Maintenance of mitochondrial homeostasis for Alzheimer's disease: Strategies and challenges

Abstract

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases, and its early onset is closely related to mitochondrial energy metabolism. The brain is only 2% of body weight, but consumes 20% of total energy needs. Mitochondria are responsible for providing energy in cells, and maintaining their homeostasis ensures an adequate supply of energy to the brain. Mitochondrial homeostasis is constituted by mitochondrial quantity and quality control, which is dynamically regulated by mitochondrial energy metabolism, mitochondrial dynamics and mitochondrial quality control. Impaired energy metabolism of brain cells occurs early in AD, and maintaining mitochondrial homeostasis is a promising therapeutic target in the future. We summarized the mechanism of mitochondrial homeostasis in AD, its influence on the pathogenesis of early AD, strategies for maintaining mitochondrial homeostasis, and mitochondrial targeting strategies. This review concludes with the authors' opinions on future research and development for mitochondrial homeostasis of early AD.

Graphical abstract

Sketch map of mitochondrial homeostasis and Alzheimer's disease (by Biorender).

High lights

• •Research on mitochondrial homeostasis is promising for the treatment of early AD.
• •Summarizing the drugs and strategies of targeted mitochondrial therapy for AD.
• •Providing ideas for drug research of mitochondrial homeostasis in AD.

1. Introduction

Alzheimer's disease (AD) is one of the most prevalent neurodegenerative diseases in the elderly population with a relatively insidious onset. AD patients are often accompanied by symptoms of cognitive dysfunction and memory impairment. Currently, more than 50 million people are diagnosed with AD worldwide, in which one in ten people over 65 and one in four people over 85 live [1]. With the increase of life expectancy and the number of the aged, AD patients are expected to reach 152 million by 2050 [2]. AD is clinically divided into three stages: the early preclinical asymptomatic stage, the mild cognitive impairment (MCI) stage, and the significant dementia stage [3]. Due to the unclear pathogenesis and the fact that the irreversible death of nerves cannot be effectively interfered in advanced AD patients recruiting by previous clinical trials [4], over 99% the development rate of AD drug failed since 2000 [5]. Individuals develop significant symptoms over a period of 20 years or more, while cellular abnormalities appear in the early stage of AD, hinting the key role of early intervention [6]. Up to 67% of the current clinical therapies in development focus on the early stage of AD, including neuroprotection (20%), synaptic function and neurotransmitters (20%), neuroinflammation (15%), mitochondrial and metabolic function (12%), etc [7].

Brain weighs only 2% of the body weight for adults but requires energy for 20% of the total energy demanded. Energy is provided by mitochondria in cells, and maintaining mitochondrial homeostasis ensures adequate energy supply [8]. Mitochondrial homeostasis constitutes the quantity and quality of mitochondria and the homeostasis of the internal environment, which is dynamically regulated by mitochondrial energy metabolism (mitochondrial oxidative phosphorylation), mitochondrial dynamics (mitochondrial fission and fusion) and mitochondrial quality control (mitochondrial biogenesis and clearance). Studies have shown that the dysregulation of mitochondrial homeostasis occurring in the early stage of AD leads to impaired energy metabolism in brain cells [9]. Therefore, regulation of mitochondrial homeostasis is a promising direction for future AD therapy [10]. In this paper, we examine how mitochondrial homeostasis contributes to the pathogenesis of AD, discuss the mechanisms of mitochondrial homeostasis in the pathogenesis of early AD, and summarize the promising drugs for maintaining mitochondrial homeostasis and new strategies for mitochondrial targeted therapy. This review will pave the way for mitochondrial therapies and new drug development for early AD.

2. Dysregulation of mitochondrial homeostasis on the onset and progression of AD

Brain cells in the central nervous system (CNS) mainly consist of neurons and glial cells. Neurons are responsible for processing information related to brain function. The synaptosome is the site of functional connection between neurons and the key site of information transmission. Different types of glial cells, including microglia, astrocytes, and oligodendrocytes, perform the formation of myelin sheaths outside neuronal axons, neuronal nutrient supply, and participation in signal transduction in the brain. In the pathogenesis process of AD, glial cells can amplify their functions by crosstalk with each other [11]. Since glial cells ensure effective electrical impulses, providing metabolic support for neurons, regulating synaptic function and providing immune surveillance, the changes of these cells have a direct impact on neural circuits [12]. Mitochondrial homeostasis is central to the maintenance of neuronal function and glial cells’ function. Mitochondrial homeostasis controls the ultrastructure of mitochondria, the organization of the mitochondrial network and the inheritance of mitochondria, which also regulate signals by the establishment of membrane contact sites with endoplasmic reticulum and other organelles. It is evident that the regulation of mitochondrial homeostasis is a complex and sophisticated process. The dysregulation of mitochondrial homeostasis results in neuronal damage, decreased neuronal support, myelin abnormalities and increased deposition of Aβ toxic plaques and Tau protein tangles, leading to the onset and progression of AD.

2.1. Influences of neuronal mitochondrial homeostasis of AD

Neurons are high-energy and high-oxygen-demanding cells that are more sensitive and irreversible to damage. It has been found that in the brain of AD patients the number of neurons is greatly reduced and there are a large number of damaged neurons [13]. Impaired inter-synaptic signaling and degeneration of synaptic function are key causes of cognitive impairment [14]. AD is a progressive synaptic failure disease, and synaptic damage caused by oxidative mitochondrial damage is an early event in the progression of AD [15].

Mitochondria are the energy-supplying organelles of neurons, which are mainly located in dendrites and axons. Mitochondrial oxidative phosphorylation is important for neuronal survival and function. Mitochondria produce ATP that is involved in synaptic vesicle recycling, exocytosis, presynaptic development and generation of synaptic membrane potential. Mitochondria store calcium ions and release them to maintain calcium levels in cells after stimulation. Normal mitochondria have homeostatic energy metabolism and quality control to maintain normal neuronal and synaptic function, whereas dysregulation of mitochondrial homeostasis in neurons occurs in AD before Aβ deposition and NFTs formation [16]. The dysregulation of mitochondrial homeostasis results in impaired mitochondrial transport, disturbed mitochondrial distribution, reduced energy efficiency, excessive reactive oxygen species (ROS), and decreased ATP content [17]. Overproduction of ROS damages cellular macromolecules, such as DNA, lipids and proteins, ultimately causing neurodegeneration and abnormal death of neurons [18]. Excessive calcium levels lead to glutamate release and excitotoxicity, ultimately leading to neuronal dysfunction and loss. Due to the high dependence of synaptosomes on mitochondria, imbalance in mitochondrial homeostasis can lead to disruption of neural activity. Cell cycle exit and neuronal differentiation 1 (CEND1) has an important role in neuronal development, cell cycle regulation and the maintenance of synaptic function. CEND1 is specifically localized in the outer membrane of the presynaptic mitochondria of neurons and is significantly reduced in AD pathogenesis, and its deletion leads to upregulation of the mitochondrial fusion protein dynamin-related protein 1 (Drp1), which in turn induces abnormal mitochondrial division and exacerbates the progression of AD [19].

The key feature of AD patients is the accumulation of highly toxic substances in the brain, i.e., the deposition of toxic Aβ plaques. A study published in Nature in 2017 demonstrated for the first time that enhancing mitochondrial protein homeostasis and repairing mitochondrial health reduced the formation of toxic Aβ plaques in the brain of AD mice and thus improving the function of brain [20]. GMC101, a worm model with Aβ toxicity, was used to retest its mitochondrial characteristics. Aβ aggregation was reduced in cellular, worm and transgenic mouse models of AD by pharmacologically and genetically targeting mitochondrial translation and mitochondrial autophagy to increase mitochondrial homeostasis. It is thus clear that dysregulated mitochondrial homeostasis induces cell abnormalities in brain and exacerbates toxic plaque deposition in early AD, and that maintaining mitochondrial homeostasis can be therapeutic from early AD intervention. Normal Tau proteins in the brain bind to microtubules, a component of the cytoskeleton, and help maintain the stability of the cell structure, whereas Tau proteins in AD brains misfold and aggregate to form NFTs. Mitochondria in neurons interact with Tau proteins, while AD brains have much lower levels of Tau-interacting proteins, including mitochondrial proteins. A study found that the dysregulation of mitochondrial homeostasis inhibits Tau-mitochondrial protein interactions, and they next intend to explore whether improving mitochondrial-Tau protein interactions can improve AD [21]. Degradation of RHOT1 is triggered by prolonged activation of the PRKN pathway-mediated mitochondrial autophagy, exacerbating the damage of synapse by disrupting mitochondrial supply. Increasing RHOT1 expression in mice compensates for synaptic mitochondrial defects, suppresses tau protein-induced synaptic deficits and protects synaptic plasticity [22].

2.2. Influences of microglial mitochondrial homeostasis of AD

Microglia cells are brain-resident macrophages that are activated by a variety of factors and receptors during AD pathogenesis, such as trigger receptor 2 (TREM2) and toll-like receptors (TLRs). Activated microglia cells exhibit migratory and phagocytic abilities to engulf and remove toxic aggregates of Aβ and Tau proteins [23]. Microglia cells are the most abundant immune cells in brain with high plasticity. M1-type microglia cells release large amounts of pro-inflammatory factors, such as IL-1β, IL-6, IFN-γ and TNF-α that trigger neuroinflammation. M2-type microglia cells release anti-inflammatory factors, such as IL-4, IL-10 and neurotrophic factors that protect neurons. AD patients' brains contain large numbers of activated microglia cells and inflammatory markers that accelerate neuronal death and the progression of AD [24]. It has been shown in genome-wide association studies that the majority of the risk loci for AD are strongly associated with microglia cells and neuroinflammation, suggesting that microglia cells play a key role in the disease. During the development of AD, microglia cells lose their ability to clear Aβ and are over-activated to cause neuroinflammation. It has been revealed in the AD model of APP/PS1 mice that blocking the proliferation of microglia and promoting their transition to the M2 type resulted in improved memory and behavioral tasks [25]. Targeted deletion of spleen tyrosine kinase (SYK) in microglia leads to increased Aβ deposition, exacerbated neuropathology and cognitive deficits in the AD model of 5xFAD mice [26]. A large number of molecules mediate the bidirectional communication between microglia and neurons in the healthy brain. On the one hand, neurons influence the function of microglia through neurotransmitters such as ADP, adenosine, γ-aminobutyric acid (GABA), glutamate, dopamine, and migration factor. On the other hand, GDNF and IGF-1 secreted by microglia contribute to the maintenance of neuronal physiological functions [27].

Mitochondria are the metabolic hub and intracellular signaling platform of microglia cells that are responsible for providing energy, secreting immune signals and growth factors. However, reduced mitochondrial mass and activities and low mitochondrial turnover rates in microglia cells have been found in AD patients. Dysregulation of mitochondrial homeostasis produces excessive ROS that polarizes microglia into M1 type, releases inflammatory factors, induces neuroinflammation, and exacerbates the progression of neuronal damage and cognitive impairment [28,29]. It has been identified in genome-wide genomics that more than 30 loci are associated with microglia and inflammatory factors in AD, among which myeloid TREM2 is a regulator of mitochondrial metabolic adaptation and only expressed in microglia cells. TREM2 is highly expressed in AD patients’ brains [30]. Blocking the activity of this pathway in mouse models of AD has shown to protect synapses by preventing synaptic degeneration and memory impairment [31,32]. Regulation of TREM2-mTOR signaling can increase the phagocytosis of microglia in AD. SHIP1 that is predominantly expressed in microglia inhibits TREM2 signaling and is the only target of the TREM2-mTOR signaling pathway for the treatment of AD at present [33]. Lack of TSPO protein on the outer mitochondrial membrane (TOM) inhibits both oxidative phosphorylation and glycolysis, resulting in overall metabolic defects in primary microglia and increased fatty acid oxidation in steroidogenic cells. In the mouse model of AD, increasing the expression TSPO protein in microglia has been shown to reduce oxidative stress, improve mitochondrial respiration, and reduce inflammatory markers [34].

2.3. Influences of astrocytic mitochondrial homeostasis of AD

The telopods of astrocytes surround almost the entire cerebrovascular system, forming the blood-brain barrier (BBB) that regulates the transport of glucose in brain. Astrocytes regulate glutamate and ion homeostasis, cholesterol and sphingolipid metabolism, and respond to environmental factors, all of which are implicated in neurological disorders [35]. Astrocytes undergo a series of morphological/biological changes during aging, disease and injury. Beneficial astrocytes can release a series of cytokine factors to promote synapse establishment and repair to maintain normal cognitive function [36]. Whereas, neurotoxic A1-reactive astrocytes are prevalent in the brain of AD patients, which release a variety of cytokines to promote chronic neuroinflammation and induce cell death [37]. The function of astrocytes is inextricably linked to the interaction of microglia [38]. On the one hand, IL-1α, TNF-α and C1q secreted by microglia induce the transcriptional responses of astrocytes, and microglia in AD release mitochondrial fragments triggering the reactive conversion of astrocytes to A1 type, which produces neurotoxic factors and large amounts of inflammatory factors, triggering neuronal death [29]. On the other hand, astrocytes regulate the clearance of Aβ plaques by microglia through the secretion of IL-3 cytokines. IL-3Rα⁺ microglia increase sensitivity to IL-3 by expressing IL-3Rα, a unique mitochondrial TREM2-dependent population. By continuously injecting IL-3 into the lateral ventricles of 5xFAD mice for one month, the number of Aβ plaques and the level of soluble Aβ were significantly reduced, and the short-term memory was improved [39].

Mitochondria in astrocytes consume 20% of the brain's oxygen, the dysfunction of which leads to insufficient energy production, calcium dysregulation, glutamate dysregulation and inflammatory responses. Levels of NADPH oxidase type 4 (NOX4) are elevated in astrocytes with damaged cortical areas in AD patients, which leads to oxidative stress and mitochondrial fragmentation through inhibition of intracellular antioxidant responses and mitochondrial respiration. Oxidative stress induces lipid peroxidation and promotes iron death in astrocytes, which is an important molecular mechanism of mitochondrial metabolic disorders in astrocytes of AD patients [40]. Studies have found that impaired mitochondrial fragmentation and autophagy in astrocytes of the hippocampal dentate gyrus induce neuronal dysfunction and memory loss in AD [41]. Researchers selected rapamycin to positively modulate the Parkin/PINK1 pathway to induce autophagy and promote the formation of autophagic vesicle, which reduced Mfn1 and Parkin levels in cells, restored mitochondrial metabolism, and corrected the pH of the mitochondrial matrix without affecting cell numbers [42]. Reduced brain glucose uptake is associated with cognitive impairment, and studies have shown that endogenous glucagon-like peptide-1 receptor (GLP-1R) signaling in astrocytes maintains mitochondrial homeostasis and is rely on FGF21 for the regulation of glucose metabolism [43]. Astrocytes can deliver mitochondria to neurons in need, therefore neuroprotection in AD can be achieved using mitochondrial therapy in astrocytes [44]. Ca²⁺-CD38-cADPR signaling pathway is necessary for the release of mitochondria-containing vesicles from primary astrocytes to neurons. By upregulating CD38 in astrocytes with CRISPR/Cas9 technology, it was possible to restore neuronal energy production and the release of mitochondria-containing vesicles [45]. Interestingly, induction of neuron-enriched expression of antioxidant mitochondrial proteins such as superoxide dismutase 1 (SOD1) in astrocytes by CRISPER technology promotes the transformation of astrocytes into neurons, and this astrocyte-neuron conversion has great potential for neuronal replacement therapy [46].

2.4. Influences of oligodendrocytic mitochondrial homeostasis of AD

Oligodendrocytes are differentiated from oligodendrocyte progenitor cells (OPCs), which form myelin to wrap around nerve axons and account for approximately 20% of the cell population in brain. Extensive myelin alterations and disruptions were observed in both the brains of patients who died of AD and in mouse models, including extensive myelin loss, reduced number of oligodendrocytes, focal demyelination due to Aβ plaques, and increased proliferation of OPCs [47]. The degeneration of formed myelin is evident in AD brains, but the formation of new myelin is significantly increased, suggesting that myelin regeneration has been compensated by extensive demyelination, but still not enough to compensate for the loss of total myelin [48]. Promoting myelin formation in the brains of AD mice can significantly improve AD-related cognitive dysfunction [49,50]. Using large-scale single-cell RNA sequencing (MARS-seq), investigators identified a disease-associated oligodendrocyte signature (DOLs) associated with brain pathological states in the AD model of 5xFAD mice. Oligodendrocytes expressing key DOL markers (SERPINA3N/SERPINA3) were enriched near Aβ plaques, and expression was associated with cognitive decline [51]. Macrophages in CNS are associated with the health of myelin. Studies have found that depletion of microglia in adult mice by the addition of CSF1R inhibitors to the diet results in myelin hyperplasia and myelin degeneration, suggesting that microglia are required for the maintenance of myelin after formation. Microglia-oligodendrocytes link through the TGFβ1-TGFβR1 axis to regulate the health of myelin. Microglia are promising therapeutic targets for dysmyelination of myelin growth and integrity [52].

Myelin degeneration and white matter loss due to death of oligodendrocytes are early events leading to cognitive impairment in AD. Mature oligodendrocytes in AD patients and AD mice exhibit NLRP3-dependent GSDMD-associated inflammatory damage with demyelination and axonal degeneration. Drp1 hyperactivation in mature oligodendrocytes inhibits glycolysis through inhibition of hexokinase 1 (HK1), which in turn activates the NLRP3 inflammasome. These findings suggest that the lack of glycolysis in oligodendrocytes plays a causal role in the development of AD, and that the Drp1-HK1-NLRP3 signaling axis in mature oligodendrocytes may be a key mechanism and therapeutic target for demyelination, white matter degeneration, and cognitive impairment in AD [53]. In addition to the important role for myelin formation, oligodendrocytes are able to provide direct glycoproducts to axons that are used as fuel to help axonal mitochondria during hyperactive phase [54].The development of AD is accompanied by abnormal axonal mitochondrial energy production, and it was found that oligodendrocytes transduce the deacetylase Sirtuin 2 (SIRT2) into mature neuronal axons by releasing exosomes that regulate the levels of axonal mitochondrial protein ANT1/2 acetylation, which affects the biological processes of axonal energy metabolism [55].

3. Mechanisms of dysregulated mitochondrial homeostasis for AD

As already mentioned in the introduction, cells maintain mitochondrial homeostasis through a variety of control mechanisms, including mitochondrial energy metabolism, mitochondrial dynamics, and mitochondrial quality control, etc. In AD, the dysregulation of mitochondrial homeostasis manifests in multiple mechanisms: increased ROS production and imbalanced calcium homeostasis due to mitochondrial oxidative stress, upregulation of mitochondrial splitting proteins and downregulation of mitochondrial fusion proteins, impaired mitochondrial biogenesis and mitochondrial clearance, etc. The following part explores the pathogenesis of dysregulation in the mitochondrial homeostasis of AD with the aim of providing new targets for AD therapy.

3.1. Mitochondrial oxidative stress in AD

As the main site of cellular material metabolism, mitochondria take the electron transport chain (ETC) as a generator set, and take nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide reduced (FADH2), the high-energy substances released from catabolic reactions such as glycolysis, as raw materials to generate ATP through oxidative phosphorylation (OXPHOS) [56]. Mitochondria also play the role of intracellular “calcium reservoir”, maintaining the dynamic balance of calcium ions in cytosol and participating in intracellular calcium signaling. Mitochondrial oxidative stress is due to abnormal oxidative phosphorylation showing as the high level of ROS and overloaded calcium ions in mitochondria, which are key factors in the dysregulation of mitochondrial homeostasis in AD [57]. Mitochondrial oxidative stress is associated with ferroptosis. The tricarboxylic acid (TCA) cycle in mitochondria is a central hub regulating fatty acid catabolism and synthesis as well as OXPHOS, which is also the main point of ferroptosis [58]. Mitochondria, the complex synthesis site of heme and Fe/S protein, are always exposed to the environment of high iron flow, and ROS produced by mitochondria will transform more toxic free radicals with iron through Fenton reaction. Mitochondrial ferritin (FtMt) is a key mitochondrial iron storage protein that acts as an iron oxidase and catalyzes the conversion of Fe²⁺ to Fe³⁺. High expression of FtMt reduces the level of free iron pool by regulating the intracellular redistribution of iron, thus inhibiting Fenton reaction, reducing ROS and reducing oxidative stress damage [59,60]. FtMt prevents ROS overproduction and maintains energy metabolism by enhancing mitochondrial bioenergy and regulating glucose metabolism through pentose phosphate pathway [61].

The progression of AD is associated with oxidative damage of brain. As the level of the key mitochondrial oxidative enzyme 3-nitrotyrosine increase with age, oxidation leads to mitochondrial dysfunction and ultimately to cognitive impairment [62]. Increased intake of antioxidant compounds can help the body reduce ROS. One of the mitochondrial ETC impairments is the elevated NADH/nicotinamide adenine dinucleotide (NAD+) ratio. Normally, electrons carried by NADH in the cytoplasm are passed to the ETC for consumption and regeneration of NAD+, while ETC shutdown, NADH continues to accumulate in the cytoplasm, resulting in an elevated NADH/NAD + ratio, which inhibits glycolysis and threatens the survival of neurons. Therefore, glycerol-3-phosphate (Gro3P) biosynthesis, the NAD + regeneration pathway can be used to alleviate ETC damage in AD [63]. Recently, more attentions are paid to calcium homeostasis regulator family protein (Calhm) in AD research. Calhm1 controls calcium homeostasis and increases neuronal resistance against Aβ toxicity, in which P86L mutations are associated with the incidence of AD. Calhm2 that regulates the inward flow of calcium ions and inflammatory activation in microglia cells may be a therapeutic target for AD [64]. By knockdown of the highly expressed Calhm2 in 5 × FAD mice, the pro-inflammatory activity is inhibited and the phagocytic activity of microglia is enhanced. Thus, phagocytosis and inflammation are restored in balance, significantly reducing Aβ deposition and alleviating cognitive impairment.

3.2. Imbalanced mitochondrial dynamics in AD

Mitochondria are highly dynamic organelles that divide and fuse continuously in response to the physiological environment, and this fission-fusion equilibrium maintains the normal function of the cell. At any time during the cell cycle, a mitochondrion may divide into two separate mitochondria. At the same time, mitochondria fuse, in which both the inner and outer membranes of the mitochondria rupture and rejoin to form a complete mitochondrion. Currently known proteins associated with mitochondrial dynamics include Drp1, mitochondrial fission protein 1(Fis1), Miro, optic atrophy protein 1 (Opa1), fusion protein 1/2 (Mfn1/2), mitochondrial dynamics proteins of 49 and 51 kDa (MiD49 and MiD51, respectively), and mitochondrial fission factor (Mff), etc.

Drp1 translocates from the cytoplasm to TOM recruiting by Fis1 located in TOM, or binding to Mff when the mitochondria send a fission signal, thus initiating the fission process. Mitochondrial fusion is controlled by three GTPase proteins, Mfn1 and Mfn2 at TOM and Opa1 at the inner mitochondrial membrane (TIM). The C-terminal part of Mfn1 mediates the oligomerization of Mfn2 molecules of adjacent mitochondria and promotes mitochondrial fusion. The fusion genes enhance the oxidative phosphorylation function of mitochondria, while the division genes induce apoptosis by promoting Cyt C release. Recent studies have found that mitochondrial fission occurs in two conditions [65], one occurs in healthy mitochondria, which break in half down the middle when they need to increase in number, and the other occurs in damaged mitochondria, which divide from the ends to create one large and one small segment, and the damaged smaller part is eliminated. Excessive mitochondrial splitting leads to the loss of mitochondrial membrane potential, ETC chain uncoupling and ROS burst, exacerbating oxidative stress in mitochondria [57]. Clinical studies have observed a decrease in the expression of mitochondrial fusion proteins such as Mfnl, Mfn2 and Opal, while a significant increase in the expression of fission proteins such as Drpl and Fisl in the brains of AD patients than in healthy people [[66], [67], [68]]. The excessive mitochondrial fission in AD contributes to mitochondrial fragmentation and is a key factor in the dysregulation of mitochondrial homeostasis. The fission protein Drp1 can interact with Aβ and lead to increased production of ROS, which in turn activates Drp1 and Fis1, causing excessive mitochondrial fragmentation, impaired mitochondrial transport, and ultimately synaptic dysfunction. Further studies revealed that the interaction of Drp1 with p-Tau protein enhanced the activity of Drp1, leading to excessive mitochondrial division in AD patients. It can be seen that the intervention of mitochondrial dynamics in the early stages of AD pathogenesis before Aβ deposition and NFTs formation, may prevent or even reverse the onset and development of AD [69].

3.3. Malfunctioning mitochondrial quality control in AD

Mitochondria, as the energy and metabolic center of cells, have a relatively independent quality control system. Mitochondrial quality control refers to the process of generation of new mitochondria and selective removal of damaged/aged mitochondria. The production of new mitochondria depends on mitochondrial biogenesis, and a series of transcription factors and transcriptional co-activators are involved in the regulation of mitochondrial biogenesis. Selective clearance of damaged/aging mitochondria relies on the coordinated operation of the molecular chaperone protein system and two protein hydrolysis systems, namely the ubiquitin-proteasome system and the autophagy-lysosome system.

3.3.1. Impaired mitochondrial biogenesis in AD

Mitochondria have a half-life of approximately 2–3 weeks and are in a dynamic state in the cell. Mitochondrial biogenesis refers to the activation of different signaling pathways for the synthesis of new mitochondria, including the tightly regulated processes of mitochondrial DNA (mtDNA) replication, transcription, the synthesis and assembly of lipid and protein [70]. Four main mitochondrial biogenesis -related genes involve peroxisome proliferator-activated receptor (PPAR), peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), nuclear respiratory factor 1/2 (Nrf-1/2) and mitochondrial transcription factor A (TFAM). PGC-1α is an important regulator of mitochondrial proliferation and oxidative function that is responsible for the induction of mitochondrial fatty acid and ferrous heme biosynthetic pathways. PGC-1α also activates the downstream Nrf-1 gene, which is involved in the regulation of mitochondrial respiratory genes. Nrf-1 and Nrf-2 bind to co-transcriptionally activate transcription factor B1 (TFB1M), transcription factor B2 (TFB2M), and TFAM, thereby regulating mitochondrial gene transcription and protein expression [71].

mtDNA encodes 37 genes, 22 tRNAs, and 13 proteins necessary for ATP synthesis in humans. It is multicopy, circular, and double-stranded with 16568 base pairs. Since mtDNA is not packaged or protected by histones, it is exposed to ROS for extensive periods of time. The dysfunction of mitochondrial biogenesis caused by mtDNA mutations will lead to AD [72]. mRNA and protein levels of the mitochondrial biogenesis factors PGC-1α, Nrf-1, Nrf-2 and TFAM were found to be reduced in brain tissue and cell models of AD patients. The silent information regulator SIRT1 is an NAD⁺-dependent histone deacetylase that upregulates PGC-1α by deacetylation to activate mitochondrial biogenesis. In animal models of AD, SIRT1/PGC1-α expression is significantly decreased and PGC-1α-Nrf-TFAM pathway is downregulated, with its alterations in mitochondrial proteins and mitochondrial network morphology, suggesting a defective mitochondrial biogenesis function in AD [73].

3.3.2. Impaired mitochondrial clearance in AD

The clearance of damaged/aging mitochondria operates primarily through the molecular chaperone protein system with two protein hydrolysis systems, namely the ubiquitin-proteasome system (UPS) and the autophagy-lysosome system. Most structural and functional proteins of mitochondria are encoded by nuclear genes and translated by ribosomes in the cytoplasm. The synthesized precursors enter the interior mitochondria via channels formed by the translocase of TOM-the translocase of TIM complex [74]. Chaperone molecules in mitochondrial matrix promote the folding of nuclear and mitochondrial proteins, and they degrade misfolded or aggregated proteins [75]. In mitochondria, UPS ensures the entry of synthesized polypeptides and removes poorly imported polypeptides and destroys mislocated proteins. Mitophagy is a form of selective cellular autophagy. After depolarization damage stimulated by factors such as cellular senescence, mitochondria are specifically wrapped by bilayer membrane structures and then fused with lysosomes for degradation. Mitophagy reduces the aggregation of damaged mitochondria and helps to balance the quantity and quality of mitochondria. There are three known mechanisms of mitophagy, namely the PINK1/Parkin pathway that regulates mitochondrial depolarization, the BNIP3/NIX pathway associated with reticulocyte maturation, and the autophagy mediated by the mitochondrial autophagy receptor protein FUN14 domain containing 1 (FUNDC1) on TOM [76]. The FUNDC1 protein located on TOM is able to interact with the intracytoplasmic molecular chaperone protein HSC70, through which damaged or misfolded proteins in the cytoplasm can be recruited to the mitochondria and subsequently enter the mitochondrial matrix through the TOM-TIM complex and be degraded by the matrix mitochondrial protease LONP1 [77]. Mitochondrial clearance and mitochondrial biogenesis synergistically regulate mitochondrial homeostasis, in which the PGC-1α-Nrf-1-FUNDC1 pathway plays a key role. The key mitochondrial biogenesis factors PGC-1α and Nrf-1 directly regulate FUNDC1 expression, while FUNDC1 deletion inhibits mitochondrial biogenesis [78].

Dysfunctional mitophagy in AD patients leads to the accumulation of mitochondrial autophagosomes in neurons, ultimately causing impaired synaptic plasticity and cognitive deficits. PINK1/Parkin mitophagy plays a leading role in reversing cognitive deficits and preventing AD [79]. The loss of Parkin protein that maintains mitochondrial homeostasis leads to neuroinflammation and cognitive degeneration. PINK1 as a key protein in the Parkin pathway, is induced by PTEN. The depolarization of mitochondrial membrane leads to the translocation of cytoplasmic Parkin and PINK1 to the membrane following mitochondrial injury. Parkin prevents the formation of extracellular plaque by ubiquitinating intracellular Aβ and induces Beclin-1, USP, USP30, and USP35, which have important roles in regulating Parkin-mediated mitophagy. The mitochondrial fusion protein Mfn2 is associated with the release of translocation signaling, and this translocation is a marker for Parkin-mediated initiation of mitophagy [80]. AMPK and mTORC1 are two key kinases involved in mitophagy initiation upstream. Energy depletion activates AMPK or nutrition deficiency inhibits mTORC1, which results in mitophagy by phosphorylating and activating the ULK1 complex. PtdIns3K is activated by the ULK1 complex and generates pre-autophagic structures by recruiting the effector proteins of PI3P. Mitophagy was found to inhibit the aggregation of Aβ and Tau proteins in AD mice to reverse their cognitive deficits [81]. However, mitophagy agonists are currently scarce and more discovery approaches for efficient agonist are needed.

As shown in Fig. 1 for a summary, the mitochondrial oxidative stress, mitochondrial dynamic imbalance and malfunctioning mitochondrial quality control and their interactions lead to the dysregulation of mitochondrial homeostasis in AD. In-depth study of the mechanism of mitochondrial homeostasis will be able to provide new ideas for AD drug development.

Fig. 1.

The dysregulation of mitochondrial homeostasis in AD brain (by Figdraw). A: Mitochondrial oxidative stress. B: Imbalanced mitochondrial dynamic. C: Impaired mitochondrial biogenesis. D: Dysfunctional mitophagy.

4. Drugs for maintaining mitochondrial homeostasis in AD

Therapeutic agents that target mitochondria for AD include antioxidants, calcium channel blockers, fission inhibitors and AMPK activators [82]. The following discusses the drugs for AD therapy that are summarized in Table 1 and classified by mitochondrial energy metabolism, mitochondrial dynamics and mitochondrial quality control.

Table 1.

Drugs classified by mitochondrial energy metabolism, mitochondrial dynamics and mitochondrial quality control for AD therapy.

Classification	Drug	Mechanism	Ref.
Improve mitochondrial energy metabolism	Vitamin E, vitamin C, alpha-lipoic acid, MitoQ, curcumin, astaxanthin, ginkgo biloba preparations	Reduce superoxide radicals and scavenge hydrogen peroxide	[[83], [84], [85], [86]]
	Cyclosporin A	mPTP inhibitor	[87]
	Nicardipine	Calcium channel blocker	[64]
	sodium rutin	Enhances the level of mitochondrial oxidative phosphorylation	[89]
	Batrachocholine	Improve mitochondrial ATP metabolism and modifying antioxidant proteins	[90]
	Ginsenoside Rb1	Inhibition of mitochondrial complex I activity	[91,92]
Balance the mitochondrial dynamics	Mdivi-1	Repairs mitochondrial breakage and distribution defects	[76]
	SS-31	Acts on the cardiolipin of mitochondrial inner membrane	[93,94]
	Butylphthalide	Balances the mitochondrial dynamics	[95]
Coordinate mitochondrial quality control	Rapamycin, Torin1 and niclosamide	Downregulation of the mTORC1 pathway	[96]
	Resveratrol	Sirtuin proteins activator	[98]
	quercetin	Promotes PGC-1α to regulate mitochondrial homeostasis	[99]
	Ginsenoside RC	Activates the SIRT1-pgC1α pathway	[100]
	Berberine	AMPK activator	[97]
	Metformin	Enhances the transcriptional activity of mitochondrial biogenesis factor Nrf-2 and thus upregulating the expression of peroxidase GPx7	[101]
Restore mitochondrial autophagy	Melatonin	Improves mitochondrial-lysosomal fusion	[102]
	Resveratrol	Upregulates Parkin-induced mitochondrial autophagy	[103]
	β-caryophyllene	Increase the expression of PINK1, Parkin and Beclin-1	[104]
	Lithium urate, tomatidine, NAD+ nucleoside	Mitophagy enhancers	[105]
	T-271	Mitophagy inducer	[106]

4.1. Drugs for improving mitochondrial energy metabolism in AD

Currently, impaired mitochondrial energy metabolism is mainly treated with antioxidants to reverse oxidative stress and restore mitochondrial function. Antioxidants that can reduce superoxide radicals and scavenge hydrogen peroxide include vitamin E, vitamin C, alpha-lipoic acid, MitoQ, curcumin, astaxanthin and ginkgo biloba preparations etc [[83], [84], [85], [86]].

Pathological persistent opening of mPTP in mitochondria is an important cause of mitochondrial dysfunction, meaning mitochondrial membrane potential collapse and uncoupling of oxidative phosphorylation. Cyclosporin A (CsA) inhibits the opening of mPTP in mitochondria by interfering with cyclophilin D (CyPD) on TIM [87], thereby reducing mitochondrial calcium overload and ROS production to decrease mitochondrial oxidative stress [88]. Calcium dysfunction leads to neuroinflammatory responses by activating microglia. Nicardipine is an effective calcium channel blocker that can significantly inhibit the activation of microglia induced by lipopolysaccharide (LPS)/IFN-γ [64].

Many natural products can improve the energy metabolic function of mitochondria. For instance, sodium rutin, a derivative of natural flavonoids, enhances the level of mitochondrial oxidative phosphorylation in microglia to promote the phagocytosis and clearance of Aβ [89]. Batrachocholine (DAU) is an isoquinoline alkaloid that protects neurons and inhibits apoptosis by improving mitochondrial ATP metabolism and modifying antioxidant proteins, therefore DAU rescues abnormal mitochondrial energy metabolism and improves synaptic function [90]. Regulation of NADH dehydrogenase activity in mitochondrial complex I may be a potential intervention to limit the production of mitochondrial ROS induced by reperfusion [91]. Ginsenoside R_b1 reduces the production of ROS under ischemia by transient inhibition of mitochondrial complex I activity, alleviates mitochondrial oxidative damage, and prevents astrocyte activation [92].

4.2. Drugs for balancing mitochondrial dynamics in AD

Mdivi-1, a mitochondrial division inhibitor, improves mitochondrial function and cell survival by enhancing mitochondrial fusion activity, repairing mitochondrial breakage and distribution defects, and increases biogenesis and synaptic protein production in AD mice [76]. D-Arg-2′6′-dimethylTyr-Lys-Phe-NH2 (SS-31) is a mitochondria-targeting short peptide that acts on the cardiolipin of mitochondrial inner membrane [93], which corrects the imbalance of mitochondrial dynamics by the down-regulation of mitochondrial fusion proteins Drp1 and Fis1 and up-regulation of the mitochondrial fusion proteins Mfn1/2 and Opa1 in AD mice [94]. A study found that butylphthalide could exert neuroprotective effects on AD mice and improve their spatial learning by balancing mitochondrial dynamics [95].

4.3. Drugs for coordinating mitochondrial quality control in AD

Regular mitochondrial function and internal environmental homeostasis require close coordination between mitochondrial biogenesis and clearance. Compounds that have biogenic and autophagic activities are expected to be effective therapeutic interventions for AD.

4.3.1. Drugs for restoring mitochondrial biogenesis in AD

Drug molecules such as rapamycin, Torin1 and niclosamide are targeted to down-regulate the mTORC1 pathway to promote mitochondrial biogenesis. Resveratrol, berberine, and quercetin are commonly used AMPK activators [96]. Resveratrol, a natural polyphenol found in a variety of plants, has been proved to inhibit phosphodiesterase PDE4 as well as activate SIRT1, and quercetin regulates mitochondrial homeostasis by clearing mitochondrial ROS and promoting PGC-1α [[97], [98], [99]]. Ginsenoside R_C is a SIRT1 activator, which can promote glucose uptake, pyruvate metabolism and mitochondrial biosynthesis by activating the SIRT1-PGC1α pathway [100].Metformin was found to delay cellular senescence by enhancing the transcriptional activity of mitochondrial biogenesis factor Nrf-2 and thus upregulating the expression of peroxidase GPx7 [101]. However, it remains to be determined whether these benefits observed in animal models can be replicated in humans.

4.3.2. Drugs for restoring mitochondrial autophagy in AD

Melatonin restores mitochondrial autophagy by improving mitochondrial-lysosomal fusion, thereby improving mitochondrial function, attenuating pathological Aβ deposition, and improving cognitive function [102]. Natural compounds such as resveratrol can up-regulate Parkin-induced mitochondrial autophagy [103], and β-asarone can increase the expressions of PINK1, Parkin and Beclin-1, and also regulate the autophagy capacity of mitochondria, playing a neuroprotective role [104]. Mitophagy enhancers can effectively improve neuronal function and learning and memory function of animal models. Currently, mitochondrial autophagy enhancers under investigation include lithium urate, tomatidine and NAD⁺ nucleoside, etc. Based on AI fused with multidimensional molecular information of virtual screening algorithm, researchers successfully identified two new neuronal mitochondrial autophagy inducers, Kaempferol and Rhapontigenin, and confirmed the effectiveness of the drugs in animal models [105]. Researchers discovered the compound T-271 that can induce mitochondrial autophagy using probes combined with high-throughput screening technologies [106].

5. Mitochondria-targeted therapeutic strategies

For the poor therapeutic effect of drugs in AD therapy, the lack of action selectivity and the difficulty in reaching the target site to regulate mitochondrial homeostasis are the key reasons. The bilayer membrane structure of mitochondria is the main barrier for drug delivery, of which TOM is permeable only to molecules with low and medium molecular weight, and the TIM is poorly permeable. Therefore, it is of great importance to explore efficient mitochondria-targeted drug delivery strategies to achieve precise drug delivery and modulation for the treatment of AD.

There are various approaches to target mitochondria, among which nanoparticles with adjustable particle size and easy surface modification have advantages of improving targeting efficiency and increasing drug bioavailabilities, becoming an effective strategy for the treatment of AD [107]. Considering that mitochondrial membranes are rich in cardiolipids, researchers have synthesized liposomal and polymeric nanoparticles. Based on the strong negative potential of the TIM, researchers have synthesized mitochondria-targeting cationic compounds such as diquantinium chloride (DQA) and triphenylphosphine (TPP) derivatives to selectively target mitochondria [108]. The FDA-approved cationic lipid DQA can be used to create DQAsomes, a liposome-like structure that attaches to pDNA via electrostatic interactions, yet DQAsomes have a low efficiency of 5% at transfecting mitochondria. Choi and coworkers combined DQA with 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphate ethanolamine (DOPE), which improved cellular uptake and endosomal escape, enhancing the expression of mitochondrial gene [72]. Lipophilic cationic TPP can enhance the electrostatic interactions between delivery carriers and mitochondria. Faria and colleagues combined TPP with poly(ethylene glycol)-poly(ethyleneimine) (PEG-PEI) via amide coupling and prepared PEG-PEI-TPP nanoparticles that significantly enhanced the mitochondrial internalization of pDNA [109]. Hu et al. designed mitochondria-targeted functionalized molybdenum disulfide quantum dots (TPP-MoS2 QDs) nanoparticles. The bifunctional nanoparticles have the ability to pass through the blood-brain barrier, escape from lysosomes, and target mitochondria, attenuating neuroinflammatory reactions by converting microglia from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype [110]. It is possible to modify nanoparticles with different molecules to form dual targeted systems for neurons and mitochondria. Researchers designed a PEG-PLA micelle system (CT-NM) co-modified by neural cell adhesion molecule-mimetic peptide C3 (neuron-targeting functional group) and TPP. CT-NM has excellent brain neuronal mitochondrial targeting that can efficiently deliver resveratrol into brain mitochondria. The AUC_0–24h value of CT-NM reached 105 times than free drugs, which can effectively reduce oxidative stress and repair mitochondrial fission/fusion imbalance, thus protecting synapses [111]. Researchers devised a strategy to neuronal mitochondria by loading functional antioxidants into red blood cell membrane (RBC) camouflaged human serum albumin nanoparticles (T807/TPP-RBC-NPs), followed by inserting DSPE-PEG₃₄₀₀-T807 (blood-brain barrier crossing and neuronal cells binding) and DSPE-PEG₂₀₀₀-TPP (mitochondrial targeting motif), and co-localization experiments demonstrated that the functionalized NPs had better mitochondrial localization [112].

Although drugs have difficulty crossing the mitochondrial bilayer structures, most mitochondrial proteins are encoded by nuclear genes and then enter into mitochondria via mitochondrial precursor proteins (MPPs) that generate signals for mitochondrial targeting. Currently, two common features are shared in known MPPs that can successfully import proteins into mitochondria, namely, essential homologous amino acid sequences (including arginine) and amphipathic N-terminal regions. Dendritic lipopeptide molecules (DLP) modified with arginine residues were developed as mitochondrial targeting agents, which amplify the interaction with mitochondria by mimicking the structural domain of basic amino acid in MPPs. The stearoyl as a hydrophobic chain segment increases lipophilicity and strengthens the affinity with mitochondrial membrane. Compared with TPP modified mitochondrial targeting system, the DLP-based delivery system exhibited 3.7-fold higher mitochondrial targeting efficiency that could be considered as a delivery strategy targeting mitochondria [113]. In mitochondria-mediated energy production and cellular functions, NAD+ in mitochondria is a key molecule, and all NAD+ is transported from the cytoplasm. Mitochondrial protein SLC25A51 (also known as MCART1) is the transport protein of NAD+ in mammalian cells [114], which can be applied in delivery system to target mitochondria. The pre-sequence translocase-associated import motor complexes (PAM) are required for the introduction of matrix targeted proteins, bind to TIM and interact with transporters to aid their introduction [115]. The mitophagy receptor protein FUNDC1 localized in TOM can interact with the molecular chaperone protein HSC70 localized in the cytoplasm. Damaged or misfolded proteins in the cytoplasm can be recruited to the mitochondria through this interaction and then enter the mitochondrial matrix via the TOM-TIM complex [77]. Small heat shock proteins (sHSP) in mammalian cytoplasm can be transported into mitochondria and localized in mitochondrial membrane space (IMS) to perform molecular chaperone functions. sHSP member HSPB1 transport into mitochondria is independent of mitochondrial membrane potential [116].

Lin et al. developed two fluorinated amphiphilic analogues, and self-assembled core-shell micellar nanocarriers with potential-independent mitochondrial targeted delivery [117]. The fluorinated amphiphilic micelles internalized by cells preferentially aggregate in mitochondria. The fluorinated amphiphilic nanocarriers achieve mitochondrial targeting through strong affinity with cardiolipin, which are electrically neutral, and their targeting to mitochondria does not depend on electric potential. This electrically neutral fluorinated amphiphilic molecule is expected to develop into a reliable mitochondrial targeting tool. Some researchers have prepared mitochondria-targeting nanoparticles by using SS-31 as the targeting ligand [118,119], SS-31 is a positively charged mitochondrial targeting short peptide under physiological conditions, with 5000 times higher affinity for the TIM than other organelle membranes. The accumulation of SS-31 in mitochondria is independent on the mitochondrial transmembrane potential with low toxicity that is a promising mitochondrial targeting material for AD [93].

It shows that the mitochondrial targeting strategies for AD is still in its infancy due to multiple barriers in vivo, including the blood-brain barrier, cellular barrier and mitochondrial membrane barrier, etc. The mitochondrial targeting delivery systems for AD are hard to design and translate to the clinic if the delivery systems are complex. Currently, TPP derivatives are mostly used as mitochondrial targeting ligands, but the enter into mitochondria of TPP derivatives is dependent on the transmembrane potential of mitochondria that can damage mitochondria [120], causing damage to brain cells. In conclusion, delivery systems should be pay more attention to carrier materials, for instance, “green” carrier materials simplify the delivery strategy and facilitate clinical translation, and safer carrier materials avoid toxic functions on neuronal cells.

6. Conclusion and future perspectives

As the dysregulation of mitochondrial homeostasis occurs in the early stage of AD, the maintenance of mitochondrial homeostasis holds great promise in AD therapy, yet clinical application is still a ways off, and more work needs to be done on the following three aspects.

First, in-depth study the mechanisms involved in mitochondrial homeostasis. Clarifying the mechanisms involved in the occurrence of dysregulation and maintenance of mitochondrial homeostasis in AD will enable the development of new targeted therapies and the delivery of new drugs that benefit AD patients. However, the specific mechanisms are still poorly understood, reminding researchers to excel in more advanced techniques and methods to accelerate the development of new targets for mitochondrial homeostasis. Traditional microscopy with a resolution of 300 nm fails to observe fine structures in mitochondria. The super-resolution technique of Stimulated Emission Depletion Microscopy (STED) has ultra-high resolution and fast imaging speed capable of observing mitochondria in living cells, the dynamic changes of mitochondria at long time range and the structure of mitochondrial cristae [121]. Mitochondria labeling by autophagy probe is hard to fully achieve precise localization, prone to false positive signals and proteins that are fully acid-resistant is required. Therefore, researchers constructed the mitochondrial autophagy probe mito-SRAI by high throughput screening of an acid-tolerant fluorescent protein TOLLES. Mito-SRAI is well suited for monitoring mitochondrial autophagy in live samples and isolated tissues, and for quantitative studies [106].

Second, develop effective mitochondria-targeted delivery strategies. Side effects commonly occurred in strategies targeting mitochondria, which require safe carrier materials and targeting ligands, and precise drug targets provided by identifying special proteins involved in mitochondrial production or degradation. Currently, biomarkers are available with AD mainly Aβ and Tau proteins while relatively few mitochondrial proteins. A total of 10 proteomic datasets were analyzed to quantify 13833, 5941, and 4826 proteins in human cortex, cerebrospinal fluid, and serum, respectively. Results demonstrated that nearly 60% of the potential protein markers of AD are mitochondrial proteins, with the most promising proteins being SMOC1, TAU, GFAP, SUCLG2, PRDX3, and NTN1 [122]. An additional way to achieve a neuroprotective effect is to replace unhealthy mitochondria with healthy ones or degrade unhealthy ones. While mitochondrial transplantation has not been tested in clinical trials for similar neurological disorders, it has shown neuroprotective effects in mice [123]. Further research is required on techniques aimed at improving mitochondrial health in neurons.

Third, treat AD patients individually. Considering the different types of AD, an appropriate therapy should be selected instead of blind treatment. AD patients can be divided into familial AD and sporadic AD according to the presence or absence of a family history. Familial AD is induced by autosomal dominant inheritance, and the onset is mostly 40–65 years old. Individuals with sporadic AD generally attack after age 65, accounting for 95% of the total AD population [124]. Mitochondrial homeostasis is the early impairment factor contributing to the pathophysiology of sporadic AD. Mitochondrial infusion has shown great potential both preclinically and clinically [125]. An increasing number of studies have attempted to improve mitochondrial function in aging and age-related diseases [126]. From these attempts, methods involving mitochondrial isolation, blood transfusion and transplantation have become the focus of attention. In particular, mitochondrial infusion refers to the injection of mitochondria into the bloodstream from healthy tissues or tissues affected by injury, disease or aging. To investigate whether healthy young mouse mitochondria extracted from the liver can improve mitochondrial function in older animals, mitochondria isolated from young C57BL/6 mice were injected intravenously into older mice of the same strain. This study demonstrated for the first time that mitochondrial infusion significantly improved mitochondrial function by upregulating the protein subunit SDHB of hippocampal mitochondrial complex II in elderly mice [127].

In the near future, breakthrough treatments for AD may be found by in-depth research on the mechanisms of mitochondrial homeostasis, development of effective mitochondria-targeted strategies, and personalized treatment.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

No data was used for the research described in the article.