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Mechanisms underlying the neuronal lysosomal
system dysfunction in Alzheimer’s Disease
A ik at erini S f y ak i , S t ud ent ID : 1279196 2, Mas t er in B rain and Co g nit iv e S c ienc es , Univ ers it y o f A ms t erd am , No v emb er 2020.
Abstract
Lysosomes serve as the cell’s degradation center, assisting in the maintenance of proteostasis by digesting internalized materials and defective organelles. This ability is of high importance for post-mitotic cells, including neurons. Aberrations in the neuronal lysosomal system are one of the earliest defects in Alzheimer’s Disease (AD). The mechanisms underlying this disruption are not yet fully elucidated. In AD, changes are observed in the lysosomal system of neurons. In this literature thesis, the most prominent lysosomal alterations encountered in AD patients and models are being examined. These include lysosomal deacidification which leads to deactivation of hydrolases, an abnormal flux of calcium ions that overactivates genes implicated in AD development and a dysregulation in the production of molecules fundamental for the normal lysosomal function. A key molecule regulating lysosomal biogenesis, autophagy and lysosomal function, the transcription factor EB (TFEB), is specifically addressed. TFEB’s activity is controlled by phosphorylation and dephosphorylation. In AD, TFEB is downregulated, suggesting that the development of drugs and methods aiming to its dephosphorylation might consist a novel therapeutic approach against the progression of the disease. Alterations in the neuronal lysosomal system are interconnected indicating that a main common pathway might be underlying them. Shedding light in these molecular pathways might reveal novel drug targets against AD pathology.
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Table of Contents
Abstract ... 1
Introduction ... 3
Critical overview of the current literature... 4
Lysosomes and the importance of their acidification via the vATPase complex ... 4
Degradation process ... 5
Types of Autophagy ... 6
The neuronal lysosomal system ... 8
AD pathology and pathogenesis ... 8
Production of Αβ peptides and amyloid plaques ... 9
Link between the lysosomal system and AD ... 10
Lysosomal acidification: a key parameter in the development of AD 11
Calcium flux implicated in AD pathology ... 12
TFEB’s important role in lysosomal functioning ... 13
TFEB’s link to AD pathology and future approaches ... 15
Interdisciplinary relation ... 17
Conclusions... 18
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Introduction
Alzheimer’s disease (AD) is one of the major neurodegenerative diseases and the main cause of dementia, responsible for half of the dementia cases, affecting more than 50 million people worldwide. The number of people suffering from dementia is expected to increase dramatically in the next 30 years according to the World Alzheimer Report 2015, Alzheimer’s Disease International. The disease owes its name to the German psychiatrist and pathologist who described it first, Alois Alzheimer. AD impairs various cognitive functions, with memory decline being the most prominent hallmark of the disease described in the elderly (Nygård, 2003; Salmon, 2011). The main parameter considered responsible for the progression of the disease is the rapid accumulation of specific proteins and protein products in the brain. The key components that have been implicated in contributing in the pathogenesis of the disease are amyloid beta plaques and intraneuronal protein tau tangles, which aggregate outside and inside the neurons respectively (Scheltens et al., 2016; Selkoe, 1991). The reason and the mechanism by which these key features assist in the development of the disease are not yet fully comprehended. Several pathways and mechanisms are suggested to be involved (J. Kim et al., 2007; Nixon, 2017; Van Acker et al., 2019). Currently there is no effective treatment or prevention strategy capable of reversing or prohibiting the progression of the disease. This problem derives from the lack of comprehension of the pathophysiology and the huge number of alterations observed in various parameters that are usually described as biomarkers of the disease. The rapidly rising number of individuals affected by AD highlights the great importance for further research and different approaches to be conducted.
Strong evidence exists supporting the hypothesis that the lysosomal and autophagy system constitutes one of the main mechanisms underlying the evolution of neurodegenerative diseases, including AD (Nixon, 2017). The lysosomal and autophagy pathways are of high importance for the maintenance of homeostasis inside the cells, and especially for neurons, which are post-mitotic non-dividing cells in need of effective clearance mechanisms and careful preservation of proteostasis (Ferguson, 2019). In neurodegeneration, disturbance of this balance is reported due to the augmented accumulation of proteins and several detrimental bioproducts that can result in neuronal death (Gao et al., 2018; Settembre et al., 2013).
The lysosome is a membrane bound organelle characterized by an acidic internal environment for its enzymes to function optimally during the degradation process (Ferguson, 2019). It is capable of receiving, and from thereon, recycling various materials that are delivered to it via autophagy, phagocytosis and endocytosis. The nature of the molecules recycled depends on the delivery method that was used. Interestingly, proteins and other key molecules implicated in the generation of AD are normally transferred via endosomes inside the lysosomes in order to be degraded (Gao et al., 2018; Peric & Annaert, 2015; Sun et al., 2015). Nevertheless, aging and neurodegeneration cause a reduction in the lysosomal function by worsening the neuronal clearance mechanisms as well as the substrate delivery to lysosomes, contributing further to the pathophysiology of the disease (Nixon, 2020). This highlights the importance of fully understanding the role of the lysosomal system in aging, neurodegeneration and longevity.
Various proteins that are affected by lysosomal defects are interestingly reported to be disturbed also in AD. Some of these proteins that gained central attention in this field are presenilins which affect lysosomal acidification and are also implicated in amyloid processing. It is suggested that disrupted lysosomal acidification causes a rise in the pH levels which subsequently leads to decreased proteolytic capabilities of the lysosomes (J. H. Lee et al., 2010, 2015; Van Acker et al., 2019). Interestingly, acidification is linked to the cellular and
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lysosomal levels of calcium, which under certain conditions can cause the activation of proteins and molecules implicated in the progression of AD (Colacurcio & Nixon, 2016; J. H. Lee et al., 2015). This indicates that a complicated link between lysosomal acidification, the genes mediating it and the ions implicated in it, exists and a full comprehension of this could mean a further step towards the unravelling of promising treatments.
In addition, another protein that has gained a lot of attention during the last years in AD research is the transcription factor EB (TFEB) which facilitates the expression of genes containing a specific but rather common sequence in their promotor region, the CLEAR motif (Palmieri et al., 2011). TFEB also plays an important role in the reciprocal signaling between the lysosomes and the molecules balancing autophagy per se. TFEB is drastically involved in various intracellular clearance pathways. This renders it a possible therapeutic target for a wide variety of diseases that are accompanied by lysosomal and autophagic malfunction. Its implication in AD was put under examination since TFEB’s activity and localization is also altered in AD (Chung et al., 2019; Cortes & La Spada, 2019; Gao et al., 2018; Lie & Nixon, 2019; Nixon, 2020). The aim of this literature thesis is to provide an overview of studies on AD and the lysosomal and autophagy pathways as well as the connection between them. Furthermore, mechanisms and molecules that affect both the function and biogenesis of lysosomes, and are themselves affected by AD, are examined in order to evaluate recent findings in the field.
Critical overview of the current literature
Lysosomes and the importance of their acidification via the vATPase complex
Neuronal survival depends on the maintenance of proteostasis, which is a term used to describe protein homeostasis. Neurons are postmitotic cells since they are terminally differentiated at the very beginning of the development of an organism (Frade & Ovejero-Benito, 2015). Due to this characteristic of the neuronal cells, their survival and health maintenance is dependent on the recycling mechanisms provided by the lysosomes rather than on other mechanisms such as exocytosis or dilution during cell division (Ferguson, 2019). There is a strong link connecting the functioning of lysosomes and the development of neurodegeneration and this linkage gives rise to more than 50 inherited lysosomal storage disorders (LSDs) that appear in the early stages of life (Lie & Nixon, 2019; Platt et al., 2012). Lysosomes degrade and recycle endocytic and autophagic substrates including membranes, proteins and lipids. Molecules destined for degradation are transferred from the cytoplasm to the lysosomal lumen, where they can be degraded by hydrolases (the lysosomes contain more than 60 different soluble hydrolases which are active in pH<7) (Finkbeiner, 2020; Samie & Xu, 2014). The vast majority of the lysosomal hydrolases operate optimally in acidic pH, although the exact optimal pH value for each hydrolase might vary considerably. For instance, regulation of pH seems to be of great importance for the functioning of cathepsins. Cathepsins are proteases mostly encountered in the lysosomes because of their acidic nature, which facilitates their activation (Stoka et al., 2016). Cathepsins acquire their mature form only after cleavage of their inhibitory compartment. This can only be accomplished in an acidic environment by the process of autocatalysis or by the contribution of another protease. In neutral pH, most of the cathepsins remain inactive and structurally unstable. Their optimal pH varies in a range from 3.5-6.0 and if they are exposed to a pH other than the aforementioned then degradation initiates, which is carried out by another cathepsin (Richo & Conner, 1994). Thus, alterations in the pH value can directly affect the proteolytic activities of the lysosomes
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and these differences are further deteriorated by aging and accumulation of non-fully degraded substrates. Together, this indicates that an abnormal increase in the intraluminal pH can affect the effectiveness of the lysosomal degradation process. What is more, alterations in the lysosomal acidification can affect the maturation process of many hydrolases. The intraluminal acidification is the result of the collaboration of various ion channels and the most important among them is mentioned to be the vacuolar ATPase (vATPase) complex (Mindell, 2012; Nixon, 2020).
The vATPase complex is known to be a ubiquitous ATP-dependent proton pump that contributes in carrying out the acidification of the lysosomes and has lately been hypothesized to also assist in sensing the intralumenal amino acid levels (Lie & Nixon, 2019). It takes advantage of the energy freed from the ATP hydrolysis and transfers H+ ions on the inside of the lysosomes creating in this way their acidic nature in the lysosomal lumen. The vATPase protein complex consists of at least 13 different subunits and can be distinct into two domains; the V0 domain which is bound to the membrane and the V1 domain that is peripherally associated (cytosolic). The V1 domain has 8 different subunits whose main purposes are to assist in the binding and hydrolysis of ATP and thus providing energy to the vATPase and to mediate the H+ ion transfer across the membrane. The V0 domain also has different subunits,
with V0a being the largest among them. V0a is an integral membrane protein that has four distinct isoforms a1, a2, a3 and a4 and a1 is the most prominent one encountered in the brain (Saw et al., 2011). Various versions of the complex have been reported to be located in the membranes of organelles including endosomes, secretory vesicles and plasma membrane. vATPase activity controls and is itself controlled by cascades relative to nutrient supply and metabolism. Its activity is terminated by a process called dissociation that separates the two subunits from each other, leading to loss of the proton-pumping capability of the complex, while a lack of amino acids causes an acute vATPase assembly until saturation is met (Colacurcio & Nixon, 2016).
Degradation process
Cargo destined for degradation located on the outside environment of the cell goes through endocytosis. Endocytosis initiates with the invagination of the membrane leading to the formation of endocytic vesicles (Lie & Nixon, 2019). These vesicles evolve through a maturation process first into early and then into late endosomes. Late endosomes contain endosomal sorting complexes that store the cargo led for degradation into intraluminal vesicles. The intraluminal vesicles sorted with the late endosomes compose the Multi-Vesicular Bodies (MVBs)(Samie & Xu, 2014). MVBs can then directly fuse with lysosomes, the final compartment of the endocytic pathway, and form hybrids called endolysosomes that mediate the degradation process (Bright et al., 2016).
In order to avoid a possible development of toxicity or even cell death, cells use another pathway, known as the autophagic pathway, to degrade intracellular organelles and misfolded proteins. This self-degradation process occurs in all cell types, including those located in the CNS. Through autophagy, malfunctioning intracellular molecules can be led to the lysosomes (Maday & Holzbaur, 2014). In more detail, autophagosomes fuse with lysosomes to create the autolysosomes which is the compartment where the autophagic cargo gets degraded to building-block molecules. Under conditions of cellular stress, the autophagic process helps in the restoration of homeostasis (Antonioli et al., 2017). Autophagy aims to provide the cell with the necessary nutrients and energy, as well as the remediation of any toxic components that might accumulate during a cellular functional lesion. Autophagy can be beneficial or harmful
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depending on its manner and its duration. In order to avoid uncontrollable decomposition, autophagy is subjected to strict regulation (Samie & Xu, 2014).
Types of Autophagy
Depending on the substrate, there are three distinct types of autophagy and the respective pathway that is followed in order to end up inside the lysosomes: a) the macroautophagy, b) the microautophagy and c) the Chaperone Mediated Autophagy (CMA) (Nassif et al., 2017; Xilouri & Stefanis, 2015)(Figure 1).
a) Macroautophagy
Macroautophagy is the most common type of autophagy and the only one that can decompose organelles among other substrates. It consists the major intracellular pathway which transports intracellular cargo to the lysosomes, and it begins with the formation of autophagophores (Finkbeiner, 2020). Studies performed in yeast helped unveil the steps followed during autophagy. In summary, during macroautophagy double membrane vesicles called the autophagosomes are formed. Autophagosomes have the ability to engulf cellular proteins and organelles. They travel along microtubules until they meet the lysosomes with which they fuse to form the autolysosomes. The cargo that is led to the lysosomes is meant to be decomposed. Macroautophagy can occur in a more holistic manner (in bulk) or in a more selective one, depending on the cargo that has been gathered (Kaushik & Cuervo, 2019). More than 30 genes related to autophagy (ATG genes) have been discovered until today in yeast models that are implicated in the different stages of autophagy. In superior eukaryotic organisms, many evolutionarily conserved genes have been discovered (Levine & Klionsky, 2004). The Atg protein complexes participate in the formation steps of the autophagosomes. In this way, Atg proteins and their homologues in superior organisms play a very important role in the initiation as well as the completion of the macroautophagy procedure. Since macroautophagy takes place in the cytoplasm, the involved proteins interact with a plethora of other secreted proteins, which seems to have a regulatory role in their expression and secretion through a variety of mechanisms which are not yet fully understood (Cavalli & Cenci, 2020). When the nutrient conditions in the environment are rich, the activity of Atg proteins is downregulated leading to suppression of autophagy and when the conditions are poor autophagy is enhanced. One parameter contributing to this is the mammalian target of rapamycin complex 1 (mTORC1) which is activated depending on the nutrition status of the cell and can inhibit autophagy by phosphorylating both directly and indirectly Atg proteins and other proteins related to autophagy. This means that there is a strong but inverse coupling of mTORC1’s activation and the induction of autophagy (Basu, 2019; Y. C. Kim et al., 2015; Noda, 2017; Switon et al., 2017).
b) Microautophagy
Microautophagy entraps the cytosolic cargo into small vesicles that are formed due to the invagination of the lysosomal membrane. The invagination can either be massive or selective. The latter includes the identification of the substrate by the protein HSC70/HSPA8 and other chaperone proteins that have not been fully defined yet (Finkbeiner, 2020; Kaushik & Cuervo, 2019; Klionsky, 2005).
c) Chaperone-Mediated Autophagy (CMA)
It is the first type of autophagy that was proven to be selectively decomposing its substrates. This pathway is capable of translocating polypeptides containing the KFERQ
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motif from the cytoplasm directly into the lumen of the lysosome. In this pathway only proteins can be degraded (Klionsky, 2005). More specifically, all the proteins of the cytosol that are targeted to be degraded via CMA include in their sequence the pentapeptide KFERQ which is the mark leading these proteins to the CMA pathway. The KFERQ motif can be found in approximately 40% of the cytosolic proteins of the mammalian proteome. In some proteins KFERQ can be the outcome of post-translational modifications, increasing in this way the possible substrates submitted to CMA (Finkbeiner, 2020). What is more, KFERQ is found in many proteins (including a-synuclein) implicated in neurodegenerative diseases. The proteins following the CMA degradation pathway when translocated to the lysosomes they need to already be degraded into monomers, which indicates that CMA might not be as effective as macroautophagy in its main purpose. CMA is completed in four steps: 1) recognition of the substrate and targeting to the lysosomes, 2) binding and unraveling of the substrate to the lysosomal receptor LAMP2A, 3) translocation of the substrate and finally 4) degradation inside the lysosomal lumen (Finkbeiner, 2020; Klionsky, 2005; Xilouri & Stefanis, 2015). This type of autophagy, when its function is disturbed it leads to protein aggregation and the expression of related diseases (Chung et al., 2019).
Figure 1 Types of autophagy. a) Macroautophagy, b) Microautophagy and c) Chaperone-mediated autophagy (CMA).
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The neuronal lysosomal system
Lysosomes can be observed in all types of neurons. However, they are more profoundly found in cell bodies, less in dendrites and very few in axons (Becker et al., 1960). The latter might be the outcome of specific mechanisms preventing the lysosomes from transferring towards the axons or mechanisms that transport the lysosomes away from the axons (Maday & Holzbaur, 2014). Autophagosomes located in the axons first fuse with endosomes in order to form a new hybrid called the amphisome. While travelling towards the soma, amphisomes become progressively more acidified and start gaining lysosomal markers such as LAMP1 (Hollenbeck, 1993). However, the low cathepsin levels found in the retrogradely trafficked immature lysosomes indicate that the final mature form might only be achieved in the cell body, since most of the proteases needed are located in the soma (Ferguson, 2019; Gowrishankar et al., 2015). Malfunctioning lysosomes can cause cell death by leaking cathepsins in the cytoplasm, which then trigger caspases (Leist & Èa, 2001). Lysosomal compromise can have a mild or strong effect on how the neurons operate and this contributes in the development of neurodegeneration (Nixon, 2017).
The lysosomal system encountered in neurons is very similar to the one encountered in other cell types. However, there is a unique neuronal characteristic making them differ extremely from other cell types and that is their structure. In neurons, the distance between the soma and the axons can extend up to 1 meter in humans. This highlights the need for an efficient transportation system through which the substrates destined for degradation can reach the lysosomes in the cell body. Neurons have a peculiar morphology considering that they have long or short dendrites, different positions where the soma might be encountered and extremely long axons. Their long axons indicate that they need to degrade cargo even at this very distant site. Nonetheless, autophagosomes are mainly produced in the axons. From there, they need travel to the cell body where they can fuse with lysosomes and eventually lead the cargo they carry to degradation (Finkbeiner, 2020).
AD pathology and pathogenesis
In AD, tau tangles and amyloid plaques are mostly encountered in the frontal and temporal lobes as well as the hippocampus, whose function is mostly related to memory. As the disease develops, more cortical regions are affected, including the occipital and parietal lobes. Neurodegeneration and synaptic dysfunction occur in the brain regions where tau tangles and amyloid plaques are abundantly deposited (Van Acker et al., 2019; Dickson, 1997).
An abnormal phosphorylation of the tau protein leads to its aggregation. Tau protein comes in 6 soluble isoforms deriving from the alternative splicing of the microtubule associated protein tau (MAPT) gene (Goedert et al., 1989; Vassar et al., 1999; Wiersma & Scheper, 2020). Normally tau’s role is to stabilize microtubules in the axons mostly of Central Nervous System (CNS) neurons and less commonly in astrocytes and oligodendrocytes (Cleveland et al., 1977; Haritani et al., 1994). In healthy individuals, tau is phosphorylated and dephosphorylated in order to function properly. However, sometimes tau is hyperphosphorylated leading to pathology. The hyperphosphorylated tau is produced as a response to the changes occurring to the kinase/phosphatase activity due to the accumulation of Αβ, which now leads to the formation of tau tangles. Recent data indicate that tau pathology in its early stages might be reversible (Chen et al., 2017; Van Der Harg et al., 2014).
Amyloid plaques are formed from the gradual accumulation of Αβ peptides inside the cells. Αβ peptides serve as the building stone of amyloid plaques (Dickson, 1997). The exact role of Αβ in the pathogenesis of the disease is not yet fully comprehended. Although insoluble
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aggregates of Αβ amyloid were first thought to be the main cause of the pathogenesis of the disease, strong evidence indicates that it is the oligomeric Αβ causing the most toxic consequences (Knobloch et al., 2007; E. B. Lee et al., 2006; Selkoe et al., 2016).
AD is multifactorial and it comes in both familial and sporadic forms. The familial form is the least encountered form, has an early onset (before the age of 65) and is characterized by dysfunction of various genes including the amyloid precursor protein (APP), Presenilin 1 (PSEN1) and Presenilin 2 (PSEN2). More specifically, DNA analyses revealed that the APP gene is located on the 21st chromosome. Various APP mutations lead to inherited AD forms (Selkoe, 1991). Mutations on the PSEN1 and PSEN2 genes were also associated with familial AD. The sporadic form is associated with a dysfunction of the Apolipoprotein E (ApoE) gene which has 3 alleles ApoE2, ApoE3 and ApoE4. ApoE4 is intertwined with an increased probability of developing the disease. In the presence of ApoE4 an augmented Aβ production and a decreased cellular clearance of the aggregating Αβ occur (Moosavi et al., 2015; Suzhen et al., 2012; Dorszewska et al., 2016). Additional data suggest that ApoE4 promotes the pathogenic cleavage of APP (Mattson, 2011).
Due to the production of amyloid plaques as well as the mutations concerning the APP, PSEN1 and PSEN2 genes, the researchers formed the amyloid cascade hypothesis in order to provide a logical explanation for the causation of AD. The amyloid cascade hypothesis suggests that the formation of amyloid plaques and the inability of the neurons to remove them cause neurotoxicity and cytopathic events leading to dementia. However, other studies claim that there is no strong correlation between amyloid plaques and the loss of synapses and neurons (Masliah et al., 1992; Terry et al., 1991). Αβ oligomers give rise to another hypothesis, the amyloid-β oligomer hypothesis, which accuses these specific amyloid plaque intermediates of causing AD’s pathogenesis (Cline et al., 2018).
Production of Αβ peptides and amyloid plaques
The different forms of Αβ that can be encountered are soluble Αβ, Αβ oligomers and Αβ present in amyloid plaques. Amyloid plaques are formed due to the aggregation of amyloid fibrils which are insoluble and larger than oligomers. The oligomers, on the other hand, might be smaller, but they are soluble and capable of spreading across the CNS. Thus, Αβ precursors show higher neurotoxicity than the amyloid plaques. Αβ can form various monomeric or oligomeric structures, however all of them result in the formation of amyloid fibrils (Benseny-Cases et al., 2007; Goldsbury et al., 2000; Nichols et al., 2002).
APP is a protein produced in the endoplasmic reticulum (ER) and it afterwards completes its maturation in the Golgi apparatus where it acquires an O-glycosyl residue and tyrosine sulfation (Sinha & Lieberburg, 1999). Its final destination is the membranes of various cells, including neurons, and it is very frequently encountered in synapses. When APP is located in the membranes, secretases act upon it and severe it. Once APP is cleaved by β- and γ-secretases, Aβ are generated, which can either remain on the membrane or can be released in the extracellular environment where they bind to receptors, membranes and metals. The human APP can be processed via two distinct proteolytic pathways; the amyloidogenic and the non-amyloidogenic. The main difference between these two pathways is the type of secretases used in the severing of APP. The amyloidogenic is processed by β-secretase (BACE), leading to the production of soluble sAPPβ, while the non-amyloidogenic is processed by α-secretase, producing sAPPα, respectively (Chen et al., 2017). The non-amyloidogenic pathway produces non-toxic products and serves as a counterbalancing mechanism that antagonizes the formation of Αβ (Sun et al., 2015). The “harmless”
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secretory cleavage of APP is abated in AD (Sinha & Lieberburg, 1999). In the amyloidogenic pathway BACE cleaves the N-termini while γ-secretase cleaves the C-termini of Αβ.
When BACE acts upon APP it produces a cell-associated β-C-terminal fragment (β-CTF). Endoproteolysis followed by C-terminal trimming from γ-secretase leads to the production of Αβ peptides with different lengths and hydrophobicity (Fernandez et al., 2016). Their size can vary from 37-49 amino acids (aa). In 90% of the cases β-CTF produces a Aβ40 and in the rest 10% of the cases a Αβ42 (Sinha & Lieberburg, 1999) which is more prone to form amyloids (Chen et al., 2017). Αβ42 accumulates into β-pleated sheets forming amyloid plaques, compared to the shorter Αβ40, which might inhibit the amyloid deposition (Kim et al., 2007; Kumar-Singh et al., 2006). Recent data suggest that the Αβ42/Αβ40 ratio, in the cerebrospinal fluid (CSF), is a more accurate AD biomarker than the levels of Αβ42 or Αβ40 per se, in the CSF (Baiardi et al., 2019; Janelidze et al., 2016). On the other hand, Αβ40 seems to play a neuroprotective role. Some studies indicate that overproduction of Αβ40 leads to plaque formation avoidance (Qiu et al., 2015). Αβ42 aggregates preferentially in both familial and sporadic AD. Interestingly, knockout of the PSEN1 gene in rodents hindered the formation of Αβ40 and Αβ42 which revealed a connection between the PSEN1 expression and the operating rate of γ-secretase (Sinha & Lieberburg, 1999).
Link between the lysosomal system and AD
Structural anomalies of the endosomes, also called endosomopathies, consist the earliest specific marker for AD. Endosomopathies are usually caused by an overactivation of components from the Rab GTPase family. Abnormal endosomes are characterized by an upregulated expression of endocytic genes, increased levels of endocytosis, decreased transportation of enlarged endosomes and even neurodegenerative events associated with abnormal cell signaling. In AD, in particular, there is an irregular overactivation of Rab5, which leads to increased levels of endocytosis, swelling of endosomes and pathologic endosomal signaling (W. Xu et al., 2019). These aberrant endosomes fuse in between them and give rise to enlarged endosomes. At the very beginning of the pathogenesis of AD, the Rab5-mediated increased levels of endocytosis cause an increased lysosomal biogenesis. The autolysosomes and amphisomes produced are enlarged, since they aggregate autophagic and endocytic substrates led to decomposition. As a result the majority of the synthesized lysosomes are dysfunctional. This abnormal enlargement can also compromise their transportation (Audano et al., 2018). Another parameter assisting in lysosomal biogenesis in the early stages of AD is the transcription factor EB (TFEB), whose levels are affected by the disease (Tiribuzi et al., 2014). TFEB will be further analyzed later in this report. As the disease progresses, lysosomes lose their functional activity as they tend to become enlarged and lose the effectiveness of the hydrolases (Nixon, 2017).
The decreased proteolytic function of lysosomes observed in AD cases strengthens the suspicion that macroautophagy plays a key role in the development of the disease. The production of autophagosomes and lysosomes is increased in the very beginning of AD. Thus, enhancing the efficiency with which macroautophagy is performed might be an effective way to mitigate the progression of the disease (Chung et al., 2019). Indeed, studies that administered rapamycin (a specific mTOR inhibitor) in AD animal models, as a way to supress the activity of mTOR, reported a reduction in the levels of Αβ and rescued cognitive functions and memory deficits through enhancement of the autophagic flux (Caccamo et al., 2011, 2014; Spilman et al., 2010). However, this was not evident in late onset AD models (Majumder et al., 2011), indicating that autophagic regulation is important during the early stages of the
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development of the disease rather than during the late stages when the pathology is more conspicuous.
In AD neurons, Αβ is encountered inside the lysosomes at high levels, indicating that -if not entirely, then at least partially- Αβ degradation is mediated by macroautophagy (Chung et al., 2019). Studies in neuronal cultures revealed that soluble Αβ42 accumulates inside the lysosomes and disrupts the lysosomal membrane proton gradient leading to ROS generation which eventually causes cell death (Ditaranto et al., 2001; Oku et al., 2017). As a response to the AD-related generation of Αβ, lysosomes accumulate in neuronal locations where Αβ plaques are being formed. However, the lysosomal retrograde trafficking and maturation from the axons towards the cell body is interrupted due to plaque formation at sites that block the normal distribution of the lysosomes (Ferguson, 2018). Evidence indicates that BACE clearance, normally executed by the lysosomes, is impaired during AD. This results into higher probabilities for BACE to encounter APP, thus provoking a greater production of Αβ (Gowrishankar et al., 2015; Tammineni et al., 2017; Ye et al., 2017). Last but not least, cathepsins and other lysosomal hydrolases have been detected in the extracellular environment near amyloid plaques, indicating that plaque formation outside of the cell could be facilitated by the release of the autophagic products nearby (Sadleir et al., 2016; Nixon, 2017).
Lysosomal acidification: a key parameter in the development of AD
Mutations in the vATPase subunits causing loss-of-function result in diminished lysosomal degradation efficacy and aging-dependent neurodegeneration. Absence-of-function of the V0a1 subunit increases the neuronal susceptibility to Αβ and tau aggregation, but only when accompanied by aging or toxic stress (Colacurcio & Nixon, 2016; Peric & Annaert, 2015). Complications in lysosomal acidification do not directly affect the autophagic/lysosomal system but rather render it more prone to fail over time.
Recent evidence links dysfunction of the vATPase complex to AD development. Mutations on PSEN1, leading to the commonest form of early onset AD, also affect the function of PSEN1 needed in lysosomal acidification (J. H. Lee et al., 2010). Interestingly, presenilins have been implied to play a central role in the successful implementation of acidification in the lysosomes. Presenilins are catalytic components of the γ-secretase complex, mediating the cleavage of proteins as well as protein deactivation (Catalog et al., 2004). More specifically, PSEN1 through the γ-secretase complex, mediates the cleavage of APP to various products. All of the uprising products are implicated in the development of AD (J. H. Lee et al., 2015). Studies using PSEN1 loss-of-function approaches, implied that the Αβ42/Αβ40 ratio increased, adding up in this way to the expression of neurotoxicity and neurodegeneration. Both presenilins, PSEN1 and PSEN2, are abundant in endolysosomal/autophagic compartments and multi protein complexes and both of them can disturb the normal functioning of the lysosomes. PSEN2 is more profoundly detected in the lysosomes. PSEN1 is a specific ligand of the V0a1 subunit (J. H. Lee et al., 2010) and plays a key role in lysosomal acidification by participating in the glycosylation, stabilization and congregation of the V0a1 subunit of vATPase. When the normal function of PSEN1 is disturbed, the V0a1 subunit is not efficiently glycosylated anymore. Consequently, it gets degraded in the ER, resulting in a less effective proton pumping action of the vATPase complex, finally causing deacidification of the lysosomal lumen. This does not apply only to neuronal cells, but also to other cell types (Nixon, 2017). Deacidification causes the hydrolases to malfunction, resulting in substrate accumulation. Restoration of acidification rescues the normal functioning of the vATPase (J. H. Lee et al.,
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2010, 2015). AD patients with mutated PSEN1 show decreased acidification rate in the lysosomes and defective V0a1 maturation. Cells lacking PSEN1 and/or PSEN2 show even greater pH values inside the lysosomes and impaired lysosomal degradation. Taken together, this information suggests that PSEN1 is essential for the maturation and efficient function of the V0a1, which is crucial for lysosomal acidification. Disruption of the above can result in the development of neurodegeneration (Nixon, 2017). Disturbance of PSEN1 functioning also affects the regulation of the Ca2+ levels in the ER as it will be discussed in the upcoming
section. Both autophagy and lysosomal Ca2+ disruptions can be reversed by reacidification of
the lumen in in vitro experiments (Colacurcio & Nixon, 2016; J. H. Lee et al., 2015).
Calcium flux implicated in AD pathology
Calcium is involved in several signalling pathways, where it acts as a second messenger. By doing so, it controls a vast variety of cellular mechanisms and events, such as neurotransmission, gene expression, proliferation, apoptosis and more. For this reason, it is very important that the cells maintain the appropriate Ca2+ levels. When the normal levels of
Ca2+ are disturbed, diseases, including AD, occur as an outcome (Feng & Yang, 2017). In
resting conditions, the cellular Ca2+ levels are kept low. Upon stimulation, cellular Ca2+ levels are increased, by allowing the entrance of extracellular Ca2+ through calcium channels as well as by release from internal calcium stores in the ER and mitochondria. Once stimulation is terminated, Ca2+ is transferred back to the extracellular space or inside the intracellular stores
(Berridge et al., 2000; Laude & Simpson, 2009).
In AD, due to the accumulation of Αβ oligomers, a tremendous and abnormal influx of Ca+2
leading to toxicity and eventually neuronal loss is caused. This outcome leads to mitochondrial dysfunction and oxidative stress. Ca+2 contributes in memory formation and learning and is implied to play a key role in neuronal death as well as survival. The AD-associated disruption of the Ca+2 homeostasis leads to neuronal death (Alberdi et al., 2010; Canevari et al., 2004).
Interestingly, PSEN1 mutations, causing early onset AD, are also accused of disrupting the ER Ca2+ homeostasis by various regulatory mechanisms, causing the levels of Ca2+ in the ER to increase (J. H. Lee et al., 2015). This information encourages future studies to focus on the development of drugs or mechanisms promoting the production and activity of PSEN1. Besides the ER and the mitochondria, also the lysosome serves as an intracellular Ca2+
storage (Raffaello et al., 2016). Various studies have revealed two major categories of Ca2+
permeable channels found in the lysosomal membrane: 1) the transient receptor potential cation channel mucolipins (TRPMLs) and 2) the two-pore channels (TPCs). Under stressful conditions, including deacidification of the lysosomes, TRPML1 is activated, leading to lysosomal Ca2+ efflux (Dong et al., 2008; Li et al., 2016). This event has two outcomes in
favour of autophagy. First, the augmented Ca2+ levels activate calcineurin (CaN), which
dephosphorylates TFEB, rendering TFEB capable of initiating the expression of genes related with lysosomal biogenesis. Second, Ca2+ efflux acts in favour of lysosomal transport mediated
by dynein, enhancing in this way their fusion with autophagosomes (Di Paola & Medina, 2019; Feng & Yang, 2017; Lie & Nixon, 2019; Medina et al., 2015). Dynein is a motor protein that mediates the translocation of autophagosomes towards regions abundant in lysosomes. Disruption of microtubules and/or mutations producing abnormal dynein obstruct the normal autolysosome formation and the proteolytic process (Settembre & Ballabio, 2014).
For homeostasis to be maintained, the Ca2+ deriving from internal stores must be refilled. It is notable that dysregulations in the refilling mechanisms in the ER can consequently exert a negative effect on the lysosomal corresponding mechanisms (Feng & Yang, 2017; W. Wang
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et al., 2017). Furthermore, the lysosomal Ca2+ levels are regulated by the lysosomal pH levels. Decreased lysosomal acidification, driven by inefficient functioning of the vATPase complex, disrupts the Ca2+ efflux, mediated by TRPML1 (J. H. Lee et al., 2015; Raychowdhury et al.,
2004). Data suggest that disruption of PSEN1 expression may cause alterations in both the lysosomal/autophagic system and the Ca2+ homeostasis and this outcome may be encountered in AD (Catalog et al., 2004). In PSEN1 knock-out cells, the increased intralumenal pH causes an irregular Ca2+ efflux through TRPML1. This event leads to
augmented calcium levels in the cytoplasm. This disruption was also reproduced in animal models upon blockade of the vATPase activity and PSEN1 or V0a1 knockdown models. Lysosomal reacidification or completion of protein degradation are not fully met, even after resolving the abnormal Ca2+ levels in the lysosomes. This observation indicates that
preservation of normal Ca2+ lysosomal levels plays a significant role in regulating lysosomal
degradation, however rescuing these levels upon dysregulation is not sufficient to restore the lysosomes normal functions (J. H. Lee et al., 2015).
Lysosomal efflux caused by inefficient function of the vATPase complex is intertwined with a following overactivation of proteins participating in the neurodegenerative cascade, resulting in AD development. For instance, the increased levels of the cytosolic Ca2+ in AD is correlated
with an increase in the activity of calpains, since the latter get activated by Ca2+ (Yap et al.,
2006). Calpains are calcium-dependent proteases localizing in all cell types, but not inside the lysosomes. Their activity is terminated by a specific endogenous inhibitor, calpastatin (Hanna et al., 2008; Hosseini et al., 2018). Aging causes calpains to act more drastically and thus the levels of the products, deriving from cleavage via calpains, are elevated (Nixon, 2020). Moreover, with aging comes a decrease in the levels of calpastatin, meaning that inhibition of the cleaving process cannot be performed as accurately and effectively as in young individuals (Rao et al., 2008, 2014). Calpain’s increased activation leads to cytotoxicity, due to the elevated cleaving rates (Yildiz-Unal et al., 2015), rendering lysosomes unstable. When lysosomal stability is lifted, lysosomes react to this by releasing cathepsins, leading to an inevitable cell death (Lie & Nixon, 2019; Nixon, 2020).
TFEB’s important role in lysosomal functioning
Two molecules of high interest in AD and lysosomal dysfunction are TFEB and mTOR, which are very connected to each other. TFEB is a highly expressed transcription factor in the CNS, that promotes gene expression related to lysosome and autophagosome biogenesis and function. TFEB belongs to the microphthalmia family of basic helix-loop-helix-leucine-zipper (bHLH-Zip) transcription factors (MiT family)(Steingrímsson et al., 1998) and assists in organelle biogenesis. Most lysosomal genes share a 10-base E-box-like palindromic sequence, known as the coordinated lysosomal expression and regulation (CLEAR) motif. TFEB binds to CLEAR elements and by doing so it promotes the transcription of the genes containing this sequence. Consequently, increased levels of TFEB cause an overexpression of these genes, resulting in augmented production of lysosomes and their enzymes, leading to an overall amplified lysosomal activity (Marco Sardiello et al., 2009). TFEB does not only promote the gene expression related to lysosomal function and biogenesis, but also to autophagy, and more specifically enhances autophagosome biogenesis and autophagosome-lysosome fusion, amplifying the overall degradation rate (Medina et al., 2015; Settembre et al., 2011). TFEB is also implicated in endocytosis, lipid metabolism, immune responses to viruses and the Ca2+ signaling in the lysosomes (Vega-Rubin-de-Celis et al., 2017).
TFEB is widely expressed in the CNS and has been detected in astrocytes (Decressac et al., 2013; Xiao et al., 2014). When in its inactive state it localizes in the cytosol. Once activated, it
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is transferred to the nucleus, where it contributes in the initiation of gene expression (Medina et al., 2015). In neurodegeneration, TFEB’s activity and localization changes (Decressac et al., 2013). The mechanism underlying TFEB’s regulation is not yet fully deciphered. Current knowledge suggests that the main events in determining TFEB’s activity are post-translational modifications, i.e. phosphorylation and dephosphorylation. More specifically, when the cell conditions are nutrient-rich, TFEB is located mainly in the cytosol in its inactive state, due to its phosphorylation mTORC1. Under conditions of starvation TFEB gets dephosphorylated and is thus activated and translocated to the nucleus (Napolitano et al., 2018; Nnah et al., 2019)
mTORC1 is a protein complex (Sabers et al., 1995) whose purpose is to control protein synthesis depending on the cellular energy-nutrient conditions. A complex machinery consisted of the vATPase complex, the mTORC1 complex and a few additional complexes, called the lysosomal nutrient sensing (LYNUS) machinery, senses the nutrient content levels inside the lysosomes. When TFEB is located on the lysosomal membrane, it interacts with the LYNUS machinery. This event contributes to lysosome’s self-regulatory ability by regulating lysosomal biogenesis under nutrient-poor or stress conditions (Settembre et al., 2013). Interestingly, when nutrients are sufficient, a mechanism including binding of mTORC1 to the vATPase complex (Zoncu et al., 2011) assists in the activation of Rag GTPases, which cause the translocation of mTORC1 to the lysosomal membranes (Zhou et al., 2013). When the environment is poor in amino acids, Rags remain inactivated. In this condition, RagA/RagB are GDP loaded and RagC/RagD are GTP loaded, respectively. Nutrient rich conditions cause a switch in the GDP-GTP loading of Rags, leading to activation of the complex (Bar-Peled et al., 2012; Sancak et al., 2010). In its active state, the Rag complex causes mTORC1 to translocate to the lysosomal membrane. TFEB is also transferred to the lysosomal membrane via activated Rag GTPases (Long et al., 2005). TFEB is now phosphorylated and deactivated by mTORC1 in the lysosomal membrane. The same pattern is observed in other transcription factors such as MITF and TFE3 that show high DNA relevance to TFEB and are also members of the bHLH family (Roczniak et al., 2012). In Drosophila melanogaster, MITF has been proven to regulate the expression of lysosomal genes, especially those responsible for the formation of the vATPase subunits as well as those inducing intracellular clearance and inhibiting the accumulation of protein aggregates (T. Zhang et al., 2015).
During starvation, mTORC1 estimates the decrease in amino acids and through various mechanisms, including signaling via the vATPase complex and the lysosomal arginine transporter SLC38A9 (an amino acid transporter and sensor for arginine and cholesterol), it gets deactivated by its detachment from the lysosomal membrane (Castellano et al., 2017; Medina et al., 2015). The whole LYNUS complex is needed for the activation of mTORC1. Deficiency of the vATPase complex prohibits the activation of mTORC1 (Settembre et al., 2013). These conditions also induce a Ca2+ efflux, causing the activation of CaN (a
calcium/calmodulin-dependent protein). CaN in turn activates TFEB by dephosphorylating it (Medina et al., 2015). TFEB is now able to translocate to the nucleus and assist in the initiation of gene’s transcription. TFEB’s activity and the consequent gene expression regulate each other. This gives lysosomes the ability to be self-regulatory and to effectively adapt to changes in the cellular environment (Cortes & La Spada, 2019; Ferguson, 2019; Lie & Nixon, 2019). TFEB has also been reported to be capable of binding to its own promoter and thus enhancing its own transcription. This indicates that the expression of TFEB is submitted in a self-regulatory feedback loop (Settembre & Ballabio, 2014).
During nutrient-poor conditions TFEB enhances the function of the endocytic pathway in order to maintain the function of lysosomes and autophagosomes by contributing in the transcription
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of several endocytic genes needed for the completion of endocytosis (Nnah et al., 2019). TFEB promotes the production of endosomes and the whole process of endocytosis, in order for the lysosomes to acquire the crucial components that form the LYNUS complex (Settembre et al., 2013). Reactivation of mTORC1 complex is of high importance since it precedes the biogenesis process of autophagosomes and lysosomes, which is crucial for the maintenance of proteostasis (Yu et al., 2010). Thus, TFEB by inducing endocytosis it also enhances the activation of mTORC1. In addition, TFEB mediates the expression of Rag-GTPases (Nnah et al., 2019). Hence, TFEB appears to have an indirect mechanism of controlling the activation of mTORC1, highlighting again its self-regulatory capabilities.
TFEB’s link to AD pathology and future approaches
Lately, TFEB has gained a lot of attention as a contributor in the development of neurodegeneration, including AD. Since TFEB is connected to autophagy, current studies are trying to mark TFEB as a possible therapeutic target.
Inhibition of the activity of mTORC1, combined with a TFEB overexpression, have a beneficial effect on the function of autophagosomes and lysosomes that are decreased in AD pathology (Decressac et al., 2013; Majumder et al., 2011; J. Zhang et al., 2019). Treatment with rapamycin induces autophagy and decreases Αβ and tau levels in the hippocampus (H. Wang et al., 2016). Furthermore, overexpressing TFEB results in an augmented autophagic flux in parallel with an increased autophagosome and lysosome generation. This leads to enhanced cellular clearance of storage materials in various cell types. On the other hand, AD is accompanied by a lysosomal malfunction, which leads to a disturbed autophagic flux and an abnormal aggregation of immature AVs and endosomes, contributing in this way to the development of the pathology (Di Paola & Medina, 2019; Medina et al., 2015; Nnah et al., 2019; Settembre et al., 2011; Yu et al., 2010). TFEB’s expression, and thus activity, is downregulated in AD (Tiribuzi et al., 2014) which might be connected to the shortfall number of lysosomes characterizing this disease. However, from AD patient tissue samples, it was revealed that only nuclear TFEB is actually decreased, especially in the hippocampus (H. Wang et al., 2016). Rodent studies proved that TFEB enhances cellular clearance in neurodegenerative diseases (Settembre et al., 2008, 2011, 2013).
The link between mTORC1 and TFEB seems to be a bit more complicated than what was previously described. Recently, it was shown that when mTORC1 inhibition is lifted, TFEB rapidly travels to the cytosol, indicating that TFEB quickly responds to alterations in the activity of mTORC1. This causes TFEB to continuously shift between the cytosol and the nucleus (Zhitomirsky et al., 2018). When mTORC1 is deactivated, it gets detached from the lysosomal membrane. The following Ca2+ flux activates CaN, which in turn dephosphorylates and
activates TFEB (Figure 2). However, recent data suggest that TFEB is not fully dependent on CaN, as blockade of CaN expression only partially inhibited TFEB’s dephosphorylation, highlighting the existence of other phosphatases capable of activating TFEB (Di Paola & Medina, 2019; Zhitomirsky et al., 2018). This evidence underlines the need for further research to be conducted concerning the mechanism mediating the activation of TFEB and the factors participating in it. Further research on this field could find novel phosphatases responsible for the activity status of TFEB.
One proposed mechanism by which TFEB deactivation is accomplished suggests the phosphorylation of TFEB on S211 and S132 by mTORC1. In more detail, the phosphorylation on S211 enables the interaction between TFEB and 14-3-3 proteins through a noncanonical mode that isolates TFEB in the cytosol (Y. Xu et al., 2019). Thus, mTORC1 inhibition favors
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S211 dephosphorylation and the subsequent translocation of TFEB to the nucleus. It is not clear yet whether the dephosphorylation on S142 contributes positively to the activation of TFEB. In reality, TFEB is phosphorylated in more than 20 sites. Recently S122 was implied to be one of them. S122 is regulated by mTORC1. Mutations at either S211 or S122 cause TFEB to dysfunction. For its function to be restored, both of them need to be in their wild type form (Laplante & Sabatini, 2013; Vega-Rubin-de-Celis et al., 2017). The rest phosphorylation sites need to be examined and their significance to the function of TFEB and lysosomal biogenesis needs to be revealed in order to understand the underlying mechanism in depth. Such findings could nominate new targets for saving lysosomal proliferation and for delaying the progress of AD. However, these observations need to be validated in neuronal cells and in neurodegenerative tissue samples to be able to draw strong and accurate conclusions and suggest possible therapeutic mechanisms.
A potential therapeutic approach would include promoting TFEB’s activity. This could be conducted by overactivating TFEB or suppressing mTORC1’s activity. However, administering rapamycin or other inhibitors of mTORC1, could hide numerous risks associated with chronic inhibition of the aforementioned. Thus, a more appealing direction would be to focus on directly increasing the activity of TFEB. A promising approach would be aiming at an increased expression of molecules capable of dephosphorylating TFEB, and thus activating it. Notably, only few TFEB activators have been mentioned in the literature. Recent data suggest curcumin analogue C1 to be acting as small-molecule activators of TFEB, leading to increased autophagy, lysosomal biogenesis and reduction of the key hallmarks of AD, in AD animal models. Another upcoming TFEB activator is the curcumin derivative E4 which enhances the activity of the lysosomal and autophagy pathway (Song et al., 2020; Z. Wang et al., 2020). These studies suggest the promising use of these substances in the treatment or prevention of AD. A drawback that should be taken into account, is that currently there is not much knowledge on TFEB’s side effects. Overactivation of TFEB and an increase of factors assisting in its dephosphorylation, might seem like a promising approach in dealing with pathogenesis, nonetheless toxic effects might arise from such an action.
Figure 2 Mechanism leading to TFEB activation. A) Cell with lysosomes under nutrient -rich conditions. mTORC1 is bound in the lysosomal membrane and phosphorylates TFEB. The phosphorylated TFEB interacts with 14-3-3 proteins causing its isolation to the cytosol. B) Cell with lysosomes in starvation. mTORC1 gets detached from the lysosomal membrane. A Ca2+ follows leading to the activation of CaN which
dephosphorylates TFEB. TFEB is now able to enter the nucleus and initiate the transcription of genes that include the CLEAR motif in their sequence.
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Interdisciplinary relation
The neuronal lysosomal system and its functional alterations in AD consist an intriguing and promising, yet very challenging, approach in an attempt to unveil new therapeutic targets against AD. AD is affecting a great percentage of the current population and in the next few years the numbers are expected to rise drastically. The biology domain alone cannot reach this goal fast enough, which highlights the great need for the assistance and knowledge that can be provided by other disciplines. The field of chemistry and biochemistry can be of great help in reaching this goal by conducting research on the properties and mechanisms of the molecules highlighted throughout this literature thesis. Acquiring more information on PSEN1, V0a1 subunit, mTORC1 and TFEB, as well as other proteins that assist the aforementioned in their functions and activation/deactivation, could help us further understand the interaction between them and their implication in the lysosomal system. A great part of this thesis was dedicated to TFEB, its importance and especially the mechanisms underlying its activation. New methods leading to its activation can help take the therapeutics applied to AD patients a step further. In the quest of novel TFEB activators, and more specifically factors that can dephosphorylate TFEB, biochemistry could play a critical role by revealing their chemical properties, the kinetics and reaction capabilities. This information could lead to the formation of novel hypotheses and approaches towards the problem under study. What is more, the revelation of new target molecules paves the way for the development and construction of novel drugs. Drugs restoring the normal function of the neuronal lysosomal system, including lysosomal acidification and the degradation process, might be able to decrease the speed by which the disease develops. For this reason, the assistance of medicine and pharmaceutical sciences is highly needed in order to develop new drugs targeting the neuronal lysosomal system. Another part that needs to be treated with care is the processing of the acquired data. Deep and thorough understanding of the data in complex studies is highly needed, in order to avoid possible mistakes that will delay the research’s progress. In such situations, the use of computational biology and bioinformatics is highly recommended, especially when the data is conflicting. These two fields can help target the system with the use of computationally intensive techniques, such as AI technology and machine learning. Moreover, with the use of computational biomodelling approaches, a neuronal lysosomal system could be digitally constructed, helping us unveil its complexity and the actual mechanisms underlying it, as well as the changes it undergoes, due to the development of AD. In order to have a more integrated picture and to efficiently focus on the altered by AD neuronal lysosomal system, the involvement and knowledge of other academic disciplines is essential. Thus, interdisciplinary research studies should be promoted and encouraged.
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Conclusions
The neuronal lysosomal system is of utmost importance for the preservation of proteostasis and neuronal survival. Disruption of the normal lysosomal function can result in the development of AD and vice versa. Mechanisms and techniques aiming at the preservation of the lysosomal functioning could unveil new therapeutic techniques against the progression of the disease. The most promising approaches include 1) saving the acidic nature of the lysosomes, 2) regulating the expression of key proteins assisting in the functioning of the lysosomes and 3) regulating the lysosomal calcium levels. In AD, the lysosomal pH levels get elevated, leading to disruption of the degradation process. Mutations of the vATPase complex and especially the V0a1 subunit render the neurons more prone to produce plaques and tau tangles. PSEN1 is a good candidate for further examination, since it can cause aggregation of the toxic Αβ42 and the formation of a destabilized V0a1 subunit. Deacidification causes an abnormal Ca2+ efflux through TRPML1, which over-activates proteins closely related to AD development. Restoration of normal Ca2+ levels is not capable of fully rescuing acidification in the lysosomes. However, Ca2+ efflux acts also in favor of autophagy by moving
autophagosomes towards lysosomes and by indirectly activating TFEB. Activated TFEB causes gene transcription, assisting in lysosomal activity and biogenesis but also in autophagy. For TFEB activation, mTORC1 must be deactivated. Deficient vATPase complex prohibits the activation of mTORC1, promoting in this way TFEB’s dephosphorylation. In AD, TFEB’s activity is downregulated, contributing in the lysosomal malfunction that characterizes the disease. In human AD patients, TFEB levels were decreased only in the nucleus. Inhibition of mTORC1 by rapamycin administration induces autophagy and decreases Αβ and tau levels in the hippocampus. However, little is known about the use of rapamycin as an AD drug. Blockade of CaN only partially inhibits the dephosphorylation of TFEB (Figure 3). Curcumin derivatives are capable of dephosphorylating TFEB. Further research aiming to discover TFEB’s activators needs to be conducted, as TFEB is a promising target for ameliorating AD’s effects. Methods and techniques aiming in the restoration of the aforementioned alterations occurring in AD could highlight revolutionary approaches focusing on delaying the process of the disease. Understanding why and how lysosomal dysfunction is implicated in AD is critical to our ability to prevent and hopefully treat this, as well as other neurodegenerative diseases. For this reason, it seems promising for future studies to involve a combination of other disciplines in their approach, in order to acquire a more complete idea of the mechanisms underlying the pathology of AD.
19 A) Normal lysosomal function in healthy cell
B) Disrupted lysosomal function in AD
Figure 3 Depiction of the lysosomal activity and its effects on the cell under normal conditions and in AD. A) Normal lysosomal activity of a healthy cell. PSEN1 is efficiently assisting in the glycosylation of the V0a1 subunit. With a normal V0a1 subunit the vATPase is normally constructed and functions normally as the lysosomal proton pump. The lysosome maintains its acidic pH and hydrolases carry out the degradation process properly. TRPML1 is closed and mTORC1 conserves its location in the lysosomal membrane. Because of this, calpains remain inactive and TFEB is phosphorylated (in nutrient rich conditions but gets activated in starvation). B) Disrupted lysosomal activity due to AD. PSEN1 is mutated leading to an unglycosylated V0a1 which causes the construction of a non-functional vATPase. The pH value in the lysosome increases, hydrolases malfunction and TRPML1 opens causing a Ca2+ efflux. The increased Ca2+ concentration in the cytosol activated calpains which cause lysosomal
instability, cytotoxicity and eventually neuronal death. Ca2+ also acts in favor of the lysosomal system
by activating TFEB, but in AD TFEB’s levels are decreased. The overall impaired degradation process leads to impaired BACE1 clearance which causes an augmented formation of Αβ.
20
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