Insights Into The Pathogenesis Of Neurodegenerative Diseases: Focus On Mitochondrial Dysfunction And Oxidative Stress Part 2

Several post-translational modifications also regulate mitochondrial dynamics. Drp1 phosphorylation can stimulate either fission or fusion depending on the phosphorylation site [100,101]. 

With the continuous development of neuroscience, more and more studies have shown that there is a close relationship between mitochondrial dynamics and memory, which has a positive and progressive meaning.

Mitochondria are an important organ in cells, with two main functions: one is to provide energy for cells, and the other is to regulate cell development and life activities. The function of mitochondria is not only related to cell health but also closely related to the physical health of the human body. Human memory is one of them.

Studies have found that in the human brain, many changes have occurred in mitochondrial dynamics, such as increased mitochondrial density, mitochondrial migration, and mitochondrial regeneration. These changes are not only related to the health of neurons in the brain but also closely related to the improvement of brain memory.

Specifically, mitochondrial dynamics can promote information transmission between brain neurons, thereby promoting the formation, consolidation, and renewal of memory and learning. In addition, extreme sports and other types of sports can promote the increase of mitochondrial density, thereby enhancing the brain's memory and cognitive ability.

Therefore, we should actively participate in some activities that are beneficial to physical health, such as exercise, fitness, developing good eating habits, etc., to promote the development of mitochondrial dynamics and enhance memory and cognitive ability. At the same time, we should also pay attention to rest, reduce stress, maintain good health, and thus actively treat future life.

In summary, there is a close relationship between mitochondrial dynamics and memory. An active lifestyle and healthy habits are beneficial to the development of mitochondrial dynamics, thereby improving the brain's memory and cognitive ability. It can be seen that we need to improve memory, and Cistanche can significantly improve memory because Cistanche has antioxidant, anti-inflammatory, and anti-aging effects, which can help reduce oxidative and inflammatory responses in the brain, thereby protecting the health of the nervous system. In addition, Cistanche can also promote the growth and repair of nerve cells, thereby enhancing the connectivity and function of neural networks. These effects can help improve memory, learning ability, and thinking speed, and can also prevent the occurrence of cognitive dysfunction and neurodegenerative diseases.

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Phosphorylation of MFF increases Drp1 recruitment, and subsequent mitochondrial fission [102], while ubiquitination of acetylated Mfn1 promotes its proteasomal degradation [103]; phosphorylation of Mfn1 by ERK inhibits mitochondrial fusion, promoting apoptosis [104]. 

Specific nutrient states also indirectly regulate the balance between these two processes. While starvation leads to fused and elongated mitochondria, a nutrient-rich environment is accompanied by fragmented mitochondria [105,106].

3.4. Autophagy

Autophagy is another essential step in maintaining the balance between protein synthesis and clearance, organelle biogenesis, and degradation, thereby promoting cellular health [107]. Based on how the targeted cargo is conveyed for degradation to lysosomes, autophagy can be classified into [107,108]:

- Macroautophagy, in which the autophagosome, a double-membraned vesicle, forms, and fuses with lysosomes after which their content is degraded by the acidic hydrolases of the lysosomes;

- Microautophagy, a process during which lysosomes wrap around various cytosolic compounds which are degraded after the involution of the membrane [109];

- Chaperone-mediated autophagy, a process during which chaperones bind to damaged proteins and receptors on the lysosomal membrane, followed by translocation of the protein into the lysosome for degradation [110].

Autophagy for damaged mitochondria is also known as mitophagy. The first step in mitophagy initiation is the formation of an isolation membrane, the autophagosome, believed to derive from MAMs, membranes of the ER, or plasma membrane [111,112], followed by activation of the pre-initiation complex, containing ULK1 (Unc-51-like kinase 1, Atg 13 and 101 (autophagy-related proteins), and FIP200 (focal adhesion kinase family interacting partner 200) [113]. The pre-initiation complex recruits class III phosphatidylinositide 3-kinase (PI3K), beclin1, Atg 14, autophagy, and beclin 1 regulator (AMBRA1), as well as vascular protein sorting 34 and 15 (Vps 34 and 15) to produce phosphatidylinositol 3-phosphate (PI3P). 

Pi3P is also known as the initiation complex [114]. After activation, both complexes translocate to the nucleation site of the phagophore [108]. PI3P is recognized and interacts with other IM-located proteins, such as WD repeat protein interacting with phosphoinositide (WIPI) and FYVE domain-containing proteins [115] and leads to a series of conjugations of Atgs on the phagophore, culminating in cleavage of pro-LC3 (light chain 3) by Atg4 to LC3-I, further transformed by phosphatidylethanolamine to LC3-II, and leading to elongation and closure of the isolation membrane [116,117]. 

Υaminobutiric acid type A-receptor-associated protein (GABARAP) and GABARAP-like 1 protein (GABARAPL1) are believed to play similar roles with LC3 in autophagosome expansion [118]. 

The signaling protein mTOR (mammalian target of rapamycin) strongly modulates autophagy. Inhibition of mTOR, as occurs in starvation, dephosphorylates and activates Atg13, igniting the mitophagy process [119]. 

When growth factors and cellular nutrients are abundant, mTOR phosphorylates Atg 13, preventing its binding to ULK1 and recruitment of FIP200 [107]. Fusion of autophagosomes to lysosomes is mediated by Rab7 and LAMP-2, a lysosomal transmembrane protein [120,121]. After fusion, lysosomal enzymes, mainly cathepsins, degrade the autophagosomal content [122]. 

In non-receptor-mediated mitophagy, the mitophagy induction process activates PTEN-induced kinase 1 (PINK1), which accumulates on the OMM and recruits and phosphorylates Parkin [123,124]. Parkin accumulates on the OMM and ubiquitinates OMM proteins, which leads to increased activity of PINK1 and more Parkin recruitment [113]. 

Among the proteins ubiquitinated by Parkin are voltage-dependent anion channels 1 (VDAC1), Mfn1, and Mfn2, as well as TOM20 (translocase of the outer mitochondrial membrane 20), which govern mitochondrial fusion. 

Ubiquitination of Mfn1/2 will block the fusion process and allow the isolation of the small and damaged mitochondria [125]. 

The ubiquitinated proteins recruit autophagy adaptor proteins, such as OPTN (optineurin), NBR1 (neighbor BRCA1), TAX1BP1 (Tax-1 binding protein), NDP52 (nuclear dot protein 52), or sequestosome-1, which interact with autophagosome proteins like GABARAP or LC3 through LC3 interacting regions (LIR) to mediate autophagosome formation and fusion with lysosomes [126,127]. Figure 2 shows schematically the mitophagy process.

Figure 2. Schematic diagram of mitophagy. The mitophagy induction process activates PINK1, which accumulates on the OMM and recruits and phosphorylates Parkin. 

The latter ubiquitinates OMM proteins, such as mitofusins 1 and 2 (Mfn), voltage-dependent anion channels 1 (VDAC1), and TOM20 (translocase of the outer mitochondrial membrane 20). 

Autophagosome formation starts with activation of the pre-initiation complex, containing ULK1 (Unc-51-like kinase 1), Atg 13 and 101 (autophagy-related proteins), and FIP200 (focal adhesion kinase family interacting partner 200), and continues with recruitment of class III phosphatidylinositide 3-kinase (PI3K), beclin1, Atg 14, AMBA1 (autophagy and beclin 1 regulator), and Vps 34 and 15 (vascular protein sorting), resulting in the production of phosphatidylinositol 3-phosphate (PI3P). 

The ubiquitinated proteins recruit autophagy adaptor proteins, such as neighbor BRCA1 gene (NBR1), optineurin (OPTN), Tax-1 binding protein (TAX1BP1), nuclear dot protein 52 (NDP52), or sequestosome-1, which interact with autophagosome proteins like GABARAP or LC3 to mediate autophagosome formation. Fusion of the autophagosome with lysosomes is mediated by LC3, Rab7, LRRK2, and LAMP-2 (see text).

However, Parkin-independent pathways of mitophagy exist as well [113], such as receptor-mediated mitophagy. The most studied proteins involved in receptor-mediated mitophagy are AMBRA1, FUNDC1 (FUN14 domain-containing protein 1), NIX (Nip3-like protein), and BNIP3, located on the OMM, as well as cardiolipin and prohibitin 2 (PHB2) on the IMM [113]. 

These receptors can bind to LC3 in a Parkin-independent manner [128] and induce mitophagy. Their transcription can be activated under various conditions. For example, transcription of BNIP3 and NIX are activated by hypoxia via hypoxia-inducible factor 1 alpha (HIF1α) [129], which, after phosphorylation has a high binding affinity for LC3 [130]. Dephosphorylation of FUNDC1 by hypoxia facilitates its binding to LC3 [131]. 

In recent years, researchers have shown that mitochondria can be extruded from cells and taken up by endocytosis or phagocytosis by neighboring cells, where they ultimately undergo mitophagy, a phenomenon termed transcellular mitophagy [113,132]. 

It is reasonable to assume that transferring mitochondria back to the cell soma from dendrites or axons would be energetically unfavorable, which is why neurons release mitochondria at synapses to be degraded by glial cells [133]. 

In turn, glial cells can transfer mitochondria to neurons and protect the latter from hypoxia and energetic failure [134]. The precise pathways for mitochondrial transfer are still being investigated, but some studies suggested an important role for a protein connecting mitochondria to cytoskeletal motor proteins, namely MIRO1 [135] while others showed involvement of astrocytic GFAP (glial acidic fibrillary protein) and neuronal UCP2 (uncoupling protein 2) [136]. Another way of disposing of damaged mitochondria has been identified in 1992 [137] and named mitoptosis. 

It enables the cells to degrade mitochondria without opening the MPTP and igniting apoptosis [113]. Mitoptosis is likely activated by mitochondrial membrane depolarization, damage of the mitochondrial DNA (mtDNA), and ROS [113]. 

The exact mechanisms of mitoptosis require further study, but several situations have been described, such as swelling and fragmentation of cristae followed by cytoplasmic extrusion of cristae fragments through bursting of the OMM [138], or deterioration of cristae through coalescence of the IMM with preservation of intact OMM [138]. 

ROS plays a key role in the regulation of autophagy and mitophagy. One pathway, mentioned above, is the mTOR pathway. An amino acid-rich environment leads to the translocation of mTOR complex 1 (mTORC1) to the lysosomal surface, where it interacts with Rheb and activates mTOR [139], while in starvation mTOR colocalizes with LC3 and initiates autophagy [140]. 

Considering that mTOR oxidation inhibits its activity, it is very likely that ROS regulates this step [141]. Further, S-nitrosation of IκB kinase β and JNK1 by nitric oxide inhibits their activity, which, in turn, prevents mTOR inactivation and release of Beclin from the Beclin-Bcl-2 complex [93,142]. 

Another pathway is the beclin-1-class III PI3K complex, with a series of cofactors such as AMBRA1, Bax-interacting factor 1 (Bif-1), or Rubicon (RUN domain- and cysteine-rich domain-containing beclin-1-interacting protein) [143]. Other regulators of autophagy include the IP3 receptor, AMPK (50 -AMP-activated protein kinase), and DAPK (death-associated protein kinase) [144,145]. 

ROS and reactive nitrogen species induce post-translational protein modifications which also regulate the activity of transcription factors. For example, in the Nrf2 (nuclear factor-erythroid 2-related factor 2)/Keap1 (Kelch-like enoyl-CoA hydratase-associated protein 1) pathway, modifications of Keap1 lead to the release of Nrf2, which binds to ARE (antioxidant-response element) and translocates to the nucleus, where it activates the transcription of antioxidant enzyme genes and proteins, such as p62 or p53. 

While p62 activates autophagy [146,147], p5 is linked to both autophagy-inhibiting and -promoting genes through TIGAR (tumor protein 53-induced glycolysis and apoptosis regulator) and DRAM (damage-regulated autophagy modulator), respectively [148,149]. 

In turn, impaired regulation of autophagy leads to increased oxidative stress and accumulation of ubiquitinated proteins, the latter causing mitochondrial dysfunction and further augmenting ROS generation in a feed-forward loop [93].

4. The Brain and Oxidative Stress

Oxygen is crucial for proper cellular functioning, being involved in the generation of ATP [61]. Unfortunately, due to the univalent metabolic reduction status of oxygen, with the two lone electrons of oxygen spinning in parallel, it can accept only one electron at a time [150], leading to the generation of species having one unpaired electron which can exist on their own, defined by Halliwell as free radicals [151]. 

The oxygen derivatives are either free radicals, such as the superoxide anion (•O2−), hydroxyl radical (HO• ), hydroperoxyl radical (HO2 • ), and peroxyl radicals (ROO• ), or non-radicals which can be transformed into radicals, such as hydrogen peroxide (H2O2) [61,152]. 

Redox signaling is extensively used in the brain [153], being involved in signal transduction and gene transcription. For example, NADPH oxidases (NOXs) regulate hippocampal long-term potentiation [154], and NOX2- derived superoxide and hydrogen peroxide regulate hippocampal progenitor cell growth in adult brain via the phosphatidyl inositol 3 kinase (PI3K)/Akt signaling pathway [155]. 

Similarly, NOX-derived H2O2 has beneficial roles in axonal regeneration and axonal pathfinding during wiring of the developing brain [156,157]. As a consequence of hypoxia,mitochondria-derived superoxide-induced signaling leads to adaptive responses [158]. 

To counterbalance the possible deleterious effects of excessive free radicals, the biological systems have a series of antioxidant defenses, which can be divided into enzymatic ones (superoxide dismutases, catalase, glutathione peroxidases, glutathione transferases, thioredoxins, and peroxiredoxin) and non-enzymatic defenses, such as vitamins A, C, E, beta-carotene, or glutathione [159]. 

Whenever the rate of free radical production exceeds the biological system's ability to neutralize them, oxidative stress ensues.

4.1. Vulnerability of the Brain to Oxidative Stress

The nervous system is very sensitive to oxidative stress, due to a series of reasons [153,160–162]: - Action potentials cause calcium influx and raise the intracellular calcium concentration from approximately 0.001 µm to roughly 100 µm [163]. 

High intracellular Ca2+ activates nNOS (neuronal nitric oxide synthase) and leads to NO (nitric oxide) formation [164], which binds to cytochrome c oxidase and inhibits mitochondrial respiration [165]. Mitochondria attempt to buffer intracellular calcium, but the subsequent calcium overload causes prolonged opening of the MPTP and inhibits ATP generation, inducing apoptosis [166].

The brain has very high energy demands to maintain the ionic gradients and support synaptic transmission [19] and relies mainly on synaptic mitochondria for the generation of required energy [167]. For example, neurotransmitter vesicle release requires 1.64 × 105 ATP/s/vesicle [19]. 

- The brain has low antioxidant defenses. Neuronal cells have 50 times less catalase than hepatocytes [168], while cytosolic glutathione is about 50% lower in neurons compared with other cells [153], and this might diminish peroxiredoxin activity [169]. - Microglia, the immune cells of the brain, are activated by H2O2 [170] and produce superoxide via NADPH oxidase isoforms, needed for bacterial killing [171]. 

- The metabolism of neurotransmitters, such as dopamine metabolism via monoamine oxidases, generates ROS [153,172]. - Neurotransmitters, such as dopamine, serotonin, or adrenaline, can auto-oxidize and generate superoxide [173,174]. 

- The brain is enriched in redox-active transition metals, such as Cu+ or Fe2+ [175]. Iron is a catalyzer in the hydroxyl radical-generating Fenton reaction, and also catalyzes peroxyl and alkoxyl radical generation, thereby contributing to ferroptosis, a form of cell death dependent on lipid peroxidation and Fe2+ [176]. Cu+ is a co-factor for Cu/ZnSOD and is important for cell signaling [177,178] but enhances copper-catalyzed Fenton reaction [175]. 

- The brain is particularly rich in cholesterol, which may undergo auto-oxidation [179] and brain cells have a higher membrane surface/cytoplasmic volume ratio, cellular membranes being rich in polyunsaturated fatty acids (PUFA), which are highly susceptible to peroxidation through free radical attack [153]. - Brain development and plasticity rely on non-coding RNAs (long non-coding RNAs and microRNAs) [180], but these molecules lack protective histones and are easily oxidized [181]. Oxidized messenger RNA results in truncated and mutated proteins, prone to misfolding [182].

4.2. Sources of Free Radicals

Excess free radicals can be generated from many sources.

4.2.1. Mitochondria and Oxidative Stress

Mitochondria are traditionally regarded as the main sources of ROS. At least 10 potential sources of ROS production have been identified [183] but complexes I (NADH dehydrogenase) and III (ubiquinone cytochrome c reductase) of the ETC [184] are the most important ones. 

Transferring electrons to coenzyme Q or ubiquinone by complexes I and II results in ubiquinol (reduced ubiquinone, QH2), which will regenerate coenzyme Q via semiquinone anion (•Q−), an unstable intermediate that can transfer electrons to molecular oxygen, resulting in superoxide formation [60]. Being a non-enzymatic reaction, higher metabolic rates lead to increased superoxide production [185]. 

Superoxide is highly unstable and is transformed by the mitochondrial superoxide dismutase 2 (SOD2, manganese SOD) and the cytosolic SOD1 (copper-zinc SOD) into the more stable hydrogen peroxide (H2O2). 

The latter can exit the IMM through aquaporin channels and diffuse through the OMM into the cytoplasm, where it serves for redox signaling, or is further reduced to water by catalase, glutathione peroxidases, and peroxiredoxins [186–189]. Other mitochondrial components contributing to ROS formation include monoamine oxidase, glycerol phosphate dehydrogenase, α-ketoglutarate dehydrogenase, and p66shc [60,183,190]. 

Mitochondrial ROS production is subject to variations induced by metabolic factors. For example, the NADH/NAD+ ratio affects the rate of ROS generation, which increases almost linearly with NADH reduction [191]. Succinate levels can fluctuate even in normal conditions between 0.3 and 1 mM [192], with increased succinate concentrations strongly increasing mitochondrial ROS generation [183,193]. 

The rate of ROS production is also influenced by the mitochondrial membrane potential [183]. Active mitochondrial phosphorylation of ADP or mitochondrial calcium uptake decreases the membrane potential, which influences the redox potential of the ETC and decreases ROS production [194]. Oxygen deprivation or ischemia significantly increases mitochondrial ROS generation [195], although this effect may not be attributed to mitochondria per se, but rather to signaling pathways triggered by hypoxia [196].

4.2.2. NADPH Oxidase as a Source of ROS

NADPH oxidase (NOX) was first described in phagocytes [197], after which seven NOX genes have been identified: NOX 1–5 and DUOX 1 and 2 [198]. The brain expresses mainly NOX2, as well as NOX4, both being described in the cortex and CA1 hippocampal areas [199]. 

The NOX2 enzyme complex has a membrane-bound cytochrome b558, several cytosolic proteins, and the Rac G-protein. Following phosphorylation of the cytosolic proteins and activation of Rac, the enzyme translocates to the membrane and forms active NOX2 with cytochrome b558 [200]. Further, NOX2 transfers protons across the membrane and leads to superoxide generation [198]. 

NOX4 produces mainly H2O2, used as a second messenger for cell proliferation and differentiation [201]. 

NOX has been described in neurons, astrocytes, and microglia [202], while at the cellular level, NOX isoforms localize to the endoplasmic reticulum, nucleus, plasma membrane, and mitochondria [203,204]. The ROS generated by activated NOX can depolarize the mitochondrial membrane and, together with calcium, can lead to the opening of the MPTP [205] as well as activate phospholipase C with subsequent changes in membrane structure [198].

4.2.3. Monoamine Oxidase as a Source of ROS

The monoamine oxidases (MAO A and B) are flavoenzymes located on the OMM which catabolize amine neurotransmitters, such as serotonin, epinephrine, and dopamine [206]. 

MAO-A is expressed in neurons, while MAO A and B can be found in glial cells [198]. They use FAD to break down monoamines, a process during which aldehydes are produced, while H2O2 results from the FAD-FADH2 cycle [198].

4.2.4. Peroxisomes and ROS Production

Although the major metabolic peroxisomal process leading to H2O2 generation is β-oxidation of free fatty acids [60], several other peroxisomal enzymes, such as xanthine oxidase, D-aspartate oxidase, acyl CoA oxidases, D-amino acid oxidase, urate oxidase, or L-α-hydroxy oxidase, can produce a variety of ROS, such as superoxide, hydrogen peroxide, nitric oxide, or hydroxyl radicals [207].

4.2.5. Exogenous Sources of ROS

In addition to the multiple sources of endogenous ROS, exogenous ROS can augment oxidative stress. The most common sources are water and air pollution, ultraviolet light exposure, alcohol and tobacco smoke, pesticides, industrial solvents, unhealthy diets (with smoked meat, high-fat diet), exposure to heavy metals or transition metals (Fe, Cr, Co, Cu, Hg, Pb, As), as well as certain drugs, such as Doxorubicin, Bleomycin, Metronidazole, or even Paracetamol [60].

4.3. Targets of ROS

The highly reactive free radicals damage proteins, lipids, and nucleic acids [152].

4.3.1. Proteins and ROS

Proteins can be oxidized by radicals, such as superoxide, hydroxyl radical, peroxyl, hydroperoxyl, or alkoxyl radicals, as well as by non-radical species or singlet oxygen [208]. 

Oxidation of amino acids such as lysine, arginine, proline, or threonine leads to carbonyl derivatives, used as markers of oxidative stress [209]. Methionine and cysteine, as sulfur-containing amino acids, are very susceptible to oxidation, leading to disulfides and methionine sulphoxide [210]. 

Protein oxidation leads to protein-protein cross-linkages, altering of function, loss of enzymatic activity, and functional modifications of receptor and transport proteins [211]. 

Moreover, hydrogen peroxide and hydroxyl radicals inhibit glutamate uptake by astrocytes, augmenting excitotoxicity [212]. As a consequence, these altered proteins must be cleared, either by the autophagy-lysosome pathway or by the ubiquitin-proteasome system [213,214]. 

The ubiquitin-proteasomal system (UPS), the main degradation pathway of ubiquitinated misfolded proteins and short-lived signaling molecules, contains a 19S subunit and a catalytically active 20S core [215]. The regulatory cap with chaperone proteins unfolds the target protein, removing the ubiquitin tag in an ATP-dependent process, after which the target protein is fed into the catalytic core and is degraded by the proteasomal enzymes. 

Under severe stress, the UPS is overwhelmed, and the autophagy-lysosome pathway compensates for the increased protein damage [107]. A series of autophagy receptors, such as p62, NDP52, or NBR1 recognize the ubiquitin moiety and target tagged proteins to the autophagosome by binding to Atg8/LC3 [216,217]. 

FOXO3 (forkhead box O3) is a transcription factor activated by oxidative stress, which regulates the transcription of genes involved in proteasomal as well as autophagic protein degradation [218]. In addition, Parkin, ubiquitin ligases, and CHIP (C-terminus of Hsc70- interacting protein) contribute to both proteasomal and autophagosomal degradation of proteins [107,219]. 

The relative levels between BAG (Bcl-2 associated transgene) 1 and BAG3, co-chaperone proteins, direct the cellular protein degradation pathway towards the proteasomal or phagosomal one [220].

4.3.2. Lipids and ROS

Free radicals or non-radical oxidative species attack the C-C double bonds of lipids, which is why polyunsaturated fatty acids are very vulnerable to oxidative attack [221]. 

In the initial phase, a free radical interacts with a methylene group in the fatty acid and generates a lipid radical by dissociating a hydrogen atom [222]. Further, the lipid radicals react with molecular O2 to form peroxyl radicals (ROO• ) [223], which initiate a chain of self-sustained reactions amplifying the process. 

This results in cyclic peroxides and hydroperoxides, which can further be degraded to aldehydes, the final products being malondialdehyde (MDA), hydroxynonenal (HNE), and acrolein [224]. 

The process is terminated either through the interaction of lipid radicals with lipid peroxides, resulting in non-reactive stable species [221], or through the intervention of endogenous or exogenous antioxidants (vitamins C and E) [222,225]. 

In low concentrations, 4-HNE plays important homeostatic roles by acting as a signaling molecule and modulating gene expression by inducing post-translational protein modifications. 

The most common targets are thiol residues [225]. By interacting with cysteine thiols in Keap1, 4-HNE leads to the release of Nrf2, which after translocating to the nucleus activates the expression of ARE genes such as glutathione-S-transferase, NADPH-dependent quinone reductase, or heme oxygenase-1 [226]. Another target of 4-HNE is NF-κB, a transcription factor for pro-inflammatory cytokines, normally maintained quiescent by binding to IκBα (inhibitor kappa B). 

Under cellular stress, IκB kinase phosphorylates IκBα facilitating the release of NF-κB, a transcription factor that mediates the transcription of antiapoptotic Bcl-2 proteins and inflammatory cytokines, such as interleukin-6 (IL-6). 

4-HNE inhibits IκB kinase, thus preventing IκBα phosphorylation and NF-κB nuclear translocation [227]. However, as a consequence of membrane lipid peroxidation, the membrane alters its permeability, increases its rigidity, and may even lose integrity [228]. 

In addition, lipid peroxidation products are involved in complex signaling pathways. Intracellular accumulation of 4-HNE can lead to apoptosis through both the intrinsic and extrinsic pathways [225]. 4-HNE increases the expression of p53, followed by activation of p21, JNK, Bax, and caspase 3 [229], leading to caspase-mediated apoptosis. 

In addition, 4-HNE initiates the binding of the death-associated protein Daxx to the intracellular surface of Fas [230], thereby being involved in the modulation of the extrinsic pathway of apoptosis through the downstream signaling proteins ASK1 and JNK [225]. 

Moreover, high levels of HNE can form conjugates with JNK, responsible for histone modification and facilitating nuclear translocation [231], or can activate JNK through SPKK1 (stress-activated protein kinase kinase-1) activation [232]. Similarly, HNE can activate ERK via activation of MEK1/2 and p38MAPK (mitogen-activated protein kinase) [233,234].

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