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

Abstract

Increased interest in aging and Alzheimer's disease (AD)-related impairments in autophagy in the brain raise important questions about regulation and treatment. Since many steps in endocytosis and autophagy depend on GTPases, new measures of cellular GTP levels are needed to evaluate energy regulation in aging and AD. The recent development of ratiometric GTP sensors (GEVALS) and findings that GTP levels are not homogenous inside cells raise new issues of regulation of GTPases by the local availability of GTP. In this review, we highlight the metabolism of GTP in relation to the Rab GTPases involved in the formation of early endosomes, late endosomes, and lysosomal transport to execute the autophagic degradation of damaged cargo. Specific GTPases control macroautophagy (mitophagy), microautophagy, and chaperone-mediated autophagy (CMA). By inference, local GTP levels would control autophagy, if not in excess. Additional levels of control are imposed by the redox state of the cell, including thioredoxin involvement. Throughout this review, we emphasize the age-related changes that could contribute to deficits in GTP and AD. We conclude with prospects for boosting GTP levels and reversing age-related oxidative redox shifts to restore autophagy. Therefore, GTP levels could regulate the numerous GTPases involved in endocytosis, autophagy, and vesicular trafficking. In aging, metabolic adaptation to a sedentary lifestyle could impair mitochondrial function generating less GTP and redox energy for healthy management of amyloid and tau proteostasis, synaptic function, and inflammation.

Keywords GTP · Energetics · Autophagy · Mitophagy · Endocytosis · Lysosomes · Alzheimer’s · Aging

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GTP, the other energy currency, in aging and AD 

Synthesis of the high-energy molecules ATP and GTP both rely on the redox state of NAD+/NADH and their sufficient concentrations. ATP synthesis from glycolysis is primarily powered by the oxidative power of NAD+ acting on reducing sugars. ATP synthesis by oxidative phosphorylation in mitochondria is powered by NADH generation in the TCA cycle for the reduction of pyruvate, glycerol, or fatty acids with oxygen as the terminal electron acceptor in the electron transport chain. Our label-free studies in live rat or mouse hippocampal neurons indicate an age-related depletion of NAD and NADH, that could impair synthesis of ATP and GTP [1–4]. This depletion was further exacerbated in neurons from mice carrying transgenes for human beta-amyloid and tau. Further, postmortem analysis of TCA cycle enzymes indicated up to 40% impairment in the TCA cycle enzymes from AD brains compared to age-matched controls [5]. Thus, age-related energetic depletion of NAD+ and NADH on top of AD-related mitochondrial impairment in the activity of TCA enzymes could locally affect ATP and GTP levels, even if their bulk concentrations were minimally affected.

The energy available from GTP hydrolysis is the same as ATP hydrolysis, but GTP is utilized for different purposes than ATP due to the selectivity of specific enzymes. GTP concentrations in cells are on average tenfold lower than the millimolar ATP. GTP is a major regulator of multiple energy-dependent cellular processes of protein synthesis and vesicular trafficking involving endocytosis and autophagy. The estimated total cellular GTP concentration in mammalian cells is in the range of 250–700 μM [6], but free GTP in cancer cell protrusions is closer to 30 μM [7]. Major proteins powered by GTP include dynamins for membrane fission and fusion, the small regulatory GTPases, and microtubules as detailed below. Besides de novo synthesis from inosine by IDMPH2 and xanthosine by GMPS, cellular sources of GTP are primarily localized nucleoside diphosphate kinases (NDPKs) from the non-metastatic genes (NME; sometimes called NM23) that convert ATP to GTP [8]. There are at least ten members of the NME gene family but only the first four have nucleoside diphosphate kinase activity. According to the Allen Brain Atlas, NME1 through 4 are strongly and selectively expressed in the mouse and human hippocampus where memory is impaired in AD. NME1–3 are cytoplasmic, while NME4 is mitochondrial. Since both distinct processes of endocytosis and autophagy are affected in aging and AD, we posit that alterations upstream affect both, possibly the age and AD-related consequence of limiting GTP levels on these amyloid-critical functions.

Measurement of GTP levels 

Although GTP regulates several crucial processes for cellular survival, measuring GTP levels in the live cell under physiological and pathological conditions is difficult because of the high turnover and the existence of both a protein-bound and free state. Bianchi-Smiraglia et al. [9] described a novel genetically encoded GTP sensor (GEVAL) based on a yellow fluorescent protein that detects ratiometric changes in GTP-free and GTP-bound concentration in vitro and in vivo. Recently, the same group reported a mechanism for the regulation of GTPase Rac1 in cell invasion by a human melanoma cell line driven by local GTP production [7] and reviewed their methodology [10]. As we will see in this review, GTP plays a crucial role during endocytosis and autophagy-related to amyloid processing. Currently, we are studying the age-related GTP changes in the processing of Aβ in primary cultures of hippocampal neurons from the triple transgenic 3xTg-AD mouse and its effect on alterations in autophagy. Figure  1 shows the preliminary results of GEVAL530 transfection into primary adult neurons from this AD mouse model compared to non-transgenic mouse neurons. As observed by Bianchi-Smiraglia et  al. [7], we see a non-uniform distribution of GTP in the processes and edges of the soma when measuring free GTP (Fig.  1Aa), with lower free GTP levels in the 3xTg-AD neurons (Fig.  1Ac). As detailed in Section 10, pretreatment of these neurons with an NAD+-precursor, nicotinamide, raised the free GTP levels (Fig.  1Ab, d). The 530-µM Kd for GEVAL530 used in these experiments suggests that the local free GTP concentration is within a few-fold of 530 µM. In Fig. 1B, we examined the bound GTP that appears to be localized in vesicles and increased by nicotinamide (Fig. 1Bf, h), especially in the 3xTgAD neurons. Preliminary evidence indicates that neurons treated with NME1 siRNA lower their free GTP levels (Santana Martinez, unpublished). Further studies with adult neurons across the age spectrum will determine the age-dependence of GTP deficits and the ability to remediate them with NAD+-precursors. Given the essential role of GTP in vesicular trafficking processes of endocytosis and autophagy and this preliminary evidence for changes in GTP with AD-like genetics, the following sections provide details of these processes and the effects of aging and Alzheimer's disease on them. The result of this review highlights the need for new methods like GEVAL probes to directly measure changes in bound and free GTP and total GTP concentrations.

Fig. 1 Ratiometric GTP measurements in primary mouse hippocampal neurons from middle-age mice transfected with GEVAL530 sensor False-colored pixel-by-pixel ratiometric images of neurons show non-uniform distributions of GTP. (a) Excess free-GTP/bound-GTP of middle-age (14 mo.) nontransgenic (NTg) at edges and apical dendrite compared to (c) middle-age (10 mo.) 3xTg-AD neuron. (b, d) Increase in free GTP in neurons treated with 2 mM nicotinamide by 24 h. (e, g) Untreated neurons exhibit vesicular bound GTP that is increased in (f, h) neurons treated with 2 mM nicotinamide by 24 h

Endocytosis: GTP dependence in aging and AD 

In the endocytosis process, the cell internalizes macromolecules and ligand-bound receptors and surface proteins including the amyloid precursor protein (APP) [11] (Fig.  2A). Of the two main endocytic pathways, plasma membrane-embedded APP uptake occurs mostly through clathrin-mediated uptake (Fig. 2B.1) rather than caveola-mediated from cholesterol-rich patches (Fig. 2B.2). However, hydrophobic Aβ, partitions into cholesterol-rich patches of lipid rafts in the plasma membrane with subsequent uptake by the caveola pathway [12] (Fig.  2B.2). In both cases, dynamin GTPase complexes assemble at the invagination to catalyze GTP-dependent membrane curvature for final fusion and release of mature vesicles from the plasma membrane [13–15]. Local GTP fueling is catalyzed by NME1 and 2 nucleotide phosphate kinases that bind to dynamin [16]. Early endosome Rab5 GTPase decision toward exocytosis and receptor recycling or transformation to late endosome Rab7 GTPase for degradation.

The Rab5 GTPase is considered a marker for the early endosome [17]. Rab5 protein is present in the plasma membrane, in clathrin-coated vesicles, and early endosomes (Fig.  2). In cell and fy models of Huntington’s disease, Rab5 participates in autophagosome formation to regulate autophagy and eliminate toxic mutant huntingtin [18]. Inhibition of Rab5 reduces autophagy-regulating proteases Atg5-Atg12 conjugation, resulting in decreased autophagosome formation. The Atg12-Atg5 conjugate promotes the lipidation of Atg8, and lipidated Atg8 facilitates autophagosome formation and selective cargo recognition during autophagy [19].

Another contributing factor to Rab5-mediated autophagy is phosphatidylinositol-3-kinase (PI3K). PI3-kinase complex inhibits autophagy by activating the Akt/mTOR-signaling pathway [20]. The P110β subunit of the PI3K complex regulates the catalytic activity of the Vps34 complex to promote PI3P generation [21]. This step is essential for autophagosome formation. P110β promotes the transition from Rab5- GDP to Rab5-GTP. P110β overexpression mitigates autophagic deficiency following activation of the macromolecular complex composed of Rab5, Vps34, and Beclin1, which in turn leads to autophagosome formation [22]. Targeting these specific proteins may elucidate therapeutic avenues to attenuate AD pathology.

Fig. 2 Endocytosis of the amyloid precursor protein (APP). The N-terminal portion of the 770 residue APP protein with green shading of the aggregation-prone Aβ requires cleavage by BACE and Gamma peptidases. B Endocytosis of APP. (1) Formation of the inward bud from the plasma membrane orchestrated by clathrin with endoproteases BACE and 훾 -secretase. Endocytosis supported by dynamin receives local GTP by NME1. (2) Microdomains enriched in cholesterol (lipid rafts) bind APP processed to Aβ or Aβ adsorbed from the parenchyma. (3) Early endosome attracts the GTPase Rab5 which is recognized by another GTPase, Rab11, to mediate (4) endocytic recycling of receptors back to the plasma membrane. Alternatively, (5) Rab7 mediates segregation early into late endosomes allowing further processing of APP and accumulation of Aβ. (6) Late endosome fusion with (7) lysosome to degrade contents. Some aggregated Aβ may be resistant to digestion, and with endosome disruption, Aβ may accumulate in the cytosol.

Membrane-bound Rab5 is a key factor in directly promoting Mon1-Ccz1-dependent Rab7 activation and Rab7-dependent membrane fusion [23]. Mon1-Ccz1 is a heterodimeric guanine nucleotide exchange factor GEF-complex which activates Rab5. Mon1-mediated displacement of the Rab5 GEF results in the displacement of Rab5 by Rab7. C‐Vps complex acts as a GEF for Rab7 and promotes Rab7 transiting from the GDP‐bound to the GTP‐bound state for Rab7 activation. Ultraviolet radiation resistance–associated gene (UVRAG) stimulates Rab7 activation by UVRAG‐ C‐Vps interaction through GDP/GTP exchange of Rab7 [24]. Another regulatory point in the autophagic process is homotypic fusion and vacuole protein‐sorting (HOPS) complex that activates the yeast vacuolar Ypt‐Rab GTPase during membrane fusion [25]. One of the 6 subunits of the HOPS complex is the Class C vacuolar protein sorting (C‐Vps) complex which contains Vps11, Vps16, Vps18, and Vps33. Rab7 late endocytic vesicles subsequently fuse with lysosomes for cargo degradation (Fig. 2).

Changes in Rab 5 and Rab 7 endosomal trafficking in mouse models and in AD

In mouse models of AD with mutations in the amyloid precursor protein (APP) or Aβ-producing presenilin, the endosome–autophagosome–lysosome pathway appears dysregulated partly because of impaired acidification of lysosomes that fails to sufficiently activate protease and lipases [26]. In mouse models of AD, large perinuclear bodies containing Aβ aggregates label with the autophagosome marker LC3 colocalized with Rab7 and late endosomal and lysosomal components. This could be due to failed protective upregulation or pathologic impairment. In neurons cultured from 3xTg-AD mice across the age span, we found aggregated vesicular Aβ to increase 30–50 fold with age, most prominently in Rab5-labeled early endosomes and mitochondria, but also within Rab7-labeled late endosomes and autophagosomes [27]. The snow-ball-like accumulation of Aβ42 and Aβ45 suggests that old neurons were unable to complete autophagic degradation of these longer aggregates of Aβ. We hypothesize that impaired energy production in old neurons limits the energetic capacity for the completion of autophagy. Compared to levels in the young hippocampus and cortex, in 7–12-month-old APP/PS1 mouse hippocampus, Rab7 levels decreased along with reductions in Beclin1 and Rubicon activators and an increase in the Rubicon inhibitor [28]. In the younger hippocampus and all ages in the cortex, Beclin1 activates Rab7, while Rubicon inhibits the activation of Rab7 via suppression on UV radiation resistance-associated gene protein (UVRAG)‐vacuolar protein sorting gene (Vps) interaction. Rab7 deficiency in yeast and fruit flies results in a massive accumulation of autophagosomes [29]. Rab7 knockdown also inactivates mTORC1/S6K1, and the localization of mTOR in late endosomes [30]. Interestingly, the inhibition of other stages of endocytic trafficking does not change the activity of mTORC1 suggesting that intact late endosomes are crucial for mTORC1 signaling in autophagy. This is an important reason why targeting mTOR may not slow aging or AD.

Regionally selective upregulation of Rab 5 and Rab7 proteins and mRNAs in AD, suggests selective contributions to disease pathology or an insufficient protective mechanism [31]. Aβ accumulates predominantly in Rab7‐positive late endosomes and autophagic vacuoles of neuronal cells [32] in an age-related manner [27] causing internalization of Aβ and subsequent increase of Rab7 triggering neuronal degeneration [33]. Blocking the late endocytic pathway by Rab7 suppression induces Aβ‐dependent amyloid fibril formation on the cell surface causing endosomal enlargement [34] resulting in accelerated recycling of Rab7 and endocytic trafficking of Aβ to lysosomes for degradation [35]. Inhibition of Iysosomal proteolysis can also affect the axonal retrograde transport of autophagic organelles causing AD‐like axonal dystrophy [36]. However, practical interventions to promote Rab7-mediated autophagy could delay or reverse AD progression. Examples include a shift from a sedentary state to exercise, a Mediterranean diet, and energy-boosting compounds like NAD precursors to create a redox shift [4] (Section  10). Another avenue is the strange discovery that guanosine monophosphate reductase 1 (NADPH-dependent GMPR1) levels are increased in AD brains which could itself lower GTP levels and raise AMP signaling [37]. Neurofibrillary tangles of tau were lowered when this activity was inhibited in AD mice.

With progression from mild cognitive impairment to AD, both endosomal Rab5 and Rab7 expressions were upregulated in hippocampal CA1 neurons as measured by transcriptional microarray [31]. Enlargement of Rab5-positive endosomes was associated with neurofibrillary tangles and amyloid deposition. Neurotrophic factors such as nerve growth factor (NGF) bind to their Trk receptors and internalize into Rab5-positive endosomes to initiate downstream signaling [38]. It is interesting to note that, the signaling endosome is retained as Rab5 early endosomes and does not progress to Rab7 late endosomes, during their transit within the long axons [39]. This difdifferentiates receptor-mediated signal delivery from endosome processing toward autophagy [40]. Following activation by NGF, TrkA recruits a Rab5‐GAP to quickly convert GTP‐Rab5 to GDP‐Rab5 to keep the level of GTP‐Rab5 in check, which prevents the Rab5 to Rab7 conversion resulting in inhibition of the NGF/TrkA signaling and premature degradation.

Rab5 function is compromised in the early phases of AD [41]. Persistent hyperactivation of Rab5 promoted the endocytic pathway toward late endosomes, lysosome fusion, and autophagy resulting in premature degradation of the neurotrophic factor signaling and neuronal atrophy. Vps35 and Vps26, two key retromer proteins, were also reduced in AD brains [42]. Vps26 binds to SorLA which is a sorting receptor that controls APP trafficking from endosomes to the Golgi. A reduction in Vps retromer proteins leads to abnormal Vps-SorLA complex that hinders APP trafficking casing APP accumulation in the endosomes where it is subject to beta-secretase. However, mimicking APP phosphorylation at S655, within the APP 653YTSI656 basolateral motif, can enhance APP retrieval in a retromer-mediated process causing decreased APP lysosomal targeting, and decreased Abeta production [43]. An increase in full‐length APP and APP fragment β‐CTF can, in turn, elevate Rab5 GTPase activity inducing enlargement of early endosomes [44]. Interestingly, neuronal atrophy induced by the APP β‐CTFs could be rescued by a dominant‐negative Rab5 mutant both in vitro [39] and in vivo [45].

The net result of aging is the depletion of late endosomes, late endocytic dysfunction, and impaired lysosomal fusion. In AD, endocytosis of Aβ increases into enlarged Rab5 early endosomes. The role of Rab7 in AD is yet to be clearly elucidated as both early upregulation and late downregulation of Rab7 have been observed affecting neuronal health in both directions. As emphasized by Bianchi-Smiraglia et al. [7], total cellular GTP that is measured in homogenates does not allow assessment of free GTP available locally for the Rab GTPases. Free GTP measures have not been reported yet, so their possible role in age and AD-related impairments in endocytosis is unexplored. Therefore, it will be important to measure free GTP in live cells and the effects of variations in free GTP on endocytosis as a function of age and AD models.

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