University of the Punjab, Lahore, Pakistan: Eman Fatima and His Research

Ketone Bodies and Autophagy: Synergistic Approach to luminating Neurotoxic Aggregates in Alzheimer Disease.

Abstract

Alzheimer disease (AD) is a progressive condition that exhibits cerebral glucose hypometabolism, accumulation of amyloid-b(ab) and tau tangles, and derailing of proteostatic responses. β-hydroxybutyrate (BHB), the major blood-borne ketone body, can replace glucose as an alternative cerebral energy source, and pleiotropic neuroprotective effects. BHB is shown to restore mitochondrial metabolism, reduce neuroinflammation, and regulate the unfolded-protein response to preserve lysosomal integrity and maintain autophagic flux. With experimental evidence. Simultaneously, BHB facilitates lysine b-hydroxybutyrylation (Kbhb) of metabolic enzymes, histone acetylation, as well as clearance of aggregation-prone proteins by both macroautophagy and chaperone-mediated autophagy. Preclinical AD models also show that BHB treatment lowers Aβ and tau pathology, enhances cognitive functions and displays synergistic effects in conjunction with pharmacologic activators of autophagy as AMPK agonists and TFEB inducers. All these findings reinforce a mechanistic model where ketones induced metabolic re-programming and autophagy regulation intersect to recover proteostasis and can serve as the disease-modifying approach in AD.

Introduction

The brain is a very energy consuming organ. Despite the fact that it comprises only 2

percent of the total body mass, it consumes approximately 20 percent-23 percent of the

total body energy, which is about 110 percent-140 grams of glucose per day. The energy is

necessary to sustain their resting potentials, develop action and post-synaptic potentials, as

well as to normalize pre-synaptic calcium levels and to restore neurotransmitter levels,

especially that of glutamate. The brain mostly utilizes glucose in the fed state but when the

body is in fasting, both ketone bodies are co-utilized with the most prominent in normal

consumption of glucose and the secondary one acetone. The lactate produced by anaerobic

glycolysis can also be seen as another energy source; it is used when there are basal and

hyperlactatemia conditions.

Alzheimer disease (AD) is a neurodegenerative condition that develops as a result of the

deposition of neurotoxic protein aggregates including amyloid-beta (Aβ) plaques and tau

tangles which cause impairment of synapses, neuronal degeneration and cognitive

impairment. As a person ages, the rate of cerebral glucose uptake becomes lower, which

frequently occurs without any clinical manifestation decades before the indicators appear

[1]. Even though there is a lot of research, there is still no disease-modifying treatment that

could be effective. New literature has highlighted cellular impaired clarification and

metabolic illness as major AD pathogenic factors [2].

The consumption of energy substrates is altered in both a diffuse and regional way around

the world and locally in the brain through ageing and neurodegeneration especially in

glucose. These modifications are either causal or consequential. It is critical to learn the

mechanisms of normal and pathological brain metabolism to come up with anti-aging and

anti-neuro degenerative measures [3].

The purpose of the current review is to discuss the therapeutic perspective of β-

hydroxybutyrate-mediated reprogramming of metabolism and activation of autophagy in

Alzheimer disease. The review, in particular, summarizes available data about the

association of cerebral glucose hypometabolism with the steps of the development of AD,

analyses the capacity of BHB as an alternative energy source and its impact on

mitochondrial and lysosomal functions, and outlines the molecular pathways by which

BHB induces Kbhb, controls the unfolded-protein response, and improves autophagic

clearance of Aβ and tau. Moreover, it critically discusses preclinical evidence that assesses

the synergies of BHB with pharmacologic autophagy enhancers and outlines current gaps

in knowledge that determine the further experimental and clinical research to translate

ketone-based interventions to AD treatment.

1.1. Reducing Glucose Metabolism and Sparing Ketone Metabolism in AD.

A decrease in glucose metabolism is coupled with the Aβ plaques. Aβ plaques has a direct

destructive effect on mitochondria via electron transport complex III, cytochrome c and

TCA cycle enzymes as well as reactive oxygen and nitrogen species (ROS / RNS) damage

of cell membranes, glucose transporters, and NMDA receptors. These molecular effects

help in reduction in body glucose usage. It is interesting to note that Aβ deposition takes

place an average of 15 years prior to the AD manifestation whereas cerebral

hypometabolism comes at around 10 years of age [4].

2. Ketone Body Metabolism and its role in the Alzheimer Disease (AD)

In the fasting process, the adipocytes release the free fatty acids which are carried to the

liver where they are converted to ketones. Diabetic patients have low insulin concentrations

that facilitate lipolysis by desensitizing the inhibition of hormone-sensitive lipase, leading

to β-oxidation in hepatocytes, and causing ketogenesis. Long chain fatty acids have to be

transported to mitochondria, so the process of ketosis might be restricted during ketogenic

diets, but medium-chain fatty acids (MCFA) could reach the mitochondria without the

involvement of transport proteins [5].

Ketogenic interventions are postulated to achieve improvements in cognitive and behavior

of AD models and some patients with significant improvements in energy deficits through

clinical and preclinical studies [6,7,8].

2.1. β hydroxybutyrate (BHB) production

The production of β-Hydroxybutyrate (BHB) occurs in the liver during the process of

metabolic acidosis. The main ketone is β-hydroxybutyrate. On decreased glucose

metabolism β-OHB ketolysis to generate acetyl-CoA, which enters the TCA cycle where

it gives rise to the production of ATP. In liver mitochondria ketogenesis is performed when

the production of acetyl-CoA using fatty acids is more than the oxidative power of the TCA

cycle and the excess of acetyl-CoA is changed into ketones [9]. The liver cells are the

primary producers of ketones with little input by the kidney and astrocytes. This process

and the production of acetoacetate are regulated by three enzymes, mitochondrial

acetoacetyl-CoA thiolase, HMGCS2 (controlled by insulin/glucagon through FOXA2), and

HMG-CoA lyase. The production of acetone occurs either spontaneously or through the

change of acetoacetate into BHB (most abundant ketone in the blood) through β-

hydroxybutyrate dehydrogenase which requires NADH as a cofactor [5].

Ketone bodies are soluble and transporter acetyl compounds (oxygenated to CO2 and

water) that act as a source of energy to the peripheral tissues, but are not carried out by

albumin or lipoproteins [9]. Ketogenic diets with calories limited stimulate the expression

of HMGCS2 in the liver and brain of rodents [5]. β-hydroxybutyric acid also enhances the

metabolism of mitochondria, signaling molecule regulation, histone acetylation, and

neuroinflammation and clearance of Aβ and Tau proteins in AD rodent models. H3K9

Kbhb and other Kbhb lysine β-hydroxybutylation (Epigenetic) regulation affects the

expression of genes and liver metabolism, but their involvement in AD progression has not

been published yet [10].

2.2. Uptake of Ketones in the Brain using Monocarboxylate Transporters (MCTs)

The uptake of ketone over the BBB through MCTs is concentration-dependent and not

dependent on the neuronal activity. BBB endothelium/astroglia expresses MCT1, the

astrocytes express the MCT4 (low affinity) and the neurons express MCT2 (high affinity).

In human beings and rodents, expression patterns are similar. MCTs are stimulated with

fasting, ketogenic diets, or exercise, and these efforts have increased the transport of

ketones [5].

2.3. The Ketone Bodies: Proteostasis Regulators

Other than fuel, BHB regulates proteostasis. Proteomic research suggests that BHB

selectively changes the solubility of pathological proteins including A beta. BHB or ketone

ester treatment induces aggregation-prone proteins turnover, presumably through

autophagy in mice [11]. BHB is also useful in promoting the lysine 1, β-

hydroxybutyrylation (Kbhb) of proteins, such as TCA cycle enzymes. Pathological stages

of APP/PS1 mice exhibit a reduction in Kbhb on citrate synthase and SUCLG1; BHB or

ketogenic diets replenish Kbhb, increasing activity, ATP generation, and decreasing Aβ-

plaque pathology and microgliosis [10].

2.4. Ketone-Uptake and Oxidation Conservation in the Alzheimer Disease

PET images depict that AD patients possess less than 25 percent down of frontal, parietal,

temporal lobes, cingulate gyrus CMRGlu combined with a reduction of glucose uptake rate

constants (KGlu) of around 15 percent when compared to healthy older people [4]. There

are no significant differences in CMRAcAc and AcAc uptake rates in healthy adults, MCI,

and AD groups [4,11]. This denotes that glucose metabolism reduces with age/with

cognitive impairment whereas there is no effect on ketone metabolism.

The whole-brain CMRAcAc and CMRGlc were found to increase by 28 and 44 percent

respectively with ketogenic diets in old 14-day-old rats [12]. CMRAcAc increased (p =

0.005) and CMRGlu reduced 20% (p = 0.014) in human adults [13]. Whether the KD

interventions can change the glucose uptake is still to be established [12].

2.5. Regulation of Ketone Metabolism during Autophagy

There is the crosstalk in the metabolism of ketones and autophagy. BHB controls the

unfolded protein response (UPR), lysosomal integrity, and autophagic flux and keeps

proteostasis valid. There is in vivo stroke research that demonstrates D-BHB inhibits

maladaptive UPR, decreases the activation of PERK-eIF2α-ATF4 arm and IRE1α

phosphorylation, suppresses chronic ER stress, and abates cell death [14]. BHB lowers the

expression of ATF6, PERK, and CHOP, alleviates the occurrence of inflammasome and

pyroptosis, and boosts the survival of SH-SY5Y neurons when confronted by Aβ and LPS,

facilitating adaptive, but not pro-apoptotic/pro-inflammatory cascade reactions [15].

D-BHB preserves lysosomal-autophagic vacuoles, inhibits cleavage of LAMP2, restores

ATG5, ATG7, Beclin 1, LC3, and p62 indicators, and decreases the quantity of lesion

volume, preserving fruitful autophagic flux [16]. Prolonged nutritional ketosis of the mouse

biosphere increases the markers and proteins of hippocampal autophagy (SIRT2-regulated

FOXO1/3a, PGC1a, TFEB), enhancing autophagy, mitophagy and lysosomal biogenesis

[17]. This coupling of elevated ketone utilization with stronger autophagy–lysosomal

machinery and fine-tuned UPR signaling supports the concept that ketone metabolism and

autophagy are part of a unified adaptive response that promotes clearance of damaged

proteins and organelles and may be especially relevant for limiting aggregate accumulation

in aging and Alzheimer’s disease [17,18,14].

BHB therapy enhances memory, reduces Aβ deposits and phospho-tau, elevates CMA

target proteins (LAMP2A, Hsp70), and suppresses NLRP3 inflammasome activation in

sporadic AD animals [19]. It enhances mitochondrial and lysosomal integrity, decreases

lysosomal acidification, decreases fibrillar vesicle accumulation, and improves lifespan

and memory in Drosophila AD models [20].

Ketone bodies, especially BHB are used as alternative fuels and signaling molecules to: (1)

support neuronal ATP in glucose hypometabolism, (2) remodel proteome solubility/ post-

translational modifications, and (3) increase autophagy/CMA and lysosomal activity. This

is because ketone metabolism is a major factor that contributes to proteostasis and

aggregate clearance in AD.

3. Autophagic Dysregulation in Alzheimer’s Disease

Quality checkpoint of proteins and organelles in neurons is dependent on autophagy-

lysosomal mechanisms. There is a malfunction of this system at several stages in AD

mechanisms: initiation, cargo trafficking, autophagosome-lysosome fusion, and lysosomal

proteolysis. It leads to impaired autophagy, intracellular 86 -amyloid and tau retention,

neurotic dystrophy, and cell death.

3.1. Abnormal Autophagy-Lysosomal Flux in AD

The electron microscopy and neuropathology show that autophagic vacuoles (AVs) and

autolysosomes are present in the dystrophic neurites, which are signs of autophagic stasis

and not normal turnover [21,22,23]. Autophagosomes and amphisomes in neurites and

synaptic areas and incompleteness of substances in autolysosomes, upsurge of LC3-

positive vesicles, and LAMP1-positive lysosomes but decrease in clearance capacity are

consistently demonstrated in post-mortem experiments and animal models [24,22,25].

As these results indicate, lysosomal clearance failure, but not complete constriction of

autophagy inductance, is a significant abnormality in AD [21,24,26].

3.2. Imbalanced Autophagosomal Presence and Formation

A number of studies portray altered autophagy initiation. In AD mTORC1 hyperactivity

(high amounts of p-mTOR, p-RPS6KA1, p-RPTOR, RRAGC) in the hippocampus

prevents the initiation of autophagy [27]. Significant constituents of autophagosome-

formation, such as Beclin 1, NRBF2, ULK1/2, are also reduced in AD hippocampus and

models, which is in line with impaired biogenesis [27,28,23].

Nonetheless, transcriptomic and protein studies of CA1 neurons demonstrate that the gene

expression and LC3 puncta increase, which suggests an increase in autophagosome

formation in vulnerable neurons [24,21]. The intensive autophagy triggering at the initial

stages may be compensated by these conflicting data, yet dysregulation of mTOR and

depletion of necessary ATG machinery eventually damage autophagy initiation

[27,24,22,23].

3.3. Alterations in Autophagosome Transport and Fusion

The autophagosomes are usually formed distally and being conveyed in a retrograde

manner to the soma where it fuses to a lysosome. The degenerative neurons in AD contain

undeveloped autophagosomes in the dystrophic neurites, especially in the dendrite and

axons of the nervous system, suggesting the loss of transport and maturation [23]. Aβ and

APP C-terminal flakes interfere with endosome-lysosome and autophagosome-lysosome

fusion. PS1/APP mice and Aβ aggregation prevent fusion and decrease the lysosomal

membrane and lysosome markers (cathepsin B and Lamp1) [29].

Accumulation of cholesterol in AD/Niemann-Pick C-like models does not change

autophagosome formation but interferes with endosomal-lysosomal vesicle fusion through

an abnormal distribution of RAB7 and SNARE, which does not allow Aβ /tau degradation

in autophagosomes, but leads to autophagosome secretion of Aβ [30]. The dysfunction of

SNARE complexes and malfunctioning lysosomal acidification negatively affect fusion

and AV accumulation even more [31,32].

3.4. Failure of Lysosomal Abnormality and Proteolysis

The lysosomal/autolysosomal stage is considered to be a prime suspect of primary blocks.

Structured analyses of CA1 neurons depict more autophagosomes produce and lysosomes

in the cells (MiTF/TFE activation), yet the LC3 II protein and p62 accumulate in the

enlarged autophagosomes and lysosomes (substrate degradation breakdown). PSEN1 and

APP mutations that cause familial AD affect the functioning of the lysosome, rendering it

unable to acidify and to degrade proteins, the lysosome is unable to clear autophagosomes,

but APP knockout can repair lysosomal/autophagy impairment in human neurons [33].

Swollen deacidified autolysosomal accumulation of Aβ/APP- βCTF, in AD mice, leads to

PANTHOS patterns in which effected neurons become key contributors of senile plaques

[34,35]. Human AD brains and models have been reported to have lysosomal PH defects,

cathepsin B/D activity decrease and membrane integrity damage, frequently prelude overt

plaque pathology [27,26,36].

3.5. Consequences for Aβ and Tau

Autophagy-lysosomal defects have direct effects on AD core proteins. AVs and

autolysosomes include APP, β-secretase and γ-secretase, which facilitates the synthesis of

Aβ and decreases its degradation [22,37,38]. Hyperphosphorylated tau is localized with

LC3-positive vesicles and aggregates of tau form with autophagy defects; LAMP1 and

cathepsin D defects suggest failures of tau turnover [25,39]. Genetic or pharmacologic

neuronal inhibition of Atg5, Atg7 and Beclin 1 causes ubiquitin aggregation, p62

aggregation, neurodegeneration and exacerbated AD pathology, highlighting the protective

properties of autophagy [27,39].

3.6. Dynamic and Stage-Dependent Dynamicity of Autophagy.

AD Autophagy is not a static process, but instead, an early period of compensatory

induction and bio-genesis, followed by chronic lysosomal impairment, AV overload and

neuritic dystrophy are observed during AD progression [27,24,22,26,35]. Autophagy

encouragement (e.g., mTOR suppression, TFEB stimulation) could be used therapeutically

to decrease Aβ/tau, if lysosomal function is intact, and as well as to enhance the cognition.

Nevertheless, in late lysosomal failure, subjecting the person to further induction may

increase substrate deposition and toxicity [22].

3.7. Epidiagnostic evidence of AD Autophagy collapse in the lab.

1) Pathology of human brain and mouse model.

The post-mortem evidence demonstrates that the pools of AVs and autolysosomes are huge

in the brains of AD and mouse models indicating autophagic flux blockage [27,28,34,39].

Functional imaging of neuron-specific LC3 pH sensors shows early autolysosome

acidification decline than extracellular plaque loss due to impaired v-ATPase activity [34].

Aβ/APP-βCTF is deposited in enlarged deacidified autolysosomes that produce PANTHOS

rosettes, one of the principal forms of senile plaques [34,35]. The broken connexon

between autophagosomes and lysosomes, defective retrograde transporting, and

dysfunctional lysosomes are verified as primary AD autophagy defects [27,39,40].

2) Molecular and mechanistic Evidances

The genetic and molecular research shows that the alterations in familial AD are

autophagy-lysosomal defects (e.g., PSEN1, APP, lysosomal genes) that disrupt lysosomal

acidification and proteolysis and result in the accumulation of APP, Aβ and tau and

neurodegeneration [27,35,39]. Aβ and phosphorylated tau disrupt autophagy/mitophagy

and decrease clearance of impaired mitochondria and protein aggregates [41]. Increase tau

accumulation in the presence of lysosomal inhibitors (chloroquine, NH 4 Cl, 3 -

methyladenine) is also a confirmatory that tau is removed via autophagy-lysosome

mechanisms [42].

3) Signaling changes within damaged autophagy.

The imbalance of nutrient-sensing and transcriptional pathways occurs in autophagy-

lysosomal collapse in AD. Constant mTORC1 stimulation in the human AD hippocampi

suppresses autophagy and lysosomal biogenesis and genetic mTOR deficit in Tg2576

results in autophagy restore and cognitive ability in mice [37]. Mutated AMPK, TFEB, and

associated signaling also worsens neuron autophagy-lysosome flux, initiating a vicious

cycle of low clearance, Aβ /Tau and mitochondrial dysfunction and cell death

[27,35,37,39,41,43].

4. Therapeutic implication of Autophagy Collapse in Alzheimer disease.

Because the autophagy impairment is mechanistically linked to Aβ and tau aggregates,

synaptic demise, and cell demise, the restoration of autophagic flux, especially the

lysosomal activity, is an important treatment approach.

4.1. Activation of mTOR-Independent Autophagy

mTORC1 inhibitors like rapamycin enhance autophagy and lower Aβ and tau pathology,

as well as learning and memory in various mouse models of AD [27,23,37,39,44]. Genetic

blockage of mTOR improves autophagy in Tg2576 mice and prevents cognitive

impairment, which is a good argument that lowering mTORC1 levels are therapeutic [37].

Moreover, memantine and carbamazepine, both used clinically, could induce autophagy

either through mTOR dependent or independent mechanism, and carbamazepine can

reduce the amyloid load and enhance cognition in 3xTg AD mice [23].

4.2. TFEB and Lysosomal Activation

The transcriptional master of lysosomal biogenesis is TFEB. TFEB can be

pharmacologically or genetically activated to increase the activity of the lysosome,

facilitate the breakdown of Aβ and tau, and improve pathology of AD models [32,27]. To

illustrate, intermittent hypoxia of APP/PS1 mice restores the nuclear TFEB positioning in

plaque-engaging microglia, augmenting autophagy-lysosomal legislation, amplifying Aβ

clearance result, decreasing load and neuroinflammation of plaques, and indicates the

functional advantages of TFEB-mediated lysosomal rehabilitation [44].

4.3. Small-Molecule Enhancers and Repurposed Drugs

Various agents, including rapalogs, metformin, resveratrol, nilotinib, spermidine,

curcumin, and more, stimulate autophagy with either mTOR-dependent or independent

pathways and reduce Aβ and tau pathology as well as improve cognition in preclinical AD

models [23,27,28,39,40,46]. Reviews of clinical trials show that the majority of clinical

autophagy-targeted therapies are at an early stage of pharmacological development with no

autophagy modulator approved specifically in AD. The serious issues are penetration of

the brain, optimal dosing option, as well as the danger of excessive autophagic activation

in circumstances of late-stage lysosomal failure [23,47,48].

4.4. Attacking the Upstream and Parallel Pathways

Activators of AMPK, PDE4 inhibitors, TRIB3 modulator, Nmnat, and BAG3 have been

shown to repair autophagic flux, minimize protein aggregates, and enhance cognitive

deficits in experimental systems [37,39,49,50]. By inducing autophagy and senolytic

approaches, the burden of senescent cells, neuroinflammation, Aβ and tau deposition, and

memory rescue in AD models in rodents, the autophagy senescence axis is of great

significance [37].

There is a strong stage dependency to autophagy at early stages of disease autophagy is a

protective process, but at late stages in AD there is evidence of severe lysosomal

impairment and autophagosome overproduction, which may increase the vacuolar

accumulation [27,34,35,39]. Therapeutic studies with co-targeted lysosomal acidification

and biogenesis (e.g., TFEB, v-ATPase) along with induced autophagy should also be tested

together, and the latter needs demonstration in human research [27,32,34,35,47].

5. Molecular Interaction of BHB with Autophagy, AMPK, β-Hydroxybutyrylation,

Lysosomal pH, and NLRP3 Inhibition.

BHB is an energy source and a signaling molecule that controls autophagy and

inflammation by converging on various mechanisms that are involved in

neurodegeneration and aging [51,52].

5.1. Autophagy and Mitophagy Plasticity.

BHB causes AMPK and ULK1 activation in brain cells and other cell types which stimulate

autophagy and mitophagy events. D-BHB enhances phosphorylation of AMPK Thr172 and

ULK1 Ser317, and raises the level of LC3-II, as well as LC3-positive autophagic vesicles

in cultured neurons, which indicates the presence of an AMPK-dependent but mTOR-

independent process of autophagic flux [17]. BHB enhances ULK1 ser317 phosphorylation

and inhibits mTORC1 activity, shown by decreased p-S6K1 Thr389 in HGPS fibroblasts,

and prevents BHB-induced p62/SQSTM1 degradation, progerin degradation is prevented

by inhibiting AMPK or ULK1 showing that BHB enhances autophagy via the AMPK-

mTOR-ULK1 pathway in order to clean up toxic intricate proteins [52].

In addition to autophagy, BHB rebates mitophagy and ATP generation in osteoarthritic

chondrocytes, and BHB antioxidative, anti-senescence and anti-apoptotic actions are

canceled by knockout of HCAR2, or AMPK inhibition, suggesting that it acts through the

HCAR2-AMPK-PINK1/PARK2 pathway [53]. D-BHB inhibits superfluous LC3-II

accumulation and p62 accumulation and suppresses AMPK–ULK1 Ser317 activation to

restore autophagic flux and mitigate neuronal death in rats with severe hypoglycemia and

coma, which is probably through the enhancement of mitochondrial energetics [54].

All these results suggest that BHB is either able to up- or down-regulate AMPK-dependent

autophagy in response to physiological conditions, but can always improve mitochondrial

energetics and mitophagy to reestablish autophagic flux in various cellular and in vivo

systems [17,51,52,53,54]. Despite the fact that this data implies mechanistic

interrelationships between ketone metabolism, mitochondrial quality regulation, and

proteostasis, they have been yet to be confirmed in human neurodegenerative systems.

5.2. Epigenetic Control and β-Hydroxybutyrylation of Autophagy Genes.

BHB is an epigenetic signaling metabolite which inhibits the activity of the class I histone

deacetylases and is a precursor of lysine β-hydroxybutyrylation (Kbhb), a histone mark

that promotes stress resistance, mitochondrial metabolism, and autophagy gene activation

[51,55]. A number of studies indicate that BHB-induced histone changes increase the levels

of autophagy-related and antioxidant genes, which adds to cellular resistance to metabolic

and neurodegeneration disorders [51,55,56].

Even though the direct mapping of the β-hydroxybutyrylated regulatory elements that

mediate autophagy has not been fully accomplished yet, there is current evidence of BHB

as an epigenetic promoter of autophagy and mitochondrial homeostasis [51,55].

5.3. Lysosomal Biogenesis, Function, and PH

BHB also regulates the autophagylysosomal system and lysosomal capacity. D-BHB uses

TFEB-dependent lysosomal biogenesis and SIRT2-FOXO1/FOXO3a-PGC 1α signaling in

neurons to synchronize autophagy, mitophagy and mitochondrial biogenesis [17]. BHB

recovers the impaired autophagy state of HGPS fibroblasts and triggers p62 degradation,

which is prevented by AMPK or ULK1 inhibition, and the effect reveals that a repressed

autophagy-lysosomal route has been reinstated [52]. D-BHB has been shown to rescue

LAMP2 expression and lysosomal membrane integrity, LC3-II accumulation and p62

accumulation, and increase autophagy and degrade lysosomal function, respectively, and

improve autophagic degradation in models of NMDA excitotoxicity and hypoglycemic

coma, which is also evidence of the stabilization of lysosomal activity and autophagy under

stress conditions [16,54].

Altogether, these data indicate that BHB promotes autophagic accumulation of the

lysosome and its functional stability and mitigates the stress-related bottlenecks of

autophagosome traffic formation instead of intensifying autophagosome formation

[16,17,51,52,54].

5.4. NLRP3 Inflammasomes and Autophagy Crosstalk

BHB has great anti-inflammatory properties that are direct inhibitors of the NLRP3

inflammasome and indirect instigators of autophagic homeostasis. BHB, but not

acetoacetate or short-chain fatty acids, dose-dependently inhibits NLRP3 activation and

IL-1β/IL-18 release in macrophages through the inhibition of potassium efflux and ASC

oligomerization and is independent of AMPK, reactive oxygen species, autophagy,

GPR109A/HCAR2, SIRT2 and glycolytic inhibition [57].

Against cellular evidence, in vivo BHB or ketogenic diets suppress caspase-1 activation

and IL-1β release in a number of NLRP3-mediated inflammatory systems and validate

systemic inflammasome inhibition [57]. Exogenous BHB in a 5XFAD AD model decreases

the plaque burden, microglial proliferation, ASC speck, and activates caspase-1, which

suggests that neuropathology is inhibited by NLRP3 inhibition [58]. On the same note,

BHB suppresses hippocampal NLRP3, cleaved caspase-1, IL-1β/IL-18, pyroptosis markers

and enhances chaperone-mediated autophagy markers LAMP2A, Hsp70 in an

unpredictable AD-like rat model, correlating the hippocampal inflammasome inhibition

with heterogenized proteostasis and resultant decreased neuroinflammation [19].

BHB also has an anti-NLRP3-activated effect in the hepatocytes (AMPK-FOXO3a-

antioxidant enzyme) in response to ER-stress, and the effect is independent of central

nervous system activity, and is another evidence that AMPK-regulates pathway [59]. Due

to the ability of BHB to protect lysosomal activity and prevent inflammasome activation,

the subsequent mechanism of its protective properties in metabolic, inflammatory, and

neurodegenerative diseases is that chronic NLRP3 activation disrupts the autophagic flux

and leads to pyroptotic cell death [19,51,57,58,59].

Combined, these data indicate that BHB mediates inflammatory suppressive and

proteostatic stabilizing responses to convergent metabolic signals as opposed to linear

response, and NLRP3 inhibition and autophagy stabilization are direct consequences of

cellular reprogramming under the influence of ketones.

6. Role of BHB-induced Autophagy and Complementary Synergies in clearing Aβ and

Tau in AD.

BHB modulates multiple autophagy arms (macroautophagy, chaperone mediated

autophagy, lysosomal function) and inflammation, which can functionally cooperate to

induce Aβ and tau clearance and can mechanistically interact with other autophagy-based

interventions.

6.1 BHB-induced Autophagy and Proteostasis in AD models.

a) Chaperone-Mediated Autophagy (CMA), Hsp70 and Aβ/Tau Clearance.

Systemic administration of BHB (125 mg/kg) enhanced cognitive functions, decreased

amyloid plaque burden and phosphorylated tau in the hippocampal in a high-fat/fructose

with LPS induced sporadic AD-like rat model, and also preserved neuronal architecture.

BHB stimulated several BHB pathway elements, which was mechanistic.

In particular, BHB elevated the level of hippocampal LAMP2A, the CMA rate-limiting

receptor on the lysosome, and Hsp70, a cytosolic chaperone that recognizes and delivers

substrates with KFERQ motifs to CMA [19]. Simultaneously, Aβ decatenation and

phosphorylated tau were found to decrease, and CMA upregulate, as expected by the fact

that tau and a subset of APP/ Aβ related proteins are CMA substrates, and that increased

CMA can directly induce their lysosomal degradation [19,27,37].

Taken together, these results suggest that BHB restores the activity of CMA and Hsp70-

dependent proteostatic quality control, which facilitates the selective degradation of

misfolded tau and Aβ related species.

b) Macroautophagy/Mitophagy: The Escher Not-AD Systems.

Non-AD models also provide the mechanistic understanding of the BHB-driven autophagy.

BPH activates the AMPK -ULK1 pathway and inhibits mTORC1 in Hutchinson Gilford

progeria fibroblasts, which reinstate the autophagic flux and facilitates the clearance of

toxic progerin protein, pharmacological inhibition of either ULK1 or AMPK ablates the

effect, which confirms the autophagic clearance [52]. This model is not AD-specific, but it

gives the mechanistic model of how BHB can trigger autophagy-induced clearance of

aggregation-prone proteins, a principle that is directly applicable to the Aβ and tau protein

clearance [27,37].

Additional evidence is provided by proteomic studies of a C99 expressing Drosophila AD

model, which supports the mitochondrial fragmentation caused by C99, enhances

lysosomal acidification, lowers the number of dense degradative vesicles, and prolongs

lifespan and memory. These effects suggest that the mitochondrial-autophagy-lysosome

homeostasis is restored at an Aβ production higher-order level [20].

6.2. Anti-Inflammatory Synergy: NLRP3, Autophagy, and BHB.

BHB also strongly inhibits the activity of the NLRP3 inflammasome, which is one of the

key contributors to Alzheimer disease (AD)-related neuroinflammation, which, in turn,

undermines both microglial autophagy and mitophagy [58,60]. Exogenous BHB

minimized Aβ plaque burden, microgliosis, ASC speck or caspase-1 activation in 5XFAD

mice, showing that not only NLRP3 but also AD pathology is attenuated [58].

Correspondingly, in a sporadic AD rat model, BHB reduced hippocampal levels of NLRP3,

cleaved caspase-1, IL-1β/IL-18, caspase-11 and gasdermin-N, and simultaneously

augmented CMA markers (LAMP2A and Hsp70) and a transition to the neuroprotective

M2-type microglial phenotype, compared to the pro-inflammatory M1-type microglial

phenotype [19]. Taken together, these results associate NLRP3 inhibition with decreased

pyroptosis and SASP-like cytokine signatures, and improved microglial functioning and

CMA, which in combination, enhance extracellular Aβ handling and minimized

phosphorylated tau aggregation [19,37,60].

At mechanistic level, recovery of autophagy and mitophagy suppresses the build-up of

dysfunctional mitochondria and mitochondrial ROS, which in turn suppresses NLRP3

further. This forms a positive feedback mechanism between BHB-induced autophagic

recovery and inflammasome-inhibition [37,39,60].

6.3. Autophagy-Mediated Tau and Aβ clearance: General mechanisms.

Several reviews manage to agree on the opinion that macroautophagy, chaperone-mediated

autophagy (CMA), aggrephagy, and mitophagy are the major intracellular activities that

lead to A1BB and tau species clearance [27,23,28,40,43]. Autophagy-lysosomal pathway

Aggregates of Aβ as well as tau inclusions are degraded by the autophagy-lysosomal

pathway and genetic expression of autophagy (e.g., knock-in of Beclin-1 F121A) or

pharmacological activation with rapamycin, metformin, and other small molecules reduces

amyloid burden, tau pathology, and improves cognitive functions in models of AD

[23,27,28,37,40,47]

On the other hand, defects in the fusion of autophagosomes and lysosomes and lysosomal

acidification are the cause of pathological accumulation in AD, since perturbation of

autophagic flux to accumulate vesicles of APP, β-secretase, Aβ, and tau accumulates the

aggregates, and thus fixing lysosomal activity is necessary to clear both proteins

effectively. Together, all those results confirm that autophagy-lysosomal pathways are the

predominant intracellular processes controlling Aβ and tau clearance, and that

reconstitution of lysosomal function and autophagic flux is a key to effective proteostasis

in AD [27,40,43]. In that regard, the ability of BHB to re-initiate lysosomal pathways such

as CMA and macroautophagy along with restoring a normal lysosomal activity is consistent

with the prevailing proteolytic processes of eliminating Aβ and tau [19,20,27,37,43].

This mechanistic overlap creates a rational basis to thinking whether BHB can cooperate

with complementary nodes of the same proteostatic network of other autophagy-based

interventions without suggesting any additive effects other than those experimentally

proven.

7. Additional Autophagy-Based Strategies that would be Complementary to BHB.

This will discuss the autophagy-modulating interventions that have been established to

converge mechanistically with the pathways that BHB has already activated, such as the

activation of AMPK-ULK1, mTORC1 inhibition, lysosomal biogenesis, and inflammatory

limitations on autophagic flux. It is meant to map mechanistic overlap and possible

compatibility and not to place therapeutic advantage or additive effect on top of the

available experimental evidence [19,23,27,37,47,52]

(a) Rapamycin, Metformin and autophagy enhancers.

AMPK-ULK1 signalling and/or mTORC1 inhibition can be stimulated by metformin and

rapamycin and can reverse autophagic flux, Aβ and tau pathology, and improve cognition

in various AD models, and early clinical trials are currently being conducted

[23,27,28,37,47]. BHB activates intersecting AMPK -ULK1 and lysosomal regulated axes

[37,52]. which implies mechanistic strengthening of autophagy initiation and degradative

power. Nevertheless, it should be carefully dosed not to cause excessive autophagosome

build-up and dysfunctional flux [27,23,47].

(b) TFEB, benimidazoles and aspirin mediate the lysosomal biogenesis.

Flubendazole decreases the Aβ burden and tau hyperphosphorylation in APP/PS1 mice by

stimulating autophagy via PPARγ and inhibiting GSK3 β, and synergistic aggregating

clearance by increasing autophagic flux. Aspirin enhances Aβ clearance through the

PPARα-TFEB axis, which activates lysosomal biogenesis and autophagy-lysosome

activity with cognitive benefit [61]. In isolation, the intermittent hypoxia treatment

enhances TFEB nuclear translocation in plaque microglia, boosts lysosomal and autophagy

genes, promotes Aβ catabolic activity and decreases the plaque load and neuronal damage

[44]. Convergence with TFEB-activation or PPAR-driven therapies due to the effect of

BHB on lysosomal functional enhancements on degradation of Aβ will subsequently

increase lysosomal capacity to degrade tau [19,20,37].

(c) Nanochaperones and targeted autophagy in case of tau.

Selective pathogenic tau binding with a tau-targeted nanochaperone, microtubules

stabilization, and local activation of autophagy by a tau-targeted nanochaperone leads to

improved autophagic flux and robust tau clearance and cognitive functions in AD mice

[62]. BHB in an integrated system promotes mitochondrial health, autophagy, and CMA

[19,20,37,52] and inhibitory autophagy by NLRP3 [19,58,60].

The BHB thus offers substrate specificity to tau, but the degradative potential and anti-

inflammatory environment needed to ensure an efficient clearance is maintained by

nanochaperones.

(d) Small molecules with autophagy and anti-inflammatory /ferroptosis properties.

Berberine induces autophagy and inhibits ferroptosis by down regulating JNK-38

MAPK signaling and reducing A beta plaques, neuroinflammation and neuron damage,

and enhancing memory in 5xFAD mice [63]. Isobavachalcone stimulates CAMKK2 -

AMPK signaling, autophagy, and inhibition of NLRP3 inflammasomes to promote

extracellular A -clearance and cognitive enhancement in 5xFAD mice [64]. These

analogs mechanistically converge the AMPK activation and inflammasome

suppression and upregulate autophagy similar to BHB in conceptual compatibility in

the same regulatory framework [19,37,58,60,62,64].

8. Mechanistic Integration: The BHB and Autophagy-Directed Therapies

Cooperatively Clear Aβ and Tau.

Altogether, the above-mentioned studies can be said to support a combined mechanistic

model:

8.1. Recovery of autophagic cell lysosome and lysosome capacity.

BHB (through CMA and macroautophagy) in tandem with metformin/rapamycin,

TFEB activators and nano-/ small-molecule autophagy enhancers all induce

autophagosome formation, intracellular trafficking, lysosomal fusion and acidification

to be restored. Such a coordinated restoration can help with the degradation of

intraneuronal Aβ and tau [19,23,27,37,43,45,61,62,].

8.2. Selective elimination of Aβ/tau species.

BHB-prompted upregulation of the CMA components (LAMP2A and Hsp70) identically

promotes cleansing of particular misfolded tau species and substrates associated with the

APP [43]. Simultaneously, aggrephagy and programmed nanochaperones can target

insoluble Aβ aggregates and tau aggregates (resistant to proteasomal degradation) with

aggrephagy and engineered nanochaperones, respectively [23,40,62].

8.3. The peripheral integration and microglial.

BHB and other NLRP3 modulators facilitate a partial re-differentiating of microglia to

phagocytic, low-inflammasome, phenotypes, which facilitates increased extracellular Aβ

uptake and trafficking of Aβ to autophagic and lysosomal degradation routes [19,44,45,60].

There is also growing evidence that hepatic autophagy plays a role in the peripheral A piece

of evidence; the previously mentioned signal of systemic metabolism could assist this axis

of inter-organ proteasolysis, but direct experimental evidence of this has not been

confirmed so far [43].

9. Translational landscape and Continued Trials. KD, Exogenous Ketones,

Combinatorial Therapeutics.

Ketone-based and autophagy-targeting approaches in the clinical translation of Alzheimer

disease are still ongoing concepts, yet in their initial development stages. Numerous

randomized controlled clinical trials and small clinical studies prove that ketogenic

interventions, such as or including classic ketogenic diets, modified Atkins diets, medium-

chain triglyceride (MCT) formulas, and ketone drinks, consistently increase blood ketone

levels, boost brain ketone uptake levels, and produce profile improvements in global and

episodic cognition in patients with mild to moderate AD or mild cognitive impairment,

especially in APOE4-negative individuals [8,65,66,67,68,69]. Cerebral ketone metabolism

is improved by ketone therapies on PET imaging, and some cerebrospinal fluid and FDG-

PET biomarkers of brain metabolism fluctuate with ketone therapies [8,66,67,68,70].

Translational reviews and systematic reviews highlight that ketogenic diets and ketone

supplement are a viable but varied option in terms of their arrangement and that

intervention periods can be as short as 45 days and as long as 180 days of intervention, the

formulations they used can be MCT oils, powders, enriched meals, and ketone jet

[8,65,68,70]. Consumption of ketogenic diet in frail AD patients is limited by adherence

issues, gastrointestinal side-effects, weight loss and dietary complexity, but exogenous

ketones and MCTs seem more acceptable to have on chronic use [8,65,66,69,70]. Ketone

therapies also are getting tested in not only symptomatic AD but also prodromal and

preclinical AD populations with high amyloid burden, and are aimed at modulating the

pathology before significant degrees of neurodegeneration [66,67].

The use of autophagy activity as a major biomarker is not yet tested in clinical trials of

ketones, but mechanistic reviews have linked ketone metabolism to processes that involve

autophagy (such as better mitochondrial function, reduced oxidative stress, reduced

neuroinflammation, and AB and tau pathology modulation) [8,23,27,38,71,72,73]. Drug

trials that target autophagy, mTOR inhibitors, AMPK activators, TFEB-related agonists

and repurposed small molecules, are currently in early phases of clinical development,

mainly as monotherapies [27,47,72,74]. No published phase II direct combination trials of

ketone therapies with autophagy modulators have been undertaken as yet; but concept

papers have explicitly outlined combined strategies of using ketogenic strategies in

combination with antioxidants, neuroprotective drugs or disease-modulating drugs to

attack multiple nodes of AD pathophysiology [47,67,71,72].

Identified Gaps, Challenges and Future Research - Dose, Specificity, Biomarkers,

Combination Therapy.

There is no definition of optimal ketone levels and diet, no real definition of duration of

intervention because different trials vary with regard to fat sources, carbohydrate levels

and aim BHB levels. As much as benefits seem to be best at early or prodromal AD grade,

there are limited robust long-term outcomes [8,65,66,67,68,69,71]. The autophagy

activators are no exception since the uncertainties with respect to dose windows capable of

increasing protein clearance without leading to excessive catabolism and toxicity have been

expressed [276,48,72,74].

The mechanism of action of mTOR-targeting drugs affects many other pathways of the

cell, such as glucose metabolism, lipid regulation and protein synthesis, and this puts

systemic safety risks when used chronically in the elderly [27,4772,74]. The reviews

identify the need of brain-targeted delivery plans and type of cell design, and more

specifically, neuronal and microglial effects to minimize off-target effects [47,72,74,75].

One of the greatest barriers to translation is the lack of validated in vivo biomarkers of

autophagy, where assays have been done based on the tissue levels of LC3 and p62, or

reporter constructs being introduced into animal models and cannot be used regularly in

humans [27,47,75]. There is an urgent necessity of PET tracers or cerebrospinal fluid

markers reflecting autophagic flux, lysosomal functioning or clearance of aggregates to

connect ketone interventions to autophagy adjustments in patients [27,47,75]. Other

standardized bioenergetic and inflammatory biomarker panel, such as plasma, and

cerebrospinal fluid ketones, inflammatory cytokines, and multi-tracer PET imaging are

also needed to establish the responders and to understand the mechanisms

[8,66,67,68,69,71].

The preclinical evidence indicates that concurrently augmenting bioenergetics by using

ketones and autophagic clearance using rapalogs, AMPK activators, and

aggrephagy/mitophagy inducers has a potential to synergistically decrease Aβ and tau

burden [23,27,40,48,72]. Nevertheless, families of ketone and autophagy modulators as

adjuncts to AD to date lack large randomized controlled trials and characteristic drug-diet

and long-term tolerability of ketone and autophagy modulators in multimorbid and

polypharmacy geriatric patients [8,72,74, (Fernandes, et al., 2025). The next round of trials

should use the factorial or multi-arm design, i.e. ketogenic diet versus autophagy drug

versus combinations, combine precision stratification with APOE genotype, metabolic

status, and disease stage, and provide a follow-up time that would capture structural and

biomarker outcomes [47,66,68,69,71,75].

10.4. Potential Future and Future Work Recommendations

There is convergent data that both ketone-based strategies and autophagy modulation show

mechanistically consistent but small effects in Alzheimer disease: ketones increase cerebral

energy metabolism and reduce oxidative and inflammatory stress, and autophagy-targeted

strategies maximize the clearance of Aβ, and damaged organelles [8,47,71,72]. Though

there is promising clinical evidence available, much is still in small fragments with small

sample sizes, shorter durations of the trials and heterogeneous design of the studies

prevailing in the literature today.

The initial step in advancing research directed at Ketone Bodies and Autophagy:

Synergistic Approaches is standardized large, multicenter randomized controlled trials with

adequately powered cohorts, and harmonized protocols to support parallel randomized

trials assessing autophagic-based or exogenous intervention based on ketones, expressly

the measurement of autophagic and lysosomal dynamics of processing [68].

The second urgent goal is the production and confirmation of noninvasive biomarkers of

autophagy and aggrophagy, which can be used in longitudinal studies of humans,

preferably on the basis of PET tracers or cerebrospinal fluid and plasma molecular

signatures [27,44,47].

Emerging combination therapies such as ketotherapies and mTOR-independent autophagy

activators, mitophagy activators, or anti-amyloid and anti-tau therapies will have to be

systematically optimized on dose, evaluated in terms of safety, and stratified based on

genetic background and metabolic phenotype [27,44,47,71,72,74]. Simultaneously, the

concept of ketone and autophagy should be applied in multidomain prevention paradigms

that combine diet, physical activity, and cognitive training, with an emphasis on early or

preclinical Alzheimer diseases, in this case the most probable target of metabolic and

proteolytic regulation [8,62,66,68,69,71].

Overall, despite the promising clinical potential, it has not yet been properly investigated,

and it needs strictly designed, mechanistically guided translational research, which must

help to establish to what degree the ketone-induced autophagy modulation can play a

significant role in the development of the Alzheimer disease.

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