Prospecting the Interplay between Mitophagy and Diabetic Neuropathy: Uncovering the hidden secrets of the disease pathology

Abstract

Mitophagy, the cellular process of selectively eliminating damaged mitochondria, plays a crucial role in maintaining metabolic balance and preventing insulin resistance, both key factors in type 2 diabetes mellitus (T2DM) development. When mitophagy malfunctions in diabetic neuropathy, it triggers a cascade of metabolic disruptions, including reduced energy production, increased oxidative stress, and cell death, ultimately leading to various complications. Thus, targeting mitophagy to enhance the process may have emerged as a promising therapeutic strategy for T2DM and its complications. Notably, plant-derived compounds with β-cell protective and mitophagy-stimulating properties offer potential as novel therapeutic agents. This review highlights the intricate mechanisms linking mitophagy dysfunction to T2DM and its complications, particularly neuropathy, elucidating potential therapeutic interventions for this debilitating disease.

Graphical Abstract.

1. Introduction

Diabetes, characterized by elevated levels of blood glucose, is a prevalent and rapidly escalating disease worldwide. Projections estimate that by 2045, approximately 693 million adults will be affected, marking a significant increase of over 50% from 2017 [1, 2]. The vascular complications associated with diabetes, affecting both macro- and microvascular systems, such as cardiovascular disease, diabetic kidney disease, diabetic retinopathy, and neuropathy, constitute the primary reason for morbidity and mortality among diabetic individuals [3, 4]. These complications pose significant health challenges and incur substantial financial burdens, with disparities in healthcare expenditure and treatment accessibility observed between developed and developing nations [5-7]. Although the intricate mechanisms underlying the vascular damage induced by hyperglycemia remain complex and not entirely elucidated, it is postulated that heightened intracellular glucose levels contribute to generating reactive oxygen species (ROS), thereby initiating various detrimental pathways. The latter include alterations in polyol pathway flux, generation and activation of advanced glycation end products, PRKC (protein kinase C) activation, and modulation of hexosamine pathway flux [8, 9].

Diabetes prevalence has been sharply increased globally, with a staggering 415 million individuals now affected worldwide [10]. This surge in diabetes cases raises significant concerns as it amplifies the occurrence of acute and chronic illnesses among people, thereby exerting profound impacts on the quality of life, healthcare requirements and needs, and economic burdens. Diabetes-related macrovascular complications, including peripheral vascular disease, coronary heart disease, stroke, and microvascular consequences such as neuropathy, retinopathy, end-stage renal disease, and lower-extremity amputations, account for a substantial portion of diabetes-related medical consequences. Additionally, there is an increasing acknowledgment of an expanding array of conditions causally associated with diabetes, encompassing aging-related consequences like dementia, infections, cancers, and liver disease. Present data indicate declining mortality associated with diabetes-related cardiovascular disease [11, 12]; however, it is foreseeable that trends in other diabetes-related complications may become more prominent in the future.

A remarkable portion of diabetes-related complications with clinical syndromes stem from the harm done to the autonomic and peripheral nerve systems. Generally encompassed under the umbrella of neuropathy, such syndromes result from damage to the diffuse and focal nervous system, affecting more than 50% of diabetic individuals [13]. Distal symmetric polyneuropathy is the most prevalent kind of diabetic neuropathy (DN), which mainly affects the hands and lower limbs. This type of neuropathy has a distinctive distribution pattern, often called "stocking and glove." Other diabetes-related neuropathies affect the autonomic nervous system, resulting in impotence, diabetic cystopathy, gastrointestinal dysmotility, and cardiac autonomic neuropathy [14, 15].

Macroautophagy/autophagy is a natural self-preservation mechanism [16, 17], which degrades and recycles parts of a cell at a constitutive basal level and in response to various stress conditions, including fasting, exercise or ketosis, thus rejuvenating cells [16, 18, 19]. Mitophagy, a form of selective autophagy, is particularly geared towards degrading superfluous, damaged or dysfunctional mitochondria. Regarding the molecular process involved, damaged mitochondria are tagged with receptors and ligands, including SQSTM1/p62 (sequestosome 1) and ubiquitin, via the action of PRKN/PARK2 (PRKN RBR E3 ubiquitin protein ligase), a ubiquitin ligase, and PINK1 (PTEN induced kinase 1), a kinase targeting PRKN. The autophagy-related protein MAP1LC3/LC3 (microtubule associated protein 1 light chain 3) is then conjugated to phosphatidylethanolamine/PE, forming phagophores that encapsulate selected mitochondria [20, 21]. The phagophores mature into autophagosomes that deliver the cargo to the lysosome. Increasing evidence indicates that mitophagy is crucial in type 1 and type 2 diabetes, potentially acting as a regulator to eliminate excessive ROS during hyperglycemic conditions [22]. Moreover, experimental diabetic models have observed decreased levels of mitophagy in diabetic retinas and increased oxidative stress in db/db diabetic mice, which are connected to altered levels of proteins relevant to mitophagy, in particular PRKN and PINK1 [23]. To address impairments in diabetic mitophagy flux, restoring mitophagy through mitophagy inducers may hold promise as a possible drug target [20].

Numerous studies have investigated the therapeutic potential of autophagy modulators in diabetic pathogenesis and progression [24-26], aiming to explore their effectiveness as mitophagy inducers. However, mitophagy induction by such autophagy modulators has limited efficiency. Further efforts are needed to delve into mitophagy induction in this context, particularly considering the significant contribution of mitochondrial dynamics to the pathogenesis and initiation of type 2 diabetes [20].

2. Diabetic Neuropathy

DN presents a significant burden on patients, contributing to increased fall risks, pain, and diminished quality of life (Figure 1) [27]. The yearly economic impact of neuropathy in diabetes and its related adverse effects exceeds $10 billion in the United States [28]. The prevalence and/or incidence of neuropathy has been investigated in several studies, although the definition varies. Neuropathy was prevalent in 1% to 4% of the population in two population-based studies that used door-to-door screening; 40% to 55% of these cases were linked to diabetes [14]. A parallel investigation found that over half of the neuropathy cases were linked to diabetes following diagnostic investigations by neurologists [29]. In the Netherlands, neuropathy’s incidence positively correlates with age [30], with rates increasing from under 50 cases for every 100,000 person-years in individuals under the age of 50 to approximately 300 per 100,000 person-years in those over 75, while 32% of all cases are attributed to diabetes [14].

Figure 1.

Diabetic neuropathy: Epidemiology, prevalence, and pathogenesis.

Key predictors of DN consist of diabetes duration and hemoglobin A1c/HbA1c levels, as an estimate of glucose control over a 3-month period of the RBC life [31]. There are often interrelationships between such predictors and other metabolic factors associated with DN, especially in type 2 diabetes mellitus (T2DM), including insulin resistance and hypertension. Population-based research in the Netherlands, Denmark, China, and the United States confirmed the prevalence of obesity among patients with neuropathy [32-36]. Regardless of hemoglobin A1c levels, the metabolic syndromes’ multiple components, including low levels of high-density lipoprotein, abdominal obesity, hypertension, and hypertriglyceridemia, correlate with DN in individuals affected by T2DM [33, 34, 37], as well as in certain groups with type 1 diabetes mellitus (T1DM) [29]. Smoking, alcohol misuse, taller stature, and advanced age constitute other autonomous risk factors for DN development [38]. While different genes have been associated with DN, ACE (angiotensin I converting enzyme) and MTHFR (methylenetetrahydrofolate reductase) polymorphisms have been the subject of many studies in a variety of populations and sizable cohorts [14].

Genetic underpinnings of DN have not been thoroughly understood; therefore, ongoing research within available populations will shed more light on this area [51, 52]. DN represents a distinctive neurodegenerative condition affecting the peripheral nervous system, with a preference for sensory axons, autonomic axons, and motor axons. The mechanism by which diabetes mellitus affects sensory neurons remains a subject of debate. In progressive DN, terminal sensory axons in the periphery undergo a process of withdrawal and “dying back” while the cell bodies remain relatively intact. The “stocking and glove” pattern indicates the initial harm to the longest sensory axons. This is seen when the loss of sensory nerves in the distant parts of the leg occurs before the loss in the limbs closer to the body. Consequently, DN is categorized as length/distance-dependent neuropathy. While not primarily classified as a demyelinating neuropathy, chronic hyperglycemia targets Schwann cells; moreover, demyelination properties may be exhibited in patients with more severe DN [53-55].

The symbiotic relationship between axons and Schwann cells underscores the potential impact of Schwann cell damage on various aspects of axonal function. Schwann cells are critical in controlling axons' cytoskeletal features, influencing protein localization at the nodes of Ranvier and parameters related to axonal trafficking [56]. When Schwann cells fail to support axons adequately. They might find it difficult to supply trophic factors, cytoskeletal support, or to help ribosomes move from Schwann cells to axons. This involves the distal axons' intra-axonal mRNA translation [57]. Schwann cells in mice have vesicles containing ribosomes that can be transferred to axons. These transferred ribosomes are critical in regulating the synthesis of axonal proteins [58]. When axons encounter harm or stress, the transfer of ribosomes maintains axonal integrity [58]. However, it remains unclear if diabetes triggers inherent programs in axons, promoting their degeneration. Intracellular signaling pathways that actively cause axon degeneration have been found during investigations into Wallerian degeneration, with NMNAT (nicotinamide nucleotide adenylyltransferase) emerging as an important controller of this process. Nevertheless, it is still an open question if these pathways are triggered in diabetes [59].

Axonal changes, particularly at their distal termini, are often mirrored by alterations in the neuronal perikarya. In cases of chronic experimental diabetes, sensory neurons in the dorsal root ganglion (DRG) undergo phenotypic modifications, which may play a crucial role in supporting distal axon branches. For instance, chronic T1DM in rats is associated with a progressive reduction in the synthesis and export of NEF (neurofilament) polymers, which serves as a crucial structural framework for the axon. This loss of NEF polymers stems from decreased expression of mRNA encoding NEF [60]. Additionally, researchers who conducted preclinical studies on diabetic rodents found a correlation between endoplasmic reticulum (ER) stress and harm imposed on the peripheral nerve by diabetes [61]. This damage can profoundly affect nerve function. Furthermore, experiments using rodent models, both in vitro and in vivo, have shown that hyperglycemia can alter the function of key molecules associated with plasticity. These comprise TUBB/β-tubulin and GAP43/neuromodulin (growth associated protein 43), as well as the expression patterns of PARP (poly(ADP-ribose) polymerase) [62, 63], and heat shock proteins in the DRG [64].

While the precise procedures underlying nerve injury in DN are still being elucidated, evidence suggests that malfunction within these pathways contributes to irregular protein processing, oxidative harm, and mitochondrial dysfunction, ultimately culminating in peripheral nerve function loss [57]. Supporting this notion, interventions targeting specific molecules within these pathways have demonstrated improvements in nerve function. For instance, modulation of HSP90 (heat shock protein 90) activity improves responses to thermal and mechanical stimuli as well as nerve conduction velocity, possibly by enhancing mitochondrial function [65]. Recent research has revealed that chronic diabetes can lead to numerous changes in mRNA and microRNA within the sensory neurons of the DRG [14]. Notably, sciatic nerves from preclinical models of both type 1 and type 2 diabetes have increased levels of pathways linked to inflammation, bioenergetics, and lipid processing [66, 67].

Moreover, gene expression patterns in peripheral nerves from diabetic patients and mouse models were compared; this analysis revealed several highly conserved pathways related to inflammation, lipid synthesis, and adipogenesis [68]. Specific alterations in DRG and nerve functions associated with DN include disrupted spliceosome function, alterations in the expression of SMN (survival of motor neuron) protein, and increased activity of GW-bodies involved in mRNA processing [69]. Examining long-term diabetic rodent models has been essential in mirroring chronic human disease states. Notably, DRGs exhibit a decrease in local blood flow; however, it is still unclear if this reduction results in neuronal damage or decreased oxygen demand [14].

3. Mitochondrial dysfunction in diabetic neuropathy

3.1. Mitochondrial structure and function

Current theories suggest that mitochondria originated from free-living bacteria and played a crucial role in developing eukaryotic cells via endosymbiosis [70]. The endosymbiotic theory suggests that after early anaerobic eukaryotic cells absorbed primitive mitochondria, a mutually beneficial relationship was established (according to contemporary theories, endosymbiosis with a non-eukaryotic archaeon organism is possible). This relationship was shaped and maintained through considerable negative selection by gene shuffling. Mitochondria significantly enhanced cellular energy production by utilizing the respiratory chain to produce adenosine triphosphate (ATP). Meanwhile, the host cell provided an environment conducive to the bacteria's growth (Figure 2) [71, 72].

Figure 2.

Mitochondrion: Structure and functions.

The outer mitochondrial membrane (OMM) and the inner mitochondrial membrane (IMM) [13] are two phospholipid membranes that surround mitochondria. These membranes form two distinct compartments within the organelle: the matrix and the intermembrane space [73]. The lipid composition, permeability, shape, and properties of transmembrane proteins differ significantly between the OMM and IMM, which indicates the organelle's endosymbiotic origin. Specifically, the lipid composition of the OMM resembles that of eukaryotic cell membranes, whereas the IMM bears similarities to bacterial membranes containing cardiolipin [70].

Unlike nuclear DNA, mitochondrial DNA (mtDNA) is found in many copies within the cell, ranging from 100 to 10,000 copies, depending on certain tissues’ energy requirements [74]. Additionally, there are slight differences between the genetic code of mtDNA and nuclear DNA with changes to the codons encoding methionine and tryptophan. Additionally, only two stop codons are used by mtDNA. Mutations in mtDNA, like those in nuclear DNA, can have significant pathological implications. Mendelian inheritance patterns are not followed by mtDNA, in contrast to nuclear DNA. First, in sexually reproducing organisms, mtDNA is inherited exclusively from the mother [75], precluding transmission of mutant mtDNA to offspring from the father. Second, while each cell has two copies of its nuclear genes, it has multiple copies of its mtDNA. These copies may have the same sequence, a phenomenon called homoplasmy. Conversely, heteroplasmy can arise from mutations in some mtDNA copies due to the inheritance of mutated copies, replication errors, oxidative stress, or insufficient DNA repair mechanisms [74]. The onset and severity of disease phenotypes are significantly influenced by the ratio of mutant DNA to the normal variant.

Mitochondria, often called the "powerhouse of the cell," are crucial in energy production. Over the past three decades, research has unveiled their involvement in various physiological functions beyond energy generation, such as producing heme and iron-sulfur clusters, apoptosis, and calcium homeostasis. Apoptosis, a type of programmed cell death, is critical for embryonic development and several physiological processes. Triggered in response to various types of harm or stressors such as immune responses, oxidative stress, DNA damage, and the absence of certain growth factors, hormones, and cytokines, apoptosis orchestrates a planned and controlled cell death. Moreover, apoptosis is a common feature of development and aging [76]. There are several distinct apoptotic pathways, but they all lead to the same ultimate execution pathway. These caspase-dependent pathways cause the activation of initiator caspases (such as CASP8 [caspase 8] and CASP9), which in turn cause the activation of executioner caspases (such as CASP3 or CASP7), ultimately leading to the disintegration of cell components [77]. The extrinsic or death receptor pathway is initiated by extracellular ligands binding to death receptors on the plasma membrane rather than directly engaging with mitochondria. The death-inducing signaling complex/DISC is generated due to this process, which activates initiator CASP8 and executioner CASP3 [78].

Calcium is a vital signaling molecule within cells, making its regulation crucial. Cellular organelles, including the ER and mitochondria, can store and secrete calcium, thereby controlling the concentration of these ions in cells. Conversely, calcium signaling also influences mitochondrial functionality, although not all molecular mechanisms are fully understood. It is commonly acknowledged that several enzymes, such as PDH (pyruvate dehydrogenase), IDH (isocitrate dehydrogenase), and 2-oxoglutarate dehydrogenases, are regulated by calcium in the mitochondrial matrix. Consequently, the induction of the mitochondrial permeability transition pore (MPTP) and mitochondrial respiration can both be affected by the regulation of calcium by the mitochondria. [79].

The mitochondria and the cytosol carry out heme synthesis [70]. This process entails the introduction of ferrous iron into protoporphyrin IX's tetrapyrrole macrocycle. This process is facilitated by the enzyme FECH (ferrochelatase) in the mitochondrial matrix. Protoporphyrin IX is synthesized from succinyl-CoA and glycine. Heme-containing enzymes are also formed by the liver, even though erythroid progenitors are the primary site of heme synthesis [80]. Although the heme production pathway is preserved in liver cells, the regulation of this process varies between erythoroid progenitor and liver cell types. The machinery for heme production in the liver undergoes quick turnover, allowing it to rapidly respond to alterations in metabolic needs. In contrast, heme synthesis for red cell development is linked to the existence of iron.

Iron-sulfur (Fe/S) clusters are significant prosthetic groups with various functions in biological systems. Fe/S centers are necessary for the activity of several enzymes, such as glycosylases, helicases, primases, and respiratory chain enzymes [81]. Proteins containng Fe/S centers and the components of the biosynthetic machinery are highly conserved in both prokaryotes and eukaryotes, suggesting that they are essential to the genesis of life [82]. It is highly likely that these cofactors originated in low-oxygen environments and coevolved with atmospheric oxygen levels, driving anaerobic electron transport chains to adjust to aerobic environments. A rhomboid (Fe₂/S₂), cuboidal (Fe₃/S₄), or cubane (Fe₄/S₄) cluster is present in the majority of Fe/S proteins [83]. Although cysteine is the most common protein-ligand, arginine, histidine, and serine can also form bonds [81].

4. Mitophagy: A cellular quality control mechanism

4.1. Understanding mitophagy

Youle et al. (2008) demonstrated that PRKN is recruited to depolarized mitochondria and promotes autophagic degradation. Since then, research on mitophagy has continued to evolve, revealing numerous mitophagy pathways [84].

The process of autophagy that governs the turnover of mitochondria is called mitophagy (Figure 3) [85, 86]. Mitochondria can be targeted for mitophagy through at least two major mechanisms: ubiquitin-mediated and transmembrane receptor-mediated pathways [87, 88]. When ROS or mitochondrial DNA damage occurs, mitochondria can become depolarized. One result is the stabilization of OMM-localized PINK1, which initiates mitophagy. Subsequently, these mitochondria attract autophagy receptors, leading to the formation of autophagosomes around the mitochondria. Parenthetically, in mammals, over 30 selective autophagy receptors (SARs) have been discovered [89, 90]. These receptors bear a resemblance to yeast Atg8 and encompass the LC3 and GABARAP families (LC3A-C, GABARAP, GABARAP L1, and GABARAP L2, also referred to as GATE-16). Through conserved regions known as the AIM-like LC3-interaction region (LIR) and the GABARAP-interacting motif (GIM), mammalian SARs engage with LC3/GABARAP proteins [91-93]. The LIR/GIM region, akin to the AIM region, contains hydrophobic residues at specific locations and is often surrounded by negatively charged amino acids, enhancing the binding [92, 93].

Figure 3.

Mitophagy and its underlying mechanisms.

Interestingly, while most yeast SARs bind to the scaffold protein Atg11, only a subset of mammalian SARs interact with its functional equivalent, RB1 inducible coiled-coil 1 (RB1CC1). RB1CC1 is instrumental in attracting and activating the ULK1 kinase complex and subsequent machinery to particular cargo, ultimately aiding in the creation of the phagophore, a precursor to the autophagosome [94-100].

Although selective autophagy in plants has not been as thoroughly investigated, several SARs have been reported to interact with ATG8 in a manner similar to yeast, as shown in [101, 102]. Besides the LC3/GABARAP-binding regions, SARs usually have cargo-binding domains. These domains frequently identify ubiquitin, a typical modification on autophagic cargo. However, cargo recognition can also take place independently of ubiquitin, with SARs directly binding to the cargo or identifying exposed sugars and lipids [103-106].

The autophagosomes then fuse with lysosomes, initiating degradation [107]. Given the risks to the cell of damaged or aging mitochondria, aberrant mitochondria must be promptly removed via mitophagy to preserve cellular integrity and mitochondrial equilibrium [108].

Because neurons primarily produce ATP from their mitochondria, it is essential to precisely degrade malfunctioning mitochondria by mitophagy to preserve both the amount and quality of mitochondria in these cells [109-111]. Research by Wang et al. demonstrated that mitophagy generated by treatment with a SIGMAR1 (sigma non-opioid intracellular receptor 1) agonist in dopaminergic neurons confers neuroprotection in the MPTP-induced animal model of Parkinson disease [112]. The study of mitophagy has advanced rapidly in recent years, leading to a deeper comprehension of the molecular regulatory mechanisms involved in certain diseases [86].

4.2. Receptor-mediated mitophagy

In a manner akin to yeast Atg32, it is anticipated that mammalian mitophagy receptors are situated on the outer mitochondrial membrane and engage with LC3 via a specific area known as the LC3-interacting region (LIR) motif in their cytosolic domain (a tetrapeptide sequence W/YXXL/I) to trigger mitophagy [113, 114]. Various kinds of mitophagy receptors or receptor-associated factors have been recognized in mammalian cells, including BNIP3L/NIX, FUNDC1, PINK1-PRKN, and BCL2L13 (BCL2 like 13).

The E3 ubiquitin ligase PRKN and phosphatase and tensin homolog-induced putative protein kinase 1 (PINK1) have critical roles in the removal of damaged mitochondria that have lost their membrane potential [114-116]. The function of PINK1-PRKN in the initiation of mitophagy will be elaborated on in a separate article within this review series.

PINK1 is a kinase, whereas PRKN is an E3 ubiquitin ligase [117]. Mutations in PINK1 and PRKN are two of the first genetic events linked to autosomal recessive early-onset Parkinson disease [118, 119]. PRKN contributes to mitophagy signaling as a PINK1 downstream component, as demonstrated by studies on gene knockout in Drosophila [120]. Subsequent investigations have delved into the direct regulatory mechanisms through which PINK1 acts on PRKN. The PINK1-PRKN regulatory pathway for mitophagy hinges on assembling ubiquitin chains on mitochondria, with these chains acting as a signal effector, PRKN as a signal amplifier, and PINK1 as a sensor of mitochondrial damage. Together, these components orchestrate mitophagy activation in reaction to damaged mitochondria [121]. Furthermore, by deubiquitinating PRKN or its targets on the mitochondria, deubiquitinating enzymes/DUBs modulate mitophagy. Additionally, PRKN can facilitate TFEB (transcription factor EB) nuclear translocation, which boosts the expression of genes linked to lysosome formation [122, 123].

Defects in the mitophagy pathway are also associated with cardiac disorders. Patients with end-stage heart failure exhibit down-regulated PINK1 expression, and PINK1 knockout mice exhibit abnormal cardiac hypertrophy and left ventricular dysfunction (as shown in [124]. Interestingly, the absence of PRKN, downstream of PINK1, does not significantly impact baseline cardiac phenotypes [125]. This implies that other E3 ubiquitin ligases might compensate for the absence of PRKN.

SQSTM1/p62 (sequestosome 1) has a crucial role in connecting aggregated proteins to LC3 [126, 127]. SQSTM1 directly interacts with ubiquitin through its ubiquitin-binding domain and with LC3 via its LC3-interacting region. PRKN ubiquitylates depolarized mitochondria, and then SQSTM1, recruited to the mitochondrial outer membrane, binds to LC3 to form an autophagosome. In this process, SQSTM1 collaborates with PRKN for the removal of damaged mitochondria [128]. However, the necessity of SQSTM1 for mitophagy is a topic of debate [129]. Notably, SQSTM1 is absent in lower eukaryotes like yeast.

Recently, CUET (coupling of ubiquitin endoplasmic reticulum degradation domain targeting) proteins were identified in yeast as a new class of ubiquitin-Atg8 adaptors, with Cue5 from yeast and its human homolog Tollip serving as examples. Overexpressing Tollip enhances the clearance of Huntingtin-derived polyQ proteins in human cell lines [130].

VDAC1, a mitochondrial protein, is a target for PRKN-mediated ubiquitination and is necessary for PINK1-PRKN-directed autophagy of damaged mitochondria [131]. PRKN is recruited to mitochondria treated with CCCP (carbonyl cyanide m-chlorophenylhydrazine) and associates with VDAC1. In VDAC-deficient cells, PRKN recruitment to damaged mitochondria and mitophagy are impaired [132]. VDACs are essential for efficient elimination of damaged mitochondria through PRKN recruitment from the cytosol. However, SQSTM1 recruitment to mitochondria by PRKN occurs independently of VDAC1 [129].

Recent research by Youle's group suggests that CALCOCO2 and OPTN, previously linked to xenophagy (autophagy of foreign materials), are the primary receptors for PINK1-PRKN-mediated mitophagy [133]. PINK1 directly activates mitophagy by recruiting CALCOCO2 and OPTN. In this process, PINK1-generated phospho-ubiquitin might act as a mitophagy signal, and ubiquitin binding by CALCOCO2 is essential for mitophagy. This suggests that ubiquitin binding could be a characteristic of mammalian mitophagy receptors.

Cardiolipin, a phospholipid on the inner mitochondrial membrane, might also play a role in mitophagy. Some stimuli for mitophagy can induce the externalization of cardiolipin to the mitochondrial surface [103]. Inhibiting this externalization reduces mitophagic activity. LC3 contains a cardiolipin-binding site that allows it to interact directly with cardiolipin. Interestingly, the LC3 deletion mutant lacking the cardiolipin-binding site abolished mitophagy but could still induce non-selective autophagy [103]. These results suggest that interaction with cardiolipin might be another characteristic of mitophagy receptor-related proteins.

4.3. Mitochondrial dynamics

The primary roles of mitochondria are the oxidation of materials and molecules and coupled phosphorylation to produce ATP. However, mitochondria also synthesize ROS via chain reactions in energy metabolism. Under normal conditions, functional mitochondria efficiently eliminate additional ROS to keep the levels low, maintaining normal cellular physiology. However, damage to mitochondria compromises their ability to clear ROS, leading to elevated ROS levels and eventual cell apoptosis. Therefore, it is important to effectively remove injured mitochondria via mitophagy without affecting healthy mitochondria. Mitochondria are dynamic structures that undergo continual fission and fusion to change their size and form [117].

In the outer membrane of mitochondria, proteins such as MFN1 (mitofusin 1) and MFN2, along with OPA1 (OPA1 mitochondrial dynamin like GTPase) in the IMM, control fusion in mitochondria through fission and fusion processes. At the same time, DNM1L/DRP1 (dynamin 1 like) primarily governs mitochondrial division [134]. Drp1-mediated fission has been believed to mediate mitophagy by dividing mitochondria into fragments amenable to autophagosome engulfment [135-137] and/or segregating damaged mitochondrial subdomains for elimination [138]. Moreover, the overexpression of DNM1L compensates for a loss of PINK1 or PRKN in Drosophila melanogaster, genetically linking PINK1 and PRKN to mitochondrial fission [139-141]. However, other studies indicate that DNM1L is not required for mitophagy [142-144].

Mitochondria may undergo asymmetric fission when damaged, resulting in depolarized and fully polarized mitochondria. Depolarized mitochondria are subsequently targeted for clearance by mitophagy to maintain normal mitochondrial activities [138]. However, mitophagy affects the body’s energy supply by reducing the mitochondrial quantity. This reduction triggers the timely activation of mitochondrial biogenesis in response to increased AMP:ATP and NAD⁺:NADH ratios [145]. Therefore, a balance between mitophagy and mitochondrial biogenesis must be maintained to preserve mitochondrial homeostasis.

The PPARGC1A/PGC-1α (PPARG coactivator 1 alpha)-NRF1-NFE2L2/NRF2 (NFE2 like bZIP transcription factor 2)-TFAM (transcription factor A, mitochondrial) pathway plays a pivotal role in regulating mitochondrial biogenesis. In response to stimuli such as cold exposure, Ppargc1a mRNA levels significantly increase in thermogenic tissues, resulting in elevated mtDNA content [146]. In transgenic mouse myocytes, overexpression of PARPGC1A significantly increases mtDNA content and mitochondrial abundance [147]. It is now recognized that the master regulator of mitochondrial biogenesis is PPARGC1A, controlling transcription factors NRF1 and NFE2L2 to express vital mitochondrial proteins. Interestingly, changes in mitochondrial protein levels occur before alterations in PPARGC1A expression [148], suggesting a quick reaction potentially mediated by PPARGC1A activation, not by elevated expression. The regions that promote multiple mitochondria-related genes, including TFAM, are bound by NRF1 and NFE2L2. NRF1 and NFE2L2 operate downstream of PPARGC1A, as evidenced by the fact that NRF1’s N-terminal deletion or NFE2L2 deficiency impedes PPARGC1A's effects on mitochondrial biogenesis [149, 150]. TFAM belongs to the high mobility group box domain family, which is necessary for mtDNA replication. Mitochondrial RNA polymerase must unwind and bend the mtDNA when it binds to promoters. To help with this process, TFAM binds upstream of the transcription start site [151, 152].

TFAM is critical in mitochondrial biogenesis by facilitating mtDNA replication and packaging. It has two DNA-binding sites that facilitate the creation of loops and cross-strand binding, which compacts mtDNA and encourages packing into nucleoids [153]. Because it contains more completely compacted mtDNA molecules, the compacted DNA aids in storing mtDNA. Conversely, transcription and replication mechanisms are active when mtDNA is in its loose state. TFAM expression is often recognized as a marker of mitochondrial biogenesis because it closely mirrors the characteristics of this process [154]. However, recent reports have highlighted uncertainties regarding TFAM levels as an appropriate marker. Specifically, the expression of polypeptides encoded by mtDNA and the amount of mtDNA may not be entirely explained by TFAM levels.

Numerous signaling cascades that affect the PPARGC1A-NRF1-NFE2L2-TFAM pathway regulate mitochondrial biogenesis. The AMP:ATP ratio, Ca²⁺ levels, and NAD⁺: NADH ratio are three of these that are especially important. An elevated AMP:ATP ratio activates AMP-activated protein kinase (AMPK) and phosphorylates PPARGC1A directly, thereby increasing the expression of both PPARGC1A and TFAM. Moreover, AMP can be converted by ADCY (adenylate cyclase) to cyclic AMP/cAMP, which then triggers the cyclic AMP-PRKA (protein kinase cAMP-activated)-CREB (cAMP responsive element binding protein) pathway and drives mitochondrial biogenesis by managing PPARGC1A [155, 156].

When Ca²⁺ drives phosphorylation of MAPK/p38 (mitogen-activated protein kinase), it activates CAMK (calcium/calmodulin dependent protein kinase), which raises PPARGC1A expression and activity and advances mitochondrial biogenesis [157]. Moreover, CREB facilitates PPARGC1A activation via CAMK, suggesting that CREB plays a role in Ca²⁺-dependent mitochondrial biogenesis.

SIRT1 (sirtuin 1) deacetylates PPARGC1A, activating it and subsequently promoting mitochondrial biogenesis in reaction to increased levels of NAD⁺ [145, 158]. It is worth noting that PPARGC1A deacetylation needs interactions with Ca²⁺, AMPK, and SIRT1, indicating potential interconnections between the major stimuli for mitochondrial biogenesis [117].

4.4. Premature aging

Premature aging, a group of rare autosomal-dominant genetic disorders, presents a unique intersection of genetics and gerontology. These conditions, characterized by the early onset of aging-like symptoms, provide a window into the biological processes of aging itself. The root cause of these disorders lies in the realm of our genetic makeup, specifically in mutations within genes that play a crucial role in DNA repair pathways. This paper delves into the intricacies of these disorders, focusing on the role of genes implicated in conditions such as xeroderma pigmentosum (XP), ataxia telangiectasia-mutated (ATM), and Cockayne syndrome group A (CSA) or Cockayne syndrome group B (CSB) [159-161].

Recent research has highlighted the fact that mitochondrial dysfunction is crucial in premature aging in certain clinical situations. Intracellular NAD⁺ levels are depleted due to the buildup of nuclear DNA damage in disorders such as xeroderma pigmentosum group A/XPA, ataxia telangiectasia, and Cockayne syndrome group B. This reduction in NAD⁺ impairs mitophagy, the process of removing defective mitochondria, leading to the accumulation of dysfunctional mitochondria [160, 162].

Furthermore, Other disorders associated with deficiencies in DNA repair, such as Fanconi anemia, have been linked to faulty mitophagy [163], suggesting a broader role of mitochondrial dysfunction in premature aging. These findings emphasize the role of nuclear DNA damage and mitochondrial dysfunction in driving premature aging and related neurodegenerative symptoms and the importance of nuclear DNA damage signaling to mitochondria/N-M signaling in controlling mitophagy [159, 162].

5. Mitophagy's role in maintaining cellular homeostasis and death

Two major types of cell death with distinct morphological features and underlying mechanisms include programmed cell death (PCD) and necrosis [86]. Necrosis is defined as unintentional, passive cell death brought on by irritants from the outside world or environmental changes, which results in an uncontrollable release of cellular contents and inflammation [164, 165].

Morphologically, necrosis is characterized by changes in the cell nucleus, including nuclear shrinkage, fragmentation, or dissolution. Under a light microscope, these changes can be observed, and staining with fluorescent dyes such as DAPI may show reduced nuclear staining in necrotic cells. Additionally, necrotic cells lose membrane integrity, releasing cellular contents into the extracellular space, which can stimulate the immune system, leading to inflammation [166].

The relationship between mitophagy and necrosis varies depending on the disease step [167]. Excessive mitophagy, or removing many mitochondria, can disrupt cellular functions, leading to cell necrosis [168, 169]. Researchers, such as Paech et al., have highlighted the fact that mitophagy is protective in removing damaged mitochondria. However, in cases where cells are exposed to strong toxins or kinase inhibitors, the ability of mitophagy to repair mitochondria may be compromised [170]. In such situations, cells may undergo necrosis due to the inability to remove damaged mitochondria adequately.

The balance between apoptosis and necrosis within a cell is heavily influenced by intracellular ATP levels: high ATP levels typically favor apoptosis, whereas low levels promote necrosis [170, 171]. In a study by Xia et al., they found that when measles virus vaccine strain Edmonston B infects non-small cell lung cancer cells/NSCLCs, mitophagy can change the mode of cell death from apoptosis to more effective necrosis [172]. This shift was attributed to continuous ATP consumption, depletion during viral replication, and increased mitophagy-induced necrosis [173]. Moreover, Dhingra et al. found that BNIP3 influences mitochondrial respiratory chain signaling, which contributes to doxorubicin-induced cardiac myocyte necrosis and death [174].

5.1. Mitophagy and Apoptosis

In 1972, Kerr, Wyllie, and Currie introduced a groundbreaking concept in cell biology, delineating a distinct form of cell demise termed apoptosis [86]. This seminal work formally introduced apoptosis to the scientific community [175, 176]. Apoptosis, recognized as a pivotal process in various physiological contexts, encompasses normal cell turnover, developmental processes, and immune system function. Moreover, the apoptotic flux is also implicated in pathological conditions where the generation of apoptotic cells is observed [177].

Apoptosis is one type of PCD in all multicellular animals. This type of PCD initiates a series of events by activating various cysteine-aspartic proteases inside the cell, eventually leading to cell death. The creation of apoptotic bodies, which include the contents of the cell, is a characteristic that distinguishes apoptosis. These bodies can be readily engulfed by neighboring cells, thereby averting the release of potentially harmful contents and subsequent damage to neighboring tissues [178].

The intrinsic or mitochondrial pathway and the extrinsic or death receptor pathway are the two primary mechanisms that make up the apoptotic process [175, 179]. Extensive research has elucidated the molecular intricacies of these pathways, unveiling key apoptotic proteins such as CASP3, which play critical roles in orchestrating the cellular demise [86].

Through mitophagy, cells manage mitochondrial stress until it reaches a critical threshold, triggering apoptosis [180, 181]. This mechanism ensures that when mitochondrial damage becomes severe, such as during myocardial infarction, apoptotic proteases are activated to cease mitophagy and initiate apoptosis, facilitating cell death [182]. In dopaminergic SH-SY5Y cells, mitophagy is essential for neuroprotection against Mn-induced apoptosis [183]. According to research by Ham et al., the PINK1-PRKN pathway causes different forms of ubiquitination on VDAC1, which controls mitophagy and apoptosis. Whereas monoubiquitinated VDAC1 prevents mitochondrial calcium absorption, protecting cells from apoptosis, polyubiquitinated VDAC1 stimulates PRKN-mediated mitophagy [184]. In particular, PRKN-induced polyubiquitination on VDAC1 triggers PRKN-mediated mitophagy by attracting SQSTM1 and LC3B to the mitochondria [131].

The oligomeric assembly of VDAC1 has been associated with the induction of apoptosis. Monoubiquitination of VDAC1 may disrupt its self-oligomerization, thereby preventing OMM permeabilization and inhibiting apoptosis [185, 186]. Prior research has emphasized the role of mitophagy as a defense mechanism that helps cancer cells survive periods of acute or prolonged stress. For example, Wei et al. found that matrine, a naturally occurring alkaloid isolated from Sophora favescens roots, causes lower expression of PINK1 and PRKN, which inhibits mitophagy and increases apoptosis. Conversely, overexpression of PINK1 reactivates mitophagy, reducing apoptosis and activating CASP9 [187, 188].

Chen et al. showed that ketoconazole worsens mitophagy in hepatocellular carcinoma, which results in apoptosis via lowering PTGS2/cyclooxygenase-2 (prostaglandin-endoperoxide synthase 2) levels [189]. Depending on the cancer subtype and setting, many molecular processes supporting mitophagy may be dysregulated in cancer or function differently as tumor promoters or suppressors. More mechanistic research is required to clarify the connection between cancer-associated mitophagy and healthy tissue and the implicated mitophagy pathways. Cancer cells must tightly regulate the homeostasis of their mitochondrial networks, and aberrant mitophagy is thought to induce apoptosis by upsetting this equilibrium [86].

5.2. Mitophagy and Pyroptosis

Pyroptosis is a kind of controlled cell death that resembles necrosis rather than apoptosis; it is characterized by cell enlargement, along with hole creation in, and breakdown of, the plasma membrane [190, 191]. Pyroptosis is classified as gasdermin-mediated programmed cell death because GSDMD (gasdermin D) is a major mediator of this process [192]. Generally, under illness situations involving the production of inflammatory entities, such as NLRP3 (NLR family pyrin domain containing 3) and PYCARD/ASC (PYD and CARD domain containing), nucleotide-binding oligomerization domain-like receptors/NLRs start pyroptosis [86].

CASP1 is recruited and activated by inflammatory substances, starting a series of events that ultimately result in the cleavage of GSDMD. When the inflammasome activates the gasdermin-N domain of GSDMD, it can create gaps in particular liposomes containing cardiolipin, phosphatidylinositol, or a combination of naturally occurring polar lipids. These GSDMD pores create many openings in the liposomal membrane, disrupting its osmotic potential, causing cellular swelling, and eventual dissolution. The release of cellular contents also triggers an amplified inflammatory reaction in the surrounding environment. Besides CASP1, other inflammatory proteases such as SCAF11/CASP11, CASP4, and CASP5 are closely associated with pyroptosis. These enzymes can induce the downstream cleavage of GSDMD, leading to pyroptosis, thus being recognized as additional pathways contributing to this type of cell death [191, 192].

While the intricacies of the interplay between mitophagy and pyroptosis remain complex, a rising number of studies underscore the significant role of mitophagy in modulating pyroptosis. Blocking mitophagy by inflammasome activation, Yu et al. showed that the critical mitophagy regulator PRKN is cleaved by CASP1, thereby exacerbating mitochondrial damage and enhancing pyroptosis [193]. In a different investigation, Yu et al. demonstrated that the GLP1R (glucagon like peptide 1 receptor) agonist liraglutide attenuates pyroptosis and NLRP3 inflammasome activation by promoting PINK1-PRKN-mediated mitophagy, which in turn improves the effects of nonalcoholic steatohepatitis. Mitophagy is blocked while NLRP3 inflammatory corpuscle activation and GSDMD expression are reversed using the autophagy inhibitor 3-methyladenine/3-MA or PINK1-specific siRNA [194].

Furthermore, research has shown that palmatine blocks the activation of NLRP3 caused by lipopolysaccharide + ATP, which mitophagy inhibitors or PINK1-siRNA may inhibit [195]. In HIV-productively infected astrocytes, blocking PRKN-mediated mitophagy restores susceptibility to pyroptosis, indicating that resistance against pyroptosis depends on mitophagy to promote cell survival [196]. Apart from the PINK1-PRKN axis, BNIP3L-mediated mitophagy also influences pyroptosis emergence and modulation. Peng et al. showed that inhibiting BNIP3L expression in murine macrophage cells results in higher amounts of mature IL1B/IL1β (interleukin 1 beta) and active CASP1 while lowering levels of LC3, indicating that BNIP3L prevents macrophage pyroptosis using mitophagy [197].

5.3. Mitophagy and Ferroptosis

A newly identified kind of cell death called ferroptosis is characterized by a substantial iron and lipid peroxidation buildup during the cell's demise wherein oxidative cell death is caused by small molecules dependent on iron ions [198, 199]. This iron-dependent form of regulated necrosis has been identified as a major cause of several illnesses [200]. Smaller-than-normal mitochondria, condensed mitochondrial membrane density, missing or diminished cristae, and rupture of the outer mitochondrial membrane are the morphological characteristics of ferroptosis [201] and the imbalance between ROS production and degradation within the cell precipitates its occurrence [202]. Through various mechanisms, ferroptosis inducers may directly or indirectly affect GPX (glutathione peroxidase), reducing cellular antioxidant capability, causing the accumulation of ROS, and eventually leading to oxidative cell death [203].

Very little has been known about the function of mitophagy in ferroptosis; much of the research has been done in vitro on cancer cells. For example, it has been discovered that PRKN-mediated mitophagy induced by carbonyl cyanide 3-chlorophenyl hydrazone/CCCP inhibits ferroptosis caused by cysteine deprivation in HT1080 cells (fibrosarcoma) [200]. While heightened ROS production in cancer cells contributes to tumorigenesis, excessively high intracellular ROS levels activate several mechanisms that cause ROS-induced cell death, offering a potential means to eradicate cancer cells [204]. Inhibitors of mitochondrial complex I, such as BAY 87-2243, can encourage the increase of ROS in melanoma cells dependent on mitophagy, leading to ferroptosis. The effects of BAY 87-2243 on mitochondrial depolarization, mitophagy activation, ROS amplification, and consequent ferroptosis are inhibited by PINK1 knockdown [86, 205]. Additionally, ferroptosis is induced in breast cancer and glioblastoma cell lines by BAY 11-7085, an inhibitor of NFKB/NK-κB activation, via an NFE2L2-SLC7A11 (solute carrier family 7 member 11)-HMOX1/HO-1 (heme oxygenase 1) pathway. This leads to HMOX1 compartmentalization in the nucleus and mitochondria, resulting in mitochondrial dysfunction and subsequent lysosomal targeting for mitophagy [206]. These investigations examining cancer cells offer laboratory evidence supporting the role of mitophagy in counteracting ferroptosis. However, there is insufficient direct in vivo data to support the anti-ferroptotic effects of mitophagy induction, and it is unclear if or to what degree the reduction of ferroptosis is a component of mitophagy activation's proven effectiveness in other disease models [86].

In regard to the effects on iron chelation on mitophagy, in a recent study conducted by Kevin et al., they explored a unique form of mitophagy induced by iron chelators such as deferiprone (DFP), which functions independently of the PINK1-PRKN/PRKN pathway. The mechanisms driving this process are not yet fully understood. Their research centered on the role of SENP3, an enzyme that removes SUMO modifications, in mitophagy induced by DFP. They discovered that DFP treatment elevates SENP3 levels and that SENP3 is vital for this form of mitophagy. Furthermore, it was pinpointed that FIS1, a critical mitochondrial protein for DFP-induced mitophagy, could serve as a novel target for deSUMOylation mediated by SENP3. The results suggested that SENP3 facilitated mitophagy in response to DFP by stripping SUMO modifications from FIS1, thereby boosting its localization to the mitochondria. These insights illuminate the mechanisms of mitophagy initiated by iron chelation and suggest potential therapeutic applications of DFP in various conditions, including Parkinson's disease [207].

5.4. Mitophagy and Necroptosis

Necroptosis, a well-studied type of controlled necrosis, exhibits characteristics reminiscent of both apoptosis and necrosis [208, 209]. Based on the variables that activate it, necroptosis may be divided into three main categories: ischemia-mediated intrinsic necroptosis; extrinsic necroptosis, which is started by TNF/TNFα (tumor necrosis factor) and intrinsic necroptosis, which is caused by ROS [210].

One of the key components of necroptosis is RIPK1 (receptor interacting serine/threonine kinase 1), a ubiquitous membrane receptor that binds TNF [211]. The 76-kDa RIPK1 has three distinct domains: an intermediate domain that contains the receptor-interacting protein homotypic interacting motif (RHIM), which may bind to other proteins that also include RHIM, an N-terminal kinase domain and C-terminal death domain [212]. RIPK1 and RIPK3 make up the essential elements of the necroptotic signaling platform [213].

In traditional necroptosis, the interaction between RIPK1 and RIPK3 through the RHIM domain is responsible for assembling the necrosome complex. Cellular ATP levels are decreased due to the necrosome's interference with the ability of SLC25A4/adenine nucleotide translocase (solute carrier family 25 member 4) to operate in mitochondria [214].

Mitochondria and necroptosis are connected primarily due to their involvement in generating ROS [205, 215, 216]. Tait et al. showed that removing mitochondria does not alter RIPK3-mediated cell death; it stops the generation of ROS linked to necroptosis [217]. However, whether mitophagy can modulate necroptosis remains unknown. The execution of necroptosis depends on RIPK3 phosphorylating MLKL (mixed lineage kinase domain like pseudokinase) at the Thr357 and Ser358 residues [213]. Research by Mizumura et al. showed that PINK1-dependent mitophagy might function as an upstream regulator of the necrosome because PINK1-knockdown cells show a decrease in the phosphorylation of MLKL produced by cigarette smoke exposure [218, 219]. Nonetheless, the role of mitochondria in necroptosis seems to vary depending on the cell type and the specific stimulus [86].

6. Mitophagy interacts with pathogenic processes through which diabetic neuropathy develops: Evidence from preclinical evaluations with emphasis on the genes or proteins involved

As mentioned earlier, mitophagy is responsible for selective elimination of defective mitochondria [220-222]. Recent studies have highlighted the connection between mitophagy and the progression of T1DM and T2DM. Mitophagy appears to play a role in modulating the removal of excessive ROS during hyperglycemic conditions [22]. Experimental diabetic models, such as diabetic retinas in db/db mice, have shown reductions in the number of mitophagic autophagosomes/mitophagosomes alongside increased oxidative stress, potentially mediated by alterations in mitophagy-related proteins such as PRKN and PINK1 [23]. Moreover, there are specific genes, proteins, and signaling axes in correlation with mitophagy during DN that have experimentally been realized to be regulated by particular medicinal agents for possible DN treatment. Addressing these signaling mechanisms and the corresponding drugs in relation to mitophagy flux in diabetes could represent a promising therapeutic strategy (Table 2). Hence, using mitophagy modulators to control the mitophagy’s well-being is emerging as a possible therapeutic focus [20].

Table 2.

Major molecules and mechanisms that affect diabetic neuropathy through regulating the mitophagy flux.

Drug/Molecule	Function	Study model	Impact on mitophagy	Effect on diabetic neuropathy	Reference(s )
Piceatannol	Activating SIRT1	Rat model of DN	Enhances mitochondrial biogenesis and promotes mitophagy	Mitigates effects of diabetic insult on peripheral neuropathy	[223]
DMOG	HIF1A agonist	Experimental studies in diabetic mice	Activates mitophagy, enhanced by elevated HIF1A and PRKN levels	Reduces mitochondrial dysfunction and alleviates hyperalgesia	[224]
PARP1 Inhibitors (PJ34, AG14361)	Decrease mitochondrial oxidative damage and restore mitophagy	In vivo and in vitro studies in PDN mouse model	Restores mitophagy function in DRG neurons	Alleviates peripheral nerve hyperalgesia in diabetes	[225]
KU-596	Neurotherapeutic (improves mtBE and reduces TXNIP)	Research on Mito-QC mice	Enhances mtBE, while it reduces mitophagy in an HSPA/HSP70-dependent manner	Reverses DPN symptoms, relies on HSAP/HSP70	[226]
TFAM	Facilitates mtDNA replication and transcription	Study on TFAM transgenic mice	Not directly mentioned, but its overexpression correlates with improved mitochondrial function	Prevents neuropathy alterations, improves mtDNA copy number, and reduces nerve damage	[227]
Astragaloside	Antioxidant; suppresses excessive autophagy	Study on Schwann cells	Decreases ROS levels; preserves mitochondrial morphology and membrane potential	Protective effect against oxidative stress; potential therapeutic strategy for DPN	[228]
Pifithrin-α	TP53 inhibitor	STZ-induced type 1 diabetic mice	Prevents PRKN downregulation	Reduces pain hypersensitivity and mitochondrial dysfunction	[229]
Methylglyoxal	Glyoxalase pathway substrate	Intravenous administration in mice	Not specified, but probably affects mitochondrial membrane potential	Causes pain hypersensitivity and neurological disorders	[229]
Photobiomodulation	Clinical intervention	Mouse model of diabetic peripheral neuropathy by STZ	Increases mitochondrial content and controls PRKN expression	Elicits antinociceptive effects and aids in nerve regeneration	[230]

6.1. SIRT1

In DN and its intersection with mitophagic flux, piceatannol (PCN), an activator of SIRT1, holds promise for regulating different oxidative stress pathways and reducing inflammation in various inflammatory situations. However, the specific function of piceatannol in mitigating the effects of diabetic insult on peripheral neuropathy is largely unexplored. Oxidative stress and mitochondrial dysfunction are recognized as pivotal contributors to DN, and numerous studies have underscored the potential of SIRT1 activation in improving nerve function through mechanisms such as enhancing mitochondrial biogenesis and promoting mitophagy [223].

In a study by Khan et al., a rat model of DN was developed, revealing that exposure to high glucose induces reactive oxygen species in N2A cells, resulting in mitochondrial superoxide accumulation and altered mitochondrial membrane potential. However, treatment with PCN restores these aberrations alongside enhancing neurite outgrowth. Notably, PCN exposure leads to increased activation of SIRT1, which in turn stimulates mitochondrial biogenesis via PPARGC1A. Furthermore, activating SIRT1 is associated with an augmentation of NFE2L2-mediated antioxidant signaling. These results imply that PCN administration may increase SIRT1 and NFE2L2 activities, thereby mitigating the decrease in mitochondrial function and antioxidant activity in diabetic rats and high glucose-exposed N2A cells [223].

6.2. HIF1A

In diabetes, two prominent features are hyperglycemia and cellular hypoxia. HIF1A/HIF-1α (hypoxia inducible factor 1 subunit alpha) is key in maintaining mitochondrial homeostasis under hypoxic conditions. However, whether mitophagy is altered and can be modulated by HIF1A in the case of diabetic neuropathic pain (DNP), which is characterized by tingling, burning, sharp, shooting, and lancinating or even as electric shock sensations, remains uncertain. In experimental studies conducted in diabetic mice, mitophagy is activated concurrently with the upregulation of HIF1A in the spinal cord of these animals.

Treatment with dimethyloxalylglycine (DMOG), an HIF1A agonist, elevates HIF1A and PRKN protein levels, leading to enhanced mitophagy, reduced mitochondrial dysfunction, and alleviated hyperalgesia. Interestingly, in diabetic mice with prkn gene knockout, hyperalgesia and mitochondrial dysfunction are exacerbated. Moreover, in diabetic mice lacking PRKN (prkn^−/−) DMOG cannot induce mitophagy. These results imply that by modifying the PRKN signaling pathway, HIF1A can stimulate mitophagy in the spinal cord of mice with DNP. This sheds light on potential mechanisms and therapeutic approaches for DNP patients [224].

6.3. PARP1

Research has shown that the pathological processes of diabetes are associated with PARP1 (poly(ADP-ribose) polymerase 1). Recognized as an essential regulatory mechanism, mitophagy breaks down damaged mitochondria in lysosomes to preserve the equilibrium of ROS. However, It is unknown how PARP1 regulates mitophagy-associated mitochondrial oxidative damage and the development of painful diabetic neuropathy (PDN). To investigate this further, scientists looked into the in vivo and in vitro mechanisms of PARP1-mediated mitophagy inhibition in a Lep (leptin) gene mutation (db/db) mouse model of PDN. Their research showed that PDN mice have decreased mitophagy, increased PARP1 activity, and mitochondrial damage in dorsal root ganglion neurons [225]. They also found that in DRG neurons from PDN mice, PARP1 mediates the impairment of mitophagy.

Treatment with PARP1 inhibitors (PJ34 or AG14361) decrease mitochondrial oxidative damage, restore mitophagy function in DRG neurons, and alleviate peripheral nerve hyperalgesia brought on by diabetes. Conversely, the lysosome deacidification agent DC661 exacerbates mitophagy impairment, accelerating diabetes-induced mitochondrial oxidative stress and injury in DRG neurons. These results point to a critical role for PARP1-induced defective mitophagy in DRG neurons in the pathogenesis of peripheral neuropathic injury caused by diabetes. Potential treatments for PDN include restoring mitophagy function and suppressing PARP1 activity [225].

6.4. HSPA/HSP70 and TXNIP

KU-596, a promising small molecule neurotherapeutic, has demonstrated efficacy in reversing symptoms of diabetic peripheral neuropathy (DPN), enhancing sensory neuron mitochondrial bioenergetics (mtBE), and reducing the pro-oxidant protein TXNIP (thioredoxin interacting protein) in an HSPA/HSP70 (heat shock protein protein family A (Hsp70))-dependent manner. Despite its therapeutic potential, it is still unclear how exactly KU-596 improves mtBE and how TXNIP contributes to its effectiveness [226].

Researchers recently tried to determine whether KU-596 treatment enhances DPN, mtBE, and mitophagy in a manner dependent on HSPA/HSP70 and TXNIP. To visualize mitophagy, the researchers used Mito-QC (MQC) mice, which express an mCherry-GFP fusion protein specifically targeted to mitochondria. They noticed that MQC mice with diabetes and MQC mice deficient in TXNIP or HSPA/HSP70 develop DPN-like sensory and nerve conduction dysfunctions. These measures improve after receiving KU-596 treatment; the improvement relies on HSPA/HSP70 but not TXNIP.

Furthermore, in MQC mice, diabetes leads to a reduction in mtBE and an increase in mitophagy, which is reversed using KU-596 therapy. However, KU-596 fails to enhance mtBE or decrease mitophagy in MQC mice lacking either HSPA/HSP70 or TXNIP. These findings indicate that TXNIP may not be required for developing diabetes-induced mitochondrial malfunction and sensory symptoms. In an HSPA/HSP70- and TNXIP-dependent manner, KU-596 therapy probably increases mitochondrial resistance to diabetic stress, which decreases mitophagic clearance [226].

6.5. TFAM

The importance of TFAM, which wraps around mtDNA to facilitate mtDNA replication and transcription, is highlighted by the role that oxidative stress-induced mitochondrial dysfunction and damage to mtDNA plays in the pathogenesis of DN. Researchers examined if TFAM overexpression could lessen experimental peripheral DN in a study using TFAM transgenic mice (TFAM Tg) expressing human TFAM. According to the study, the DRG of TFAM Tg mice show roughly two-fold higher levels of mouse mtDNA and total TFAM (mouse TFAM + human TFAM) than that of control (wild-type) mice. Several neuropathy endpoints were measured, such as the intraepidermal nerve fiber density/IENFD, mechanical allodynia, thermal nociception, and motor and sensory nerve conduction velocities. In DRG neurons, western blot was used to measure TFAM levels, and qPCR was used to quantify mtDNA copy number and damage [227].

Mice with established diabetes show behavioral abnormalities, intraepidermal nerve fiber loss, and deficits in motor and sensory nerve conduction. Remarkably, regardless of changes in blood parameters, these alterations are largely prevented in diabetic TFAM Tg mice. Crucially, the mtDNA copy number in TFAM Tg diabetic mice attains values similar to those of wild-type nondiabetic mice. Additionally, in 6-week-old diabetic mice, mtDNA and TFAM are upregulated, indicating the potential therapeutic efficacy of TFAM activation in treating peripheral neuropathy associated with diabetes [227].

6.6. MAP1LC3, PINK1 and PRKN

In an insightful investigation, Wei et al. clarified the precise regulatory actions of astragaloside on the mitophagy in Schwann cells that is induced by hyperglycemia, offering valuable insights for potential clinical trials involving astragaloside intravenous (AS-IV) therapy for DPN. According to their findings, AS-IV demonstrates antioxidant properties and suppresses excessive autophagy activation in Schwann cells. These effects result in a notable decrease in ROS levels and downregulation of PRKN, PINK1, and LC3, autophagy-related proteins that act in hyperglycemia. This intervention contributes to preserving mitochondrial morphology and membrane potential, thus exerting a protective effect against excessive mitophagy and oxidative stress on mitochondrial function in Schwann cells during hyperglycemia. The study’s findings provide compelling evidence supporting the potential of a therapeutic strategy based on AS-IV to treat DPN [228].

6.7. TP53 and PRKN

In a comprehensive investigation, the development of neuropathic pain and mitochondrial dysfunction in T1DM were examined concerning the functional roles of PRKN and TP53/p53 (tumor protein p53; TRP53 in mice, denoted as TP53 for simplicity) in mitochondrial quality control. Mice with T1DM were given streptozotocin (STZ), and the effects of the selective TP53 inhibitor pifithrin-α were assessed regarding PRKN expression, mitochondrial function, and diabetic pain hypersensitivity. Additionally, intravenous methylglyoxal was administered to mice to evaluate TP53 and PRKN expression in the DRG and pain hypersensitivity. Furthermore, the mitochondrial membrane potential of cultured DRG neurons treated with methylglyoxal was examined [229].

Following STZ treatment for three weeks, the mice display hypersensitivity to pain, and in the DRG there is a marked decrease in PRKN expression and a considerable increase in TP53 expression. PRKN downregulation is avoided, and STZ-induced pain hypersensitivity is lessened by inhibiting TP53 with pifithrin-α. Furthermore, STZ-induced reductions in mitochondrial number and buildup of mitochondrial ROS are mitigated by pifithrin-α. Methylglyoxal therapy also causes pain hypersensitivity and changes the expression of TP53 and PRKN, much like STZ does. In cultured DRG neurons, treatment with methylglyoxal also reduces mitochondrial membrane potential. These results imply that changes in TP53 and PRKN expression cause mitochondrial malfunction and ROS build-up, which in turn causes pain hypersensitivity in diabetic mice treated with methylglyoxal. Moreover, methylglyoxal directly affects DRG neurons to cause neurological disorders similar to diabetes [229].

Photobiomodulation (PBM) has emerged as a promising clinical intervention due to its ability to facilitate early nerve regeneration, leading to remarkable improvements in peripheral nerve function. The therapeutic effects of PBM on DPN-induced pain and nerve damage were examined in a mouse model of DN created by STZ. The study revealed that PBM elicits antinociceptive effects in mice with neuropathic pain, and this effect is contingent upon the release of central opioids. Following 21 consecutive applications, PBM treatment leads to increased levels of NGF (nerve growth factor). In the sciatic nerve of DPN-affected animals, PBM aids in structural repair, as evidenced by increased mitochondrial content and control of PRKN expression. These results provide insight into the fundamental workings of PBM treatment and underscore its therapeutic potential in managing DPN [230].

Mitophagy and mitochondrial dynamics are crucial in maintaining mitochondrial quality and quantity by eliminating damaged mitochondria and replenishing them with healthy ones. However, in diabetic conditions, these processes can become dysregulated, leading to either the accumulation of defective mitochondria or the loss of functional ones. Both scenarios can exacerbate nerve damage and dysfunction associated with DN. Therefore, modulating mitophagy and mitochondrial dynamics shows a promising treatment approach for this condition.

Improving the clearance of damaged mitochondria and restoring the balance of mitochondrial biogenesis and turnover may protect nerve cells from oxidative stress, inflammation, and apoptosis, which otherwise contribute to DN progression. Several pharmacological agents and natural compounds have demonstrated beneficial effects on mitophagy and mitochondrial dynamics in DN models, highlighting their potential as therapeutic interventions in managing this debilitating condition.

6.8. Anti-muscarinic agents may affect mitochondrial function through AMPK signaling to aid in DPN therapy

New insights into the mechanisms of neurite growth offer an exciting potential therapeutic pathway for DPN management. It has been found that cholinergic restrictions, mediated by muscarinic receptors (MRs), inhibit neurite growth in adult sensory neurons. Remarkably, mice without the M1R exhibited increased neurite growth and showed significant resistance to diabetic neuropathy following streptozotocin (STZ) treatment [231].

In rodent studies, neuropathy symptoms such as slowed nerve conduction, thermal hypoalgesia, and loss of intra-epidermal nerve fibers (a DPN indicator) were prevented or reversed by targeting M1R with selective antagonists like pirenzepine, VU0255035, or the snake venom toxin MT7 [231]. These antimuscarinic drugs did not show significant side effects in animal models, including no changes in cardiac function and structure based on echocardiogram studies. They also did not alter the overall diabetic condition, indicating that they do not reverse DPN by affecting pancreatic function or insulin levels.

Investigations across T1DM and T2DM rodent models have shown that the nerve-protective and repair-promoting effects of M1R antagonists are linked with AMPK deactivation [231]. Further evidence of the central role of mitochondrial function comes from studies on ciliary neurotrophic factor and C-terminal inhibitors of heat shock protein 90, which are known to enhance mitochondrial function and have shown improved neuropathy in diabetic rodents [65, 232].

The impact of mitochondrial dysfunction in diabetes may not be limited to the nervous system. Reduced AMPK activity and decreased expression of mitochondrial complex proteins have been observed in kidney mesangial cells of diabetic nephropathy, suggesting that antimuscarinic therapy could have benefits beyond DPN [233].

The M1R restriction mechanism may have wider implications. Pirenzepine and MT7 have been shown to prevent peripheral neuropathy in models related to chemotherapy or HIV [231]. Furthermore, the inhibition of mitochondrial activity driven by muscarinic receptors could be beneficial in conditions with reduced energy capacity under stress, not just in neuropathies closely related to DPN. For example, antimuscarinics were identified as potential myelination enhancers in multiple sclerosis during a screening for remyelination promoters [234].

Interestingly, some antimuscarinics have been clinically used in Europe for various conditions for over two decades. In addition, topical pirenzepine for childhood myopia has a well-documented safety profile [235]. This existing safety data could fast-track clinical trials for antimuscarinics in the treatment of DPN and other peripheral neuropathies. Early DPN indicators might be the best targets for clinical trials. Antimuscarinics could be more effective during the initial stages of distal neuronal degeneration, rather than at later stages when complete fiber loss has occurred [236].

7. Mitophagy modulation as a potential therapeutic approach against diabetic neuropathy

Inflammatory stress is a pivotal factor in the development of various metabolic disorders, such as diabetes [237-239]. An underlying problem shared by all kinds of diabetes is inadequate function or bulk of pancreatic β cells to supply the body's needs for insulin, often accompanied by inflammatory damage to these cells [237, 240, 241]. While the exact molecular mechanisms remain elusive, the excessive production of free radicals, such as nitric oxide/NO and ROS, results in β-cell inflammatory injury [242].

Inflammatory signaling negatively affects mitochondrial function in β cells, leading to compromised bioenergetics, decreased insulin production in response to hyperglycemia, and activated apoptotic pathways [243]. Thus, strategies aimed at mitigating inflammation and preserving mitochondrial health hold promise as potential therapies for diabetes [221, 222, 244]..

It is still unknown if β cells are protected from inflammatory damage via mitophagy or the CLEC16A pathway. However, research has indicated that in human β cells subjected to inflammatory stimuli, CLEC16A-mediated mitophagy has a protective role. This suggests that targeting this pathway for therapeutic intervention may be possible. Recent investigations have identified several compounds that activate mitophagy and enhance metabolic tissue respiratory activities, including in β cells [245-247]. Notably, in clinical studies, the mitophagy activator urolithin A has shown encouraging outcomes that support overall metabolic health [248]. Hence, enhancing mitophagy pharmacologically may provide a new way to combat inflammatory stress and stop the loss of β cells in diabetics. Further research is needed to comprehensively evaluate the effectiveness of targeting mitophagy in treating various forms of diabetes.

To meet the cellular energy demands, Continuous cycles of fusion and fission occur in mitochondria, spreading populations of mitochondria throughout various tissues in response to different nutritional circumstances [249]. Fusion allows for the mixing of mitochondrial contents, which can help mitigate damage and maintain a healthy mitochondrial population. It involves the merging of both the outer and inner mitochondrial membranes, a process facilitated by large GTPases, including mitofusins (MFN1 and MFN2) and optic atrophy protein 1 (OPA1). On the other hand, fission is a process that divides a single mitochondrion into two. This is particularly important for removing damaged mitochondria through a process called mitophagy, and for distributing mitochondria to parts of the cell with high energy demands. DNM1L is a key player in mitochondrial fission. The balance between these two processes—fusion and fission—is critical for cellular health. Disruptions in this balance can lead to a variety of diseases, including neurodegenerative diseases, cardiovascular diseases, and metabolic disorders. Moreover, the distribution of mitochondria within cells is not random. Mitochondria move along the cell’s cytoskeleton to areas where energy demand is high. This movement is particularly evident in neurons, where mitochondria can be transported along axons and dendrites to synapses. Nutritional status also impacts mitochondrial dynamics. For instance, during nutrient abundance, mitochondria tend to be more fused, which promotes ATP production. Conversely, under nutrient scarcity, mitochondria tend to undergo fission, which facilitates the removal of damaged mitochondria and helps the cell adapt to metabolic stress [250, 251].

The availability of nutrients in T2DM promotes mitochondrial fission while concurrently downregulating mitochondrial fusion. Consequently, this imbalance results in uncoupled respiration, wherein the oxidative phosphorylation rate decreases, thereby reducing mitochondrial ROS production [252]. Apart from its regulatory function in controlling mitochondrial fission and fusion, mitochondrial morphology is crucial in advancing T2DM. In diabetic patients, there is evidence of altered mitochondrial morphology, characterized by decreased levels of CKMT (creatine kinase, mitochondrial) activity and smaller mitochondria in skeletal muscle compared to individuals without diabetes [253]. Hyperglycemia, a hallmark of diabetes, further exacerbates mitochondrial fragmentation across various tissues, including the heart, liver, cardiovascular system, and pancreas [253, 254]. Oxidative stress is crucial in regulating pancreatic β cells, affecting their function and insulin secretion. MFN1 and MFN2, two mitochondrial fusion proteins, have been discovered to positively correlate with DNM1L, which is involved in mitochondrial dynamics. Reduced mitochondrial membrane potential and ATP generation due to DNM1L dysregulation can eventually lead to a decrease in glucose-stimulated insulin release [255, 256]. In light of this pathogenic process, antioxidants may help reduce insulin resistance. According to a new study, SS-31, an antioxidant peptide, regulates mitochondrial membrane potential and ATP levels to cure DN. In mesangial cells under hyperglycemic circumstances, this effect is linked to the inhibition of NOX4 (NADPH oxidase 4) and TGFB1/TGF-β1 (transforming growth factor beta 1) expression, which results in greater activation of MAPK/p38 (mitogen-activated protein kinase) and NADPH oxidase activity [257].

Furthermore, obese individuals display reduced mitochondrial oxidative properties, such as a decrease in mtDNA and mtDNA-mediated translation systems compared to their lean counterparts. This downregulation affects the tricarboxylic acid cycle, ketone body synthesis and breakdown, and fatty acid oxidation, leading to insulin resistance, obesity, and the release of pro-inflammatory cytokines [258]. Under the conditions such as T2DM, over-nutrition impairs mitochondrial function and leads to fragmentation, inhibiting autophagic flux and increasing ROS generation [259, 260]. Natural products have garnered attention for their potential to modulate mitophagy and improve mitochondrial dysfunction associated with T2DM. Before natural products can be used in clinical applications for treating T2DM, evaluating their therapeutic effectiveness in correcting mitochondrial dynamics via the autophagosome-lysosome pathway is crucial, including understanding any potential side effects on tissues or cells. This evaluation should be done using T2DM-related models and laboratory experiments. The next paragraph in conjunction with Table 3 summarizes how natural products are commonly used to treat T2DM in the lab and real-world scenarios. These natural products regulate mitophagy, thereby restoring the dynamics of mitochondria through different processes [20].

Table 3.

Key mitophagy-related therapeutic targets/agents against diabetic complications, particularly diabetic neuropathy.

Process	Impact on mitochondria	Resulting condition	Potential Treatment	Reference(s)
CLEC16A-mediated mitophagy	Controls the flow of mitophagy in β cells	Linked to damaged function of β cells and glucose regulation	Targeting CLEC16A pathway	[245, 246, 263, 264]
Urolithin A	Activates mitophagy	Supports overall metabolic health	Clinical studies are being conducted for therapeutic intervention	[247, 248]
SS-31 antioxidant peptide	Affects β-cell function and insulin secretion	Decreased insulin release	Mediates oxidative stress	[257]
Salvianolic acid B	Inhibits mitophagy via reducing fission proteins	Prevents endothelial cell death	Salvianolic acid B extracted from Salvia miltiorrhiza with possible anti-diabetic (neuropathy) potential	[262]
Ginseng-Sanqi-Chuanxiong extracts	Encourages mitophagy through AMPK pathway activation	Prevents cardiovascular problems	GSC extracts for cardiovascular protection	[261]

NOTE: For natural products targeting mitochondrial dysfunction in diabetes models, see reference [265] (Table 1).

Salvianolic acid B, a substance isolated from Danshen (Salvia miltiorrhiza), inhibits ROCK (Rho associated coiled-coil containing protein kinase)-mediated mitophagy in traditional Chinese medicine. This inhibition is achieved through the reduction of expression levels of fission proteins, such as FIS1 (fission, mitochondrial 1) and DNM1L, ultimately preventing endothelial cell death when exposed to elevated glucose and oxidized low-density lipoproteins [261]. Similarly, Ginseng-Sanqi-Chuanxiong (GSC) extracts, a combination of ginseng, Sanqi, and Chuanxiong, have demonstrated preventive benefits against high-glucose- and palmitate-induced cardiovascular problems associated with diabetes. Through AMPK pathway activation, GSC extracts increase autophagosome formation, reduce mitochondrial ROS, and encourage mitophagy [262].

These results demonstrate how natural compounds can help restore mitochondrial dynamics via modulation of mitophagy, offering promising therapeutic strategies for managing T2DM and associated complications. However, before being clinically translated, more research is necessary to evaluate their therapeutic effectiveness and any potential adverse effects, including in vitro and in vivo investigations in T2DM models [20]. Although research conducted in vivo and in vitro has shown that natural products can have therapeutic effects on diabetes by modulating autophagy, their efficacy as anti-diabetic drugs in clinical trials remains unclear. Before proceeding to clinical trials, several important questions need to be addressed. First, various clinical limitations need to be considered. Second, researchers' use of different experimental parameters has led to contradictory results in animal studies, further complicating the understanding of the effects of these natural products. Third, another challenge is the lack of suitable tools to quantify autophagic flow precisely in human research.

Moreover, natural products frequently have several metabolic benefits, including improving insulin sensitivity, encouraging adipogenesis, lowering oxidative stress, and maintaining the integrity of the mitochondria. This complexity underscores the need for tissue-specific approaches when studying the effects of natural products on diabetes. In summary, while natural products promise to modulate autophagy and combat diabetes, further research is needed to address these challenges before their efficacy can be fully understood and translated into clinical practice.

8. Current gaps in knowledge and future directions

Effective management of DN presents a multifaceted challenge due to its complex etiology and diverse clinical presentation. The precise mechanisms of nerve damage remain elusive, varying based on individual factors such as diabetes type, duration, and comorbidities. This heterogeneity is further compounded by overlapping symptoms with other diabetic complications, rendering diagnosis difficult and requiring a multi-pronged approach involving medical history, physical examination, and various diagnostic tests. Unfortunately, no curative therapy exists for the underlying nerve damage, leaving symptom management as the primary focus. However, this is hindered by the potential side effects and contraindications of available treatments, particularly for older patients or those with comorbidities. Pharmacological options may induce drowsiness, dizziness, nausea, or allergic reactions, while non-pharmacological therapies such as acupuncture or electrical stimulation may be invasive or costly. Surgical interventions such as nerve decompression or amputation are even riskier and irreversible.

Further complicating the matter is the potential for treatment resistance and progression of neural damage. Additionally, access to and adherence to treatment vary significantly, creating disparities in outcomes. Therefore, a personalized and holistic approach is crucial, requiring collaboration between patients, healthcare providers, and multidisciplinary teams. This approach should be informed by the latest research, patient preferences, and a careful evaluation of the benefits and risks of each treatment option. Furthermore, integration with overall diabetes management is essential, including maintaining optimal glycemic control, blood pressure, and lipid profiles alongside healthy lifestyle modifications like smoking cessation, weight management, and physical activity. Regular monitoring and adjustments based on individual response and overall condition are vital for optimizing treatment outcomes and improving quality of life for patients with DN [266].

Mitochondrial ROS production contributes to the long-term deterioration of insulin sensitivity as seen during type 2 diabetes. Mitochondria-mediated regulation of cellular energy dynamics also appears crucial in promoting insulin resistance within renal tissue. Moreover, molecular factors including AMPK, SIRT1, MTORC1 (mechanistic target of rapamycin kinase complex 1), ATGs, and so forth that selectively regulate autophagy-mediated responses hold promise for being targeted as therapeutic modalities during DN. As DN is a chronic disease and its symptoms appear at the advanced stages, an improved knowledge about the mechanism that regulates mitophagy may be helpful in negating the progression of the disease. Studies are warranted for factors influencing mitophagy-facilitated quality control and pathways that are altered in the diabetic ambience causing impaired mitochondrial function. Due to more dependency of renal cells on mitochondria due to ATP requirements, minimal changes in the mitochondrial dynamics and mitophagy can prove to be lethal. Newer diabetic therapies shall strategize on molecular agents regulating mitochondrial energetics to check ROS production and mitophagy for regulating metabolic disturbances. Pharmacological interventions must also focus on mechanisms regulating mitochondrial hormesis which show a compensatory response through activating AMPK, SIRT1, SIRT3, and PPARGC1A pathways. Based on the current reports on mitochondrial functions in disease conditions, effective pharmacological agents are needed that may selectively enhance the mitophagy-derived cell survival processes [267].

To meet cells' metabolic needs, mitochondria adjust their dynamics, including biogenesis, mitophagy, and fission/fusion balance, to align with cellular energy demands and nutrient availability. In conditions of nutrient excess, such as hyperglycemia, elevated glucose levels can disrupt mitochondrial function, leading to fragmentation. These fragmented mitochondria impair autophagic flux and promote ROS generation.

The molecular processes of mitochondrial dynamics implicated in the development of insulin resistance and T2DM have been discussed in this review. Additionally, natural substances that improve mitophagy and restore mitochondrial dynamics have been covered. Importantly, natural products offer advantages over conventional drugs, with potentially fewer side effects when used appropriately [268]. Numerous natural compounds have shown promising antidiabetic effects in both clinical trials and animal studies, indicating their potential as new treatments for diabetes. Combining these natural products with conventional drugs could offer a novel approach for managing T2DM. However, clinical trials face challenges such as potential conflicts between conventional medications and natural compounds and unknowns about the safety, therapeutic window, bioavailability, and side effects of natural substances.

To fully grasp the potential of natural products in T2DM therapy, further study is required, especially in animal studies concentrating on pharmacokinetics and pharmacodynamics. In the future, researchers investigating novel mitophagy-modulating drugs for diabetes treatment might refer to this review as a helpful overview.

9. Concluding remarks

Mitophagy, the cellular process of selectively eliminating damaged or superfluous mitochondria, appears to play a critical role in the development and progression of DN. Studies suggest that impaired mitophagy in diabetic conditions leads to an accumulation of dysfunctional mitochondria, contributing to increased oxidative stress, reduced energy production, and ultimately, neuronal damage.

Emerging evidence indicates that targeting mitophagy enhancement holds promise as a potential therapeutic strategy for DN. Several approaches are being explored, including the use of natural compounds, pharmacological agents, and gene therapy. While preclinical and initial clinical trials are ongoing, further research is crucial to validate these approaches and translate them into effective treatments for patients. It is important to acknowledge that the precise mechanisms linking mitophagy dysfunction to DN are still under investigation. Individual patient factors and variations in diabetes subtypes likely influence the specific role of mitophagy. Addressing these complexities will be essential for developing personalized and targeted treatment strategies.

Overall, targeting mitophagy represents a promising horizon for therapeutic intervention in diabetic neuropathy. Continued research and development in this field offers hope for improving the lives of individuals living with this debilitating condition.

Figure 4.

Mitophagy and cellular homeostasis versus cell death. GSH: reduced glutathione; GSSG: glutathione disulfide; LPS: lipopolysaccharide; SCAF11: SR-related CTD associated factor 11; TNFRSF: TNF receptor superfamily.

Table 1.

A summary of the most substantial mechanisms leading to diabetic neuropathy.

Mechanism	Role in diabetic neuropathy progression	Reference(s)
Hyperglycemia	Chronic hyperglycemia can damage nerve fibers throughout the body, leading to various types of DNs	[39, 40]
Oxidative stress	Increases mitochondrial production of free radicals due to hyperglycemia-induced oxidative stress	[41, 42]
Advanced glycation end-products	Formation of advanced glycation end-products affects neuronal activity and mitochondrial function	[41, 43]
Polyol pathway activation	Activation of ALDRL/polyol aldose reductase signaling contributes to nerve damage, and thus DN progression	[40, 41]
Mitochondrial dysfunction	Altered mitochondrial dynamics due to nutrient excess in hyperglycemia, leading to fragmentation and impaired autophagic flux	[41, 44]
Inflammatory cytokines	Inflammation and immune system dysregulation principally involved in nerve damage, and thus promoting DN expansion	[39, 45]
ER stress	Hyperglycemia-induced ER stress triggers apoptotic processes in neurons, resulting in DN	[41, 46]
Impaired nerve perfusion	During diabetes, dyslipidemia and altered redox status lead to reduced nerve blood flow and function, triggering the development of DN	[41, 47, 48]
Calcium balance perturbation	Disruption in calcium homeostasis affects nerve function in diabetic conditions, leading to DN	[41, 49]
Mitophagy disruption	Impaired removal of damaged mitochondria due to dysfunctional mitophagy	[41, 50]

Highlights.

• Mitophagy is crucial for maintaining metabolic balance and preventing insulin resistance, key factors in T2DM and the development of associated complications.

• Malfunctioning mitophagy in diabetic neuropathy leads to a cascade of metabolic disruptions, including reduced energy production, increased oxidative stress, and cell death.

• Targeting mitophagy to enhance the process may be a promising strategy for treating T2DM and relevant defects.

• β-cell protective and mitophagy-stimulating agents may hold potential as novel therapeutics against diabetic neuropathy.

Abbreviations

AMPK

AMP-activated protein kinase

AS-IV

astragaloside intravenous

ATP

adenosine triphosphate

DMOG

dimethyloxalylglycine

DN

diabetic nephropathy

DNP

diabetic neuropathic pain

DPN

diabetic peripheral neuropathy

DRG

dorsal root ganglion

ER

endoplasmic reticulum

Fe/S

iron-sulfur

GSC

Ginseng-Sanqi-Chuanxiong

IMM

inner mitochondrial membrane

MPTP

mitochondrial permeability transition pore

MQC

Mito-QC

mtBE

mitochondrial bioenergetics

mtDNA

mitochondrial DNA

OMM

outer mitochondrial membrane

PBM

photobiomodulation

PCD

programmed cell death

PCN

piceatannol

PDN

painful diabetic neuropathy

RHIM

receptor-interacting protein homotypic interacting motif

ROS

reactive oxygen species

STZ

streptozotocin

T1DM

type 1 diabetes mellitus

T2DM

type 2 diabetes mellitus