GTP Energy Dependence Of Endocytosis And Autophagy in The Aging Brain And Alzheimer’s Disease Ⅲ

GTP‑dependence of tubulin assembly, vesicle transport, and protein synthesis 

Microtubules are key players in axonal growth and provide structural support to axodendritic vesicular trafficking. Microtubule ends undergo a repeated process of polymerization/depolymerization, called dynamic instability. Microtubule polymerization requires the addition of GTP-bound α/β heterodimers to their ends (Fig.  8A). Tubulin heterodimers bind to two molecules of GTP at two separate sites. The N-site is located at the intradimer interface, between α- and β-tubulin at which GTP is not hydrolyzed and exchanges at a slow rate. The E-site is at the intra-dimer interface formed by the β-subunit of one heterodimer and the α-subunit of a neighboring heterodimer. GTP at the E-site is hydrolyzed to GDP and exchanged for a new GTP nucleotide. Since microtubules exhibit a high rate of dynamic equilibrium between polymerization states, cytoplasmic levels of GTP must be high enough to power the demand of the dynamic instability in the microtubes (Fig. 8A).

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Microtubules form the cytoskeletal tracks on which lysosomes travel to their target endosomes and phagosomes (Fig.  8B). The kinesin motor powers transport of the lysosomes toward the positive-end of axonal microtubules with ATP, but the connection of the kinesin motor to the lysosome requires Arl8 GTPase with bound GTP [101]. Conversely, the transport of late endosomes on microtubules toward lysosomes requires Rab7 GTPase and bound GTP to attach the dynein motor

In AD, the microtubule-associated protein tau polymerizes with hyperphosphorylation into insoluble filaments of axodendritic neurofibrillary tangles (NFT) [112, 113]. Once hyperphosphorylated, tau loses it affinity for the microtubules. Wide-ranging studies have approached the alterations in microtubule dynamics, but have failed to identify a clear cause of failure in tau function. Regulation of tau involves GTPases. In AD brains, Rac1 GTPase protein levels decline 50% [114]. In vitro, Rac1 GTPase activation caused hyperphosphorylation of tau181, increased Abeta42 production and decreased actin stability in spines [114]. This group also saw a biphasic rise in Rac1 GTPase in the young 3xTg-AD hippocampus, followed by a later decline, as in late-stage human AD brains. In another study of tauopathy mice, increased farnesyl transferase activity to translocate the Rhes GTPase to autophagic vesicles was associated with low tau hyperphosphorylation, while inhibition of Rhes promoted tau hyperphosphorylation [115]. Thus, GTPase-dependent autophagy effectively clears pTau [116] and impaired autophagy from low GTP levels or oxidative redox state would impair clearance of pTau.

GTP is also essential for protein synthesis, the synthesis side of proteostasis, which requires the hydrolysis of two GTP molecules for each amino acid incorporated into a polypeptide. While ATP is used for charging aminoacyl-tRNA, for RNA helicase, and recycling GDP to GTP, GTP itself is essential for the activity of elongation factors at initiation (IF-2), elongation (EF-Tu, EF-G) and ribosome release factor for termination (RF1) [117]. This group used a cell-free reconstituted system to measure an overall Km for ATP of 27 µM and 14 µM for GTP. These high affinities suggest prioritization of protein synthesis during energy limitation to maintain high capacity, perhaps at the expense of endocytosis and autophagy which likely have higher Km’s. Consequently, a large amount of GTP is required at synaptic plasticity where metabolic demands are the highest to maintain ionic homeostasis for synaptic function and protein turnover. Protein synthesis is crucial for presynaptic neurotransmitter release as well as consolidation of post-synaptic plasticity [118]. Nevertheless, protein synthesis was decreased along with ribosomal RNA and tRNA levels, while RNA oxidation increased in the early phase of AD [119]. Altered protein synthesis leads to a constant accumulation of oxidized proteins leading to misfolding and aggregation. Protein aggregation, in turn, impairs the activity of cellular proteolytic systems resulting in further accumulation of oxidized proteins [120]. Thus, a vicious cycle results in excess protein ubiquitination and dysproteostasis. Several other genes that encode ribosomal proteins are abnormally regulated leading to altered protein levels of elongation factors eIF2α, eIF3η and eIF5 in AD [121]. Increased eIF2α and decreased eIF3η and eIF5 levels were observed in the hippocampal CA1 region of AD brain. Persistent eIF2α phosphorylation at Ser51 through prolonged overactivation of regulatory kinases inhibits the delivery of initiator methionyl‐tRNA preventing the general translation initiation of a subset of mRNAs. Thus, proteostasis is could be impaired at severely limiting GTP concentrations.

Impaired GSH‑Trx system as a trigger of autophagy failure in aging and AD 

Age is the major risk factor in AD and is associated with an imbalance between a decrease in redox buffer protection and an oxidative shift that increases ROS production. However, the proposal that ROS damage is a causal factor in the pathogenesis of AD has not been validated by antioxidant therapy and an oxidative redox shift appears to be upstream of ROS damage [122]. An epigenetic oxidized redox shift (EORS) has been proposed to precede ROS-mediated oxidative damage to account for lasting metabolic alterations in aging and AD [123]. This manifests as a decrease in reductive intracellular redox ratios from the critical systems that maintain redox homeostasis: cysteine/cystine, GSH/GSSH and NAD(P)H/ NAD(P) [3]. Across the age span of non-transgenic mouse brains, an oxidative glutathione redox state precedes an Akt metabolic shift and old-age buildup of aggregated proteins which are greatly accelerated in the 3xTg-AD mouse model before the intracellular hippocampal accumulation of Aβ or extracellular plaques [124]. Mutations in enzymes related to GSH metabolism have also been associated with the regulation of autophagy such as glutathione reductase gene (grs-1) in a C. elegans model that abolishes the nuclear translocation of HLH-30 transcription factor (orthologue of mammalian TFEB) and triggering reductions in the transcription of genes related to clearance of protein aggregates by autophagy [125]. Subcellular free NADH concentration decreases with age in mitochondria, nuclei and cytoplasm in live wild-type and AD-like neurons [2]. Could this redox shift stimulate the logarithmic increase in intracellular Aβ-aggregates with age [27]?

Although it seems evident that some combination of an increase in the rate of formation of intracellular Aβ or their rate of autophagic clearance decreases with age (the main risk factor), the mechanisms that trigger these alterations remain unclear. We propose that the redox state of cells and possibly a systemic redox shift are essential drivers of aging [123]. GSH and thioredoxin (Trx) are the most important thiol redox system in the cells against oxidative stress. However, their distribution in the cell is very different. The physiological GSH concentration is in the range of mM,~1000-fold higher than Trx (in the μM range). Despite this, the Trx system regulates a broader range of proteins than the GSH system [126]. Proteomic analysis identified the involvement of Trx in the regulation of pathways mainly linked to glycolysis/gluconeogenesis and cytoskeletal remodeling, while pathways affected by both Trx and GSH are related to insulin regulation of translation, lipid metabolism and cell adhesion [126]. In the same study, Trx was also associated with the regulation of other proteins involved in handling GTP and autophagy, such as (i) the IQ motif contained in GTPase activating protein 1 (GAP), a scaffolding protein that integrates Rho GTPase and Ca2+/calmodulin signals in the maintenance of cytoskeletal integrity; (ii) GDP dissociation inhibitor-1 (GDI-1), a protein that maintains Rab proteins in the GDP-bound conformation; (iii) ubiquitin-activating enzyme E1(UBA1), a protein required for Atg7- and Atg3-independent autophagy [127]; and (iv) cofilin-1, an actin-depolymerizing factor whose function is crucial for maintaining synaptic spines and instigating mitochondrial fission and mitophagy [128]. Interestingly, cofilin-1 levels are elevated in AD model mice and human AD, indicating its unbalanced regulation [129]. These data suggest that Trx could have a possible role in maintaining and handling of GTP and autophagy.

The N-terminus of the Rab5 and Rab7 GTPases contain single or double cysteines that should be subject to oxidation–reduction to cystine or farnesylation to anchor in membranes or nitrosylation, perhaps as these Rab's aggregate. Other redox-sensitive proteins in endocytosis such as TXNL1 [130] could accelerate or impede transitions between steps in the pathway.

The low redox potential in the disulfide bond in Atg4 is efficiently reduced and activated by Trx, suggesting a role as a redox regulator of autophagy mediated by Atg4 [131]. Other components in the thioredoxin system are involved in autophagy regulation. Deletion of thioredoxin reductase-2 (TrxR2, mitochondrial isoform) caused mitochondrial degeneration accompanied by overexpression of LC3, p62, LAMP1, and accumulation of autophagic bodies in cardiomyocytes [132]. But a TrxR1 deficiency enhanced oxidative stress, interrupted early autophagy, and decreased protein degradation in lysosomes [133]. These data suggest that several key proteins of the autophagy machinery, such as Atg in yeast, may be regulated in response to changes of intracellular redox conditions, in addition to potential regulation by GTP levels.

Boosting energy to rescue AD pathology 

While stimulation of the overall autophagic flux is beneficial for cell and tissue fitness, without sufficient energy to execute autophagy, the system will be inhibited. Increasing evidence shows that a decrease in NAD+/NADH availability with age [2, 134] plays a critical role in age-related neurovascular and cere bro microvascular dysfunction [135, 136]. Thus, the rejuvenation of cellular oxidative and reductive energy may benefit the age-related increased demands of autophagy. Restoring cellular NAD+ and NADH levels by nicotinamide mononucleotide (NMN) supplement in aged mice rescued neurovascular function, increased cerebral blood fow, and improved performance on cognitive tasks [135, 136]. Nicotinamide riboside (NR), a precursor to NMN, improves mitochondrial and adult stem cell function in aged mice as well as extends overall lifespan [137]. These NAD precursors can also alter autophagy. NR can prevent the blockage of autophagic flux and reduce oxidative stress in doxorubicin-treated cardiomyocytes, leading to enhanced autolysosome clearance via the NAD+/ SIRT1 signaling pathway [138]. NR treatment also increased autophagic function and restored mitochondrial health in animal models of Parkinson’s disease, suggesting possible treatments in other neurodegenerative diseases [139]. Since the product of Sirtuins and PARP is nicotinamide, cells need the NAD salvage pathway to recycle nicotinamide back into NMN and ultimately NAD+. Two labs have treated 3xTgAD mice with nicotinamide and observed beneficial improvement in memory, DNA repair, autophagy, accumulation of Aβ, phosphorylated tau and improved bioenergetics [140, 141]. Similarly, an improvement in cognitive functionality on multiple behavioral tests in 3xTgAD mice treated with nicotinamide riboside suggests a pivotal role for cellular NAD+depletion upstream of neuronal degeneration in AD [142]. Therefore, we treated adult 3xTgAD and non-transgenic neurons with nicotinamide to see that the bioenergetic improvement extended to improvement in GTP levels, both bound and free (Fig.  1). Overall, increased NAD+ levels may beneft numerous downstream functions such as NAD-dependent SIRT1 activated genes which rejuvenate mitochondria and inhibit inflammation and apoptosis.

Another class of molecules benefitting old neurons are Nrf2 activators [143, 144]. One example is the ester of epigallocatechin and gallic acid, (−)-Epigallocatechin-3-Gallate (EGCG), the most bioactive polyphenol found in green tea extract. EGCG significantly increased mRNA expression of the key autophagy adaptor proteins NDP52 and p62 and enhanced the clearance of AD-relevant phosphorylated tau species in primary neurons [145], as well as improving cerebrovascular tone [146]. EGCG also activated autophagic pathways by inducing Sirt1, exerting protective effects against human prion pro-tein-induced neurotoxicity [69]. Similar effects were observed using other Nrf2 electrophiles sulforaphane [147], fsetin, and urolithin A from pomegranate [148].

Conclusion and future studies

A surprising array of specific GTPase control macroautophagy (mitophagy), microautophagy, chaperone-mediated autophagy (CMA), and endocytosis, and vesicle and mitochondrial trafficking. Since they all depend on local GTP levels, it will be important to determine their binding constants for GTP. Zala et al. [51] demonstrated functional significance for mitochondrial fission GTPases Drp1 and Fis1 with Km around 100  μM, while the Opa1 GTPase with a Km around 500  μM, would require higher GTP levels to promote mitochondrial fusion. In AD, the endocytic and exocytic processing of the synaptic adhesion protein beta-APP may be impaired by lower local GTP levels and GTPases as well as local oxidative redox states. More clear in AD is the accumulation of autophagic vesicles from either blockage in the long pathway or failure to upregulate a sufficiently robust response to internal cellular damage coupled with failure to sufficiently acidify lysosomes for activation of cargo degradation. Less clear is how much of these deficits are caused by age-related degeneration, known to be associated with age-related oxidative redox shifts. Age-related changes in GTP levels or capacity to upregulate bioenergetic foxes will be an important aspect of future investigation, especially if NAD+ precursors are able to boost capacity and promote more autophagic clearance. The metabolic adaptation to a sedentary lifestyle could downregulate mitochondrial function with less energy for healthy management of amyloid and tau proteostasis, synaptic function, and inflammation. In subjects with deficits, certain energy precursors and redox modulators may add to the benefits of exercise and a healthy diet to extend and promote maximum health span.

Acknowledgments 

Figures were created on Biorender.com. 

Funding This work was supported in part by the National Institutes of Health grant RF1 AG058218 to GJB. RAS receives support from the Secretaría de Educación, Ciencia, Tecnología e Innovación  (SECTEI) for the elaboration of the products derived from this postdoctoral work.

Data availability Raw image data for Figure 1 is available on reasonable request to the corresponding author.

Declarations Conflict of interest The authors declare no competing interests.

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