 (NBR1), has been shown play several roles in maintaining cell survival (Mardakheh et al., 2010; Kim et al., 2019; Marsh et al., 2020), including the autophagy-dependent degradation of peroxisomes, known as pexophagy (Deosaran et al., 2013). Furthermore, NBR1 has also been shown to play an important role in aggrephagy as a compensatory receptor given the absence of p62 (Kirkin et al., 2009). Evidence has shown that NBR1 may be cleared through the endosomal rather than the autophagosomal pathway (Mardakheh et al., 2010), however, given that NBR1 bears the LIR and UBA domains necessary for autophagy-associated degradation, it may be plausible that NBR1 is preferentially degraded through the endosomal pathway, but has increased involvement in the autophagy-lysosomal pathway as p62 availability decreases.

Mitochondria are vital in the maintenance of the cell survival. Mitochondria are spread out through the cell as highly dynamic network; undergoing fission and fusion reactions as necessary. Fusing to facilitate efficient oxidative phosphorylation as well as protecting and maintaining mitochondrial DNA and undergoing fission to distribute mitochondria across the cell and segmenting depolarized portions to ensure their subsequent clearance (Parone et al., 2008; Twig et al., 2008; Wang et al., 2009; Perciavalle et al., 2012; Rana et al., 2017). To this end, mitophagy; the autophagy-dependent degradation of mitochondria, is yet another key autophagy subtype. Indeed, failure to eliminate dysfunctional/depolarized mitochondria results in the continued production of ROS, therefore threatening cell viability (Quintanilla et al., 2009; Wu et al., 2011). Several mitophagy receptors have been identified, presumably demonstrating the importance of mitochondrial health and turnover. These include receptors such as BNIP3 and NIX, optineurin (OPTN), optic atrophy 1 (OPA1) and the PINK/Parkin system (Zhang and Ney, 2009; Shi et al., 2014; Marceau et al., 2015; Diot et al., 2018; Padman et al., 2019; Evans and Holzbaur, 2020). The variety of identified receptors for mitophagy is a true testament to the importance of maintaining a healthy mitochondrial network and minimizing oxidative stress produced by dysfunctional mitochondria.

Of the various subtypes of autophagy that exist, aggrephagy is the one most often studied and best characterized. This is due to its critical role in re-establishing disrupted proteostasis, especially when the activity of the UPS has been impeded. Other types of autophagy, such as lysophagy, reticulophagy, xenophagy and several others have also been described along with their potential receptors (reviewed in Galluzzi et al., 2017). It is important to note that the selective nature of autophagy has only recently been identified and investigations into the exact mechanism through which cargo is sequestered are ongoing.

At a molecular level, neurodegenerative diseases are characterized by the increased presence of soluble oligomers, dysfunctional mitochondria, deacidified lysosomes and ER stress (Figure 5). Which proteinaceous cargo is problematic is what defines the neurodegenerative disease and will be discussed in subsequent sections. Due to the conformation taken on by misfolded protein monomers (Hamano et al., 2008; Chiti and Dobson, 2009), these proteins become increasingly aggregated and the resulting oligomers cause a proteostatic imbalance (Tanaka and Matsuda, 2014; Martínez et al., 2017). Additionally, due to the extent to which these proteins are aggregated, they become unable to pass through the proteasome component of the UPS. Basal autophagy has been shown to be upregulated in response to the accumulation of proteinaceous components, demonstrating a shift in protein degradation that is reliant upon autophagy (Nixon, 2007; Lim et al., 2015).

The ER is crucial in maintaining proteostasis and a disruption of that proteostasis, due to the overproduction of misfolded proteins, has been shown to induce ER stress; a major contributor toward the overall cellular stress that results in increased cytoplasmic calcium (Ca²⁺) (Goussakov et al., 2010; Hetz and Mollereau, 2014; Villegas et al., 2014; Kaushik and Cuervo, 2015; Martínez et al., 2017). Furthermore, the misfolded proteins associated with neurodegenerative diseases have been shown to co-localize with and disrupt the electron transport chain complexes of mitochondria, thereby disrupting neuronal bioenergetics (Manczak et al., 2006; Chinta et al., 2010; Lasagna-Reeves et al., 2011; Shirendeb et al., 2011). This mitochondrial disruption carries a knock-on effect as the lack of ATP generation leads to a decrease in the acidified lysosomal pool size. The loss of lysosomal acidity results in an increase in the number of autophagosomes and a decrease in the numbers of autolysosomes in the neuron as the disease progresses (Lee et al., 2011; Burbulla et al., 2017). The combined effects of increased intracellular Ca²⁺ along with dysfunctional mitochondria lead to an increased production of ROS within the cell, priming it toward death (Kishida and Klann, 2007; Loos et al., 2013). Subsequently, abundant cell death causes interruptions between neuronal connections within specific regions of the brain, thereby contributing to clinical symptoms that manifest in patients with the disease. Additionally, many neurodegenerative diseases, such as Alzheimer’s and Parkinson’s disease, have both genetic as well as non-genetic origins, indicating that these diseases may have familial roots or be a result of lifestyle, environment or aging (Andersen, 2003; Winklhofer and Haass, 2010; Genuis and Kelln, 2015; Kell and Pretorius, 2015; Xu et al., 2016; Colacurcio and Nixon, 2017; An et al., 2018). In short, the molecular pathway through which neurodegenerative diseases are manifested in patients is no simple matter and should be approached in a genetic as well as physiological and biochemical manner to reach a consensus on the best possible treatment approach.

Aggregated proteins continue to accumulate throughout the progression of the disease as the pool size of acidified lysosomes continues to decrease. As a result, there is disrupted cellular energetics and death that leads to decreased synaptic firing and consequently, disrupted neuronal networks (Figure 5). The respective insoluble inclusion bodies are used as histological markers of neurodegenerative disease, especially at late stages where autophagy function has been mostly lost due to the loss of acidified lysosomes and neurons have begun to undergo cell death. The region of the brain impacted by the build-up of these insoluble protein aggregates varies according to the particular disease. However, there are often overlapping clinical symptoms, such as dementia or decreased fine motor capabilities that are common between diseases.

It becomes clear that the soluble oligomers are problematic and reducing their expression at a cellular level is key to restoring cellular homeostasis. The autophagy pathway relies heavily upon the availability of acidified lysosomes which in turn only achieve a sufficient acidification status through the ATP produced through oxidative phosphorylation. Mitochondria in turn depend on the substrates released from the autophagy pathway, i.e., glucogenic and ketogenic amino acids, following cargo degradation. Here we will collectively term this cross talk the “autophagy triad” (Figure 6).

Autophagy activity is crucial in providing substrates for oxidative phosphorylation, and mitophagy is important for the maintenance of a healthy mitochondrial network by eliminating depolarized mitochondria (Burman et al., 2017; Rana et al., 2017). Furthermore, loss of autophagy cargo recognition has been shown to result in an increase in aggregate formation (Bhattacharjee et al., 2022) and contributes toward neurodegenerative disease manifestation. Indeed, a study investigating a p62 mutant in amyotrophic lateral sclerosis (ALS) showed that p62 behavior was changed in such a way that autophagy activity and mitochondrial respiration were both significantly decreased (Bartolome et al., 2017). Additionally, studies in which PINK1 and Parkin have been knocked out, respectively have demonstrated a decrease in mitochondrial activity, highlighting the key role of mitophagy in maintaining mitochondrial health (Song et al., 2017; Zhi et al., 2019).

Highlighted in section 6, mitochondria are dynamic organelles, undergoing constant fission and fusion events in response to the metabolic requirements of the cell. Fusion events occur through mitofusin-2 (Mfn2) and these events are shown to result in the increase of ATP, with cases of hyperfusion occurring due to a loss in ETC complex IV activity (Rolland et al., 2013). Fission events on the other hand, occur primarily through the dynamin-1-like protein (Drp1), which is responsible for separating the mitochondria in response to metabolic stress for the purpose of eliminating harmful, ROS producing mitochondria from the system (Rana et al., 2017; Fonseca et al., 2019). Indeed, Fang et al. (2019) have shown an accumulation of dysfunctional mitochondria in the neurons of AD patients, demonstrating mitophagy dysfunction early in disease progression (Fang et al., 2019). It becomes clear therefore that mitochondrial network health is vital for the maintenance of mitochondrial quality and for enabling acidification of lysosomes (Yagi et al., 2021; Huang et al., 2022).

Studies in which ATP6AP2, a key component of the lysosomal acidification machinery, had been knocked down in Drosophila and mouse models demonstrated an increase in autophagosomes along with diminishments in cognitive performance (Dubos et al., 2015; Hirose et al., 2019). Likewise, the loss of lysosomal acidity has been shown to be accompanied by an accumulation of autophagosomes as a result of a decline in autophagy flux (see section 3). The restoration of lysosomal acidification through novel nanoparticles and small molecules has been shown to decrease the numbers of autophagosomes present in the cell, demonstrating an increase in autophagy flux while also enhancing the presence of healthy mitochondria, likely through the induction of mitophagy (Vest et al., 2022; Brouillard et al., 2023; Zeng et al., 2023).

Dysfunction within any of these three entities may hence contribute toward the onset of neurodegenerative disease (Figure 6). Likewise, it is clear that therapeutic interventions that lead to the increased activity of one entity (as reviewed by Lumkwana et al., 2017; Lo and Zeng, 2023) will have subsequent knock-on effects that will enhance the functioning of the other.

AD, PD and HD constitute the most prevalent neurodegenerative diseases. Whilst all three have been shown to exhibit genetic inheritance, it is concerning to note that sporadic AD and PD are commonplace in individuals within aging populations and present without genetic factors (Greenamyre and Hastings, 2004; Alzheimer’s Association, 2022). As such, the incidence of AD and PD is of increasing concern as it reflects lifestyle as well as the environmental factors as contributing causes for neurodegenerative disorders (Winklhofer and Haass, 2010; Genuis and Kelln, 2015; Kell and Pretorius, 2015; Xu et al., 2016; Colacurcio and Nixon, 2017; An et al., 2018).

Arrasate et al. (2004) have demonstrated that a greater level of mHtt inclusion bodies is positively correlated with greater cell survival. In PD, Lewy bodies are shown to consist of iron as well as misfolded α-synuclein. By removing misfolded α-synuclein from the cytoplasm, it may be that there is less dopamine dysregulation, whereas removing iron prevents a reaction with dopamine that would result in the production of ROS (Castellani et al., 2000; Lotharius et al., 2002; Ulusoy et al., 2012). Aβ oligomers are released to the extracellular microenvironment, by a mechanism which is poorly described, but is likely to involve mitochondria-associated ER membrane, where they aggregate into senile plaques, thereby potentially eliminating the harmful oligomers from the cytoplasm (Ray et al., 2010; Nilsson et al., 2013; Bhattacharyya et al., 2021). However, prior to the formation of senile plaques, these proteins have been shown to trigger an immune response which leads to neuroinflammation in the extracellular milieu. Additionally, their presence in the extracellular millieu enables them to interact with NMDA receptors in such a way that induces excitotoxicity through excessive Ca²⁺ entry into the neuron (Kitazawa et al., 2004; Shankar et al., 2007; Goussakov et al., 2010; Ma et al., 2014). The immune response in particular may be exacerbated by increased hydrophobicity leading to the formation of senile plaques, thereby further increasing the neurotoxicity produced. Inclusion bodies are the hallmark characteristic used to histologically identify and distinguish between neurodegenerative diseases. They are composed of oligomeric forms of misfolded proteins which increase in hydrophobicity as they aggregate over time, forming the insoluble inclusion bodies that are deposited in regions of the brain that correspond with the symptoms of the disease at later stages of the disease. In addition to the characteristic oligomers, inclusion bodies have also been shown to consist of p62 and ubiquitin, demonstrating that autophagy plays a critical role in the formation of insoluble aggregations (Hara et al., 2006; Komatsu et al., 2007; Jackson et al., 2017). It is therefore plausible that inclusion bodies are not the root cause of neurotoxicity per se, but rather that they arise due to autophagy dysfunction brought about by the effects of the oligomeric constituents of the inclusion bodies that cause damage to intracellular organelles (Figure 6).

It is however of vital importance that their production be minimized in a timely manner so as to avoid disruptions of the mitochondrial ETC (Hansson Petersen et al., 2008; Orr et al., 2008; Parihar et al., 2008; Rao et al., 2014; Di Maio et al., 2016) or ER stress (Timmins et al., 2009; Gupta et al., 2010), that can lead to increased intracellular Ca²⁺ levels (Yu et al., 2009; Affaticati et al., 2011; Zündorf and Reiser, 2011; McBrayer and Nixon, 2013), both of which contribute toward a cytotoxic environment; therefore driving the likelihood of cell death onset. Additionally, inducing autophagy with mTOR inhibitors such as rapamycin has been shown to decrease the availability of these proteins (Caccamo et al., 2010; Malagelada et al., 2010; Spilman et al., 2010) and other inducers of autophagy such as spermidine, resveratrol and metformin, to name a few, have also been shown to result in reduced levels of oligomers and thus contributing toward the survival of the cell (Eisenberg et al., 2009; Shi et al., 2012; Ai et al., 2015; Pietrocola et al., 2015; Wang et al., 2018) although this should be performed with caution so as to avoid – albeit unlikely – autosis onset. It becomes clear therefore that the misfolded conformations of these key proteins are what drives cytotoxicity as their effects occur prior to the appearance of inclusion bodies, an important consideration when developing effective therapies against neurodegenerative disease in order to avoid consequential neuroinflammation.

The mild hydrophobicity of soluble oligomers creates a challenge to the autophagy system which increases autophagosome production as a means of increasing flux as a stress response to decrease the protein load (Nixon et al., 2005; Fujita et al., 2008a; Menzies et al., 2015; Bordi et al., 2016). Although basal autophagosome counts in neurons are generally low, treatment with lysosomal inhibitors such as bafilomycin A1 or chloroquine cause an accumulation of autophagosomes, lysosomes and autolysosomes demonstrating that neuronal autophagy flux is in fact very high (Boland et al., 2008; du Toit et al., 2018a). Regardless, patients still suffer from symptoms of neurodegeneration. This is likely because there is an overproduction and oligomerization of misfolded proteins, generated at a greater rate than the rate of their degradation through autophagy (Martinez-Vicente et al., 2010; Wu et al., 2015; Bordi et al., 2016). A possible reason for this may be due to the interaction between oligomers and mitochondria, resulting in decreased ATP output (Kim et al., 2001; Manczak et al., 2006; Hansson Petersen et al., 2008; Chinta et al., 2010; Di Maio et al., 2016). Since lysosomes require ATP to be reacidified after the degradation of cargo (Forgac, 2007; Zoncu et al., 2011), the decreased availability of ATP will lead to a decrease in protein degradation in spite of cargo sequestration by autophagosomes (Figure 6). Indeed, Lee J. H. et al. (2022) and Lee S. E. et al. (2022) have recently demonstrated the importance of acidified lysosome pool size in the context of AD and neuronal health (Colacurcio and Nixon, 2017; Lee J. H. et al., 2022). Additionally, this will result in dysfunctional mitophagy, leading to the accumulation of dysfunctional mitochondria and their continued production of ROS and pro-apoptotic signaling molecules (Narendra et al., 2008; Orr et al., 2008; Zhang and Ney, 2009). Taken together, it is plausible that the stress produced by mitochondria and the subsequent loss of lysosomal acidification lies at the heart of neurodegeneration as autophagy flux is dependent upon adequate mitochondrial output (Figure 6). It is important to note that these interactions occur intracellularly but create extracellular stressors that lead to knock-on effects to surrounding cells. Increasing the autophagy flux seems to be the gold standard to decreasing ND toxicity, however, as it is known that dysfunctional mitochondria do continue to accumulate during the pathogenesis of the disease, it is conceivable that increasing autophagy flux will work to a degree but reach a point where it is no longer effective as the lysosomal pool becomes increasingly deacidified. If our suggested “autophagy triad” is relevant (Figure 6), then it may be the case that inducing mitochondrial biogenesis or mitochondrial fusion, through treatments such as resveratrol or metformin, might be more beneficial than previously anticipated, as these will contribute toward lysosomal acidification and subsequently autophagy flux that effectively aids in the clearance of depolarized mitochondria as well as NDD proteins. This notion would strongly support a combined therapy that makes use of upregulated mitochondrial biogenesis paired with autophagy flux induction, therefore impacting lysosome function and subsequent mitophagy and proteinaceous cargo capacity. This deserves further study.

SdW conceptualized, wrote the manuscript, and developed the figures. BL conceptualized. BL and RT edited the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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