Insights Into The Pathogenesis Of Neurodegenerative Diseases: Focus On Mitochondrial Dysfunction And Oxidative Stress Part 3
Jul 16, 2024
4.3.3. DNA
ROS can induce many types of DNA damage, such as base modification, deoxyribose modification, single-strand breaks (SSBs) and double-strand breaks (DSBs), DNA crosslinks, or abasic sites [235].
As we learn more about biology, we are finding that base modifications can affect human memory. This idea is very exciting because it means that we can use these modifications to improve and enhance our brain function.
Studies have shown that adding methylation to exon DNA can affect memory formation and spatial learning ability. In human brain tissue samples, the DNA base 5-Hydroxymethylcytosine (5hmC) has been identified and its expression in the human adult cerebral cortex area has increased.
Further research has shown that acquiring new information and consolidating memories requires at least two different brain functions, namely "recording" and "reproduction". In the recording stage, cholinergic neurons release acetylcholine to transfer information from short-term memory to long-term memory. In this process, methylase (DNA methyltransferase) plays an important role because it can play an important role in regulating gene expression when new memories are formed.
In the next stage, the reproduction stage, neurons replay previous experiences. This means that the consolidated memory is placed in long-term memory, and some experts believe that the methylation of these genes is converted to a form that is very easy to reverse, such as 5-Hydroxymethylcytosine. That is, yutian acid plays a key role in consolidating long-term memory.
Therefore, we can see that base modification and memory are closely related. This provides us with great hope to design some new treatments to improve brain efficacy, such as cognitive enhancement, and avoid cognitive deficits. Although there is still a lot of work to be done, this is one of the breakthrough studies in our understanding and exploration of more nervous systems. It can be seen that we need to improve memory, and Cistanche can significantly improve memory because Cistanche can also regulate the balance of neurotransmitters, such as increasing the levels of acetylcholine and growth factors, which are very important for memory and learning. In addition, Cistanche can also improve blood flow and promote oxygen delivery, which can ensure that the brain obtains sufficient nutrition and energy, thereby improving brain vitality and endurance.
Oxidized DNA is probably the most common DNA lesion in neurons [236]. Under normal conditions, ROS can cause up to 50,000 DNA lesions/cell/day [237], and delays in the repair of DNA damage can cause genomic instability and induce signaling cascades leading to cell death [238].
The cells have various repair mechanisms, such as base excision repair (BER), mismatch repair (MMR), nucleotide excision repair (NER), and single- (SSBR) and double-strand break repair (DSBR) mechanisms [239].
Oxidized DNA bases are removed mainly by the BER/SSBR mechanism and involve the removal of the damaged base by a specific DNA glycosylase (DG), incision of the abasic site by an AP-endonuclease (APE1), filling of the resulting gap by a DNA polymerase, and sealing of the damaged DNA strand by a DNA ligase [240].
Several DGs have been identified, such as uracil-DNA glycosylases (UDGs), thymine DNA glycosylase (TDG), or oxidized base-specific DGs, such as 8-oxoGuanine (8-oxoG) DNA glycosylase (OGG1) [241,242]. OGG1-1a is involved in nuclear DNA repair, while OGG1- 2a contributes to mitochondrial DNA repair [243].
APE1 cleaves the sugar-phosphate backbone of DNA at the abasic site. Phosphorylation of the enzyme, as happens after 1-methyl-4-phenylpyridinium (MPP+) exposure, reduces its enzyme activity and results in the accumulation of damaged DNA in neurons [244].
Mitochondrial DNA is even more prone to oxidative damage because the mitochondrial genome is situated close to the IMM, the site of mitochondrial ROS generation, and because it lacks protective histones [245]. As mitochondrial DNA quality control is crucial for communication with the nucleus, mitochondria contain antioxidant and DNA repair enzymes, such as OGG1 and MUTYH (mutY DNA glycosylase) [246].
Mitochondrial dysfunction, as happens after mtDNA deletions, can lead to activation of NF- κB through a calcineurin-dependent signaling pathway [247] and can, in turn, be induced by various signaling molecules. For example, PPAR-γ coactivator 1 and SIRT1 regulate mitochondrial function by activating the expression of mitochondrial transcription factor A (TFAM) [248].
In addition to increased oxidative damage caused by oxidative stress, defective DNA repair mechanisms have been described in neurodegenerative diseases, such as AD or PD [235].
Dopaminergic neurons of PD patients showed upregulation of mitochondrial OGG1 and higher levels of phosphorylated APE1 [249]. Neurons from AD patients had higher levels of oxidized DNA bases in nuclear as well as mitochondrial DNA [250], exhibited a decrease of OGG1 activity [251], and lower levels of UDG [252] in comparison with neurons from healthy individuals.
4.3.4. RNA and Oxidative Damage
RNA is more abundant, accounting for 80–90% of the nucleic acid in cells [253], and is more vulnerable to oxidative damage due to its single-stranded structure, proximity to the mitochondria, lack of oxidized RNA repair mechanisms, and less protection from proteins as compared to DNA [60,254].
The functions of RNA molecules in the cell are diverse, comprising the ribosomal RNA (rRNAs), which together with the transfer RNA (tRNA) and messenger RNA (mRNA) is responsible for protein synthesis, microRNAs (miRNAs), which are post-translational regulators of gene expression, and small nuclear and nucleolar RNAs.
Traditionally, mRNA was regarded as the coding RNA [255]. The non-coding RNAs play important roles in mRNA splicing regulation and mRNA translation [256]. The most aggressive radical is the hydroxyl radical, produced in the Fenton and Haber–Weiss reactions [257].
It reacts with guanine to form 8-hydroxyguanosine (8-OHG), one of the most commonly used biomarkers of RNA oxidation [253]. The oxidative attack-induced damage to RNA leads to modified bases and nucleosides, cleavage, and fragmentation of tRNA, aminoacylation, or defects in codon-anticodon pairing [258] and causes direct RNA strand breaks [259], base mismatches on tRNAs, translation errors [182], and disordered protein synthesis [260] with significant impact on cell viability.
Although cells have several mechanisms to degrade altered transcripts, such as nonsense-mediated mRNA decay (NMD) [261], these mechanisms tend to be overwhelmed with aging. As such, altered misfolded proteins accumulate [262].
5. Selective Neuron Vulnerability in Neurodegenerative Diseases
Although the many pathways leading to increased ROS generation and their consequences apply to all cells, each neurodegenerative disease leads to the degeneration of particular groups of neurons, which led researchers to look for explanations for this selective vulnerability of neuronal populations
5.1. Selective Neuronal Vulnerability in Alzheimer's Disease
The memory loss and cognitive decline characteristic of AD are caused by atrophy of the entorhinal cortex (EC), mainly of neurons in layer II (ECII), and hippocampus, particularly the CA1 region [72].
Research has shown that neurons in these areas have high energetic demands and are very sensitive to decreased oxygen and glucose supply [263]. In addition, CA1 and EC II pyramidal neurons are glutamatergic and thereby more vulnerable to NMDA excitotoxicity and the damaging effects of increased intracellular calcium concentrations [264], as opposed to neocortical inhibitory interneurons, which have high levels of Ca2+-binding proteins [265].
A series of molecular hallmarks of the vulnerable neurons have been recently described. For example, ECII pyramidal neurons have an impaired activity of a regulator of tau splicing, likely linked to disturbed microtubule dynamics [266], which may facilitate tau spreading to other brain regions via CA1 neurons [267].
Selectively vulnerable excitatory neuron subpopulations were shown to express RORB (RAR-related Orphan Receptor B), while also exhibiting differences in the expression of genes encoding synapse- versus axon-localized proteins, subunits of the potassium channels, G-protein signaling molecules, and neurotransmitter receptor signaling molecules [268].
However, the connections between RORB expression, accumulation of phosphorylated tau, and neural degeneration remain to be further characterized.
5.2. Selective Neuronal Vulnerability in Parkinson's Disease
The motor symptoms of PD are caused by the loss of nigral dopaminergic neurons leading to dopamine depletion in the dorsal striatum [269]. Structurally, these neurons have very long and branched axons (up to 4.5 m), being connected to a large number of neurons (up to 2.4 million synapses) [270,271], which requires a high density of axonal mitochondria and challenges mitochondrial bioenergetics [272].
At the molecular level, nigral dopaminergic neurons have low Ca2+-buffering capacity despite high activity-dependent Ca2+ loads, which increase OXPHOS and ROS generation [273] and may promote mtDNA damage [274]. In addition, the metabolism of dopamine itself leads to ROS generation and causes the accumulation of mtDNA deletions [275].
5.3. Motor Neuron Vulnerability in Amyotrophic Lateral Sclerosis
The pathological hallmark of ALS is degeneration of upper and lower motor neurons, which both have very long axons and, thus, depend on proper mitochondrial function and trafficking [72].
The large motor unit size imposes high energetic demands on spinal motor neurons to maintain neurotransmission and muscle contraction [276].
The relative preservation of motor neurons in the oculomotor, trochlear, and abducens nuclei may be linked to the small number of innervated muscle fibers (up to five muscle fibers as opposed to at least 300 fibers innervated by spinal motor neurons) [277], the particular grape-like structure of the neuromuscular junction [278], as well as the high Ca2+ buffering capacity of these neurons [279].
6. Oxidative Stress in Neurodegenerative Diseases
Research has increasingly shown the presence of oxidative stress markers in the nervous system of patients having neurodegenerative diseases, but the molecular pathways are just beginning to be elucidated.
The description of genetic defects leading to AD, PD, HD, or ALS helped in identifying downstream effects of the mutant proteins, with similar pathways being subsequently demonstrated in idiopathic forms of the diseases. However, the pathophysiology is still incompletely elucidated, despite accumulated knowledge having already led to therapeutic attempts.
6.1. Oxidative Stress in Alzheimer's Disease
Familial cases of AD with mutations of Presenilin 1 (PS1) and 2 (PS2) have linked AD pathogenesis with disturbed calcium homeostasis [280]. PSs interact with the ryanodine receptors [281] and influence ER–mitochondria coupling [282].
Indeed, high MAM numbers have been described in animal models of AD and fibroblasts or brain tissue of AD patients [283]. In addition, Aβ aggregates can mediate Ca2+ transfer from ER to the mitochondria through the MCU [284], while tau inhibits mitochondrial calcium efflux [285].
Moreover, Aβ can form calcium-permeable channels in membranes [286,287], while tau can form non-selective ion channels in lipid bilayers [288]. The deleterious effects of increased mitochondrial calcium in inducing mitochondrial dysfunction and oxidative stress have been described in the previous sections.
In astrocytes, Aβ interacting with the calcium-sensing receptors (CaSRs) induces their downregulation, leading neighboring neurons to secrete newly synthesized Aβ, nitric oxide, and peroxynitrite [289].
In the mitochondria, one of the earliest alterations linked to AD is epoxidation of ATP synthase with consequent reduction of its function, described in EC neurons as early as Braak stages I-II [290,291]. Located inside the IMM, inserted in a PUFA-rich lipid bilayer, and close to the mitochondrial matrix, the enzyme is an easy target for free radicals generated by complexes I and III [292].
In addition to energetic failure, ATP synthase modification further increases ROS production with subsequent potentiation of oxidative modifications of biological molecules [293].
In this cascade, most modified lipoxidation products are involved in energy metabolism, increasing energy failure, followed by proteins involved in neurotransmission, antioxidant defenses, and ion channels, as shown in Table 1.
The communication between mitochondria and nucleus and the import of nuclear-coded mitochondrial subunits are impaired by the bioenergetic defects and dysfunctional mitochondrial proteins, leading to altered expression of regulatory and structural mitochondrial complexes and enzymes [299]. The lipid metabolism is also altered, with changes in lipidomic profiles described in AD, especially in the EC [300].
The oxidative damage to PUFAs leads to the reduction of these fatty acids in the lipid rafts in EC from early stages of AD [301], with consequences on membrane thickness, fluidity, curvature, as well as activity of membrane-bound proteins, which favor amyloidogenic processing of amyloid precursor protein.
It appears that mitochondrial oxidative stress, altered lipid metabolism, lipid peroxidation, and bioenergetic defects are all part of a self-sustained loop that contributes to augmentation of the altered mitochondrial dynamics, mitochondrial trafficking, impaired mitophagy, and impaired ER–mitochondrial interaction [290].
The intracellular build-up of amyloid beta (Aβ) and phosphorylated tau induces much of the altered mitochondrial dynamics, expressed as excessive mitochondrial fragmentation, with increased number and decreased size of mitochondria [302]. Both Aβ and phosphorylated tau increased the GTPase activity of Drp1, which leads to excessive mitochondrial fragmentation [303,304].
In vitro, mutant APP cellular lines exhibited high concentrations of mRNA and proteins of mitochondrial fission and diminished levels of mRNA and proteins of mitochondrial fusion [305]. Phosphorylated tau has also been shown to downregulate Opa1 and upregulate Mfn1 and Mfn2 [306].
In addition, cytoplasmic accumulation of Aβ leads to depletion of Parkin and PINK1 levels, thereby interfering with the mitophagy pathway. Defective mitophagy results in the accumulation of autophagic vacuoles in the neuronal soma and dysfunctional neurites [307], a process potentiated by the particularities of mitophagy in neurons, where mature lysosomes are concentrated in the cell body whereas mitochondria extend along the axons and dendrites of neurons, making neuronal mitophagy a slower process [308].
In addition, Aβ oligomers interact with autophagic vacuoles in the distal parts of axons, leading to inhibition of mitochondrial axonal transport [309]. In addition, in laboratory models, mutations in the PS1 gene altered lysosomal acidification [310] and led to diminished expression of autophagy-related genes through the ERK/CREB signaling pathway [311].
Protein tau has also important contributions to the protection of the cellular genome in physiological conditions, by binding chromatin [236]. The hyperphosphorylated state of tau interferes with this function, creating the premises for the infliction of more oxidative DNA damage and the longer time needed to repair these DNA lesions [236].
One must not overlook the role of microglial activation and chronic inflammation in the pathogenesis of AD. Microglia, the innate immune macrophage-like cells of the nervous system, account for about 10% of the cellular population in healthy adult brains [312].
In AD, microglia have increased expression of complement receptors leading to upregulation of the NF-κB signaling pathway, Aβ-activated Fc receptors (which induces the expression of MIP-macrophage inflammatory protein-1α), and increased expression of scavenger receptors A-1 and B (SCARA and SCARB) [313,314]. Binding of Aβ to SCARB-2 activates microglia, to produce proinflammatory cytokines, chemokines, and ROS [315].
Increased levels of tau, by inducing the expression of toll-like receptors, also lead to the release of pro-inflammatory cytokines IL-1β, IL-6, and IL-8 through the NF-κB signaling pathway [316]. Although inflammation activates autophagy in cells [108], since impairment of autophagy affects microglial cells as well, the chronic inflammatory microglial phenotype supplementary contributes to neuronal damage in AD [317].
6.2. Oxidative Stress in Parkinson's Disease
PD is the second most common neurodegenerative disease, exhibiting both cognitive and neuromuscular impairments.
Although most cases are sporadic, several mutations have been described in familial and early-onset forms of PD, such as mutations in PARK1 (encoding PINK1), PARK2 (encoding for Parkin), PARK1/4 (α-synuclein), PARK7 (DJ1), PARK8 (LRRK2), PARK9 (ATP13A2) PARK17 (Vsp35), FBX07, GIGYF2, or HTRA2, highlighting the involvement of the ubiquitin protein degradation pathway, oxidative stress, cell survival pathway, mitochondrial function, and apoptosis in PD pathogenesis [113,318].
The pathological hallmark of PD is the accumulation of insoluble inclusions consisting predominantly of synuclein, (Lewy bodies) mainly in the nigral dopaminergic neurons [61].
The study of toxin-induced Parkinsonian syndromes has helped in elucidating PD pathogenesis. In the 20th century, exposure to 1-methyl-phenyl 4-phenyl-1,2,3,6-tetrahydr pyridine was shown to cause symptoms resembling severe parkinsonism [319] by interfering with complex I of the ETC.
Further, in an experimental setting, rotenone, a complex I inhibitor, led to apoptosis in human neuroblastoma cells, and this finding was expanded to discuss the involvement of pesticide exposure in the etiology of PD [320].
Subsequently, several researchers consistently reported a defect of complex I of the mitochondrial ETC leading to a 30–40% decline in its activity [48,321], caused by a diminished rate of production of complex I subunits, destruction of its structure, and oxidative damage [322]. α-synuclein targets mitochondria and leads to a decrease in complex I activity [323].
LLRK2 (leucine-rich repeat kinase 2) mutations can increase α-synuclein levels [324]. PINK1 and Parkin regulate mitophagy, as described earlier. DJ1 is important for mitochondrial integrity and dynamics; overexpression of DJ1 has been shown to reduce mitochondrial fragmentation induced by rotenone, independently of PINK1 [325].
Convincing evidence has shown the accumulation of increased lipid, protein, and DNA oxidation products in the degenerating neurons in PD [326–328] together with a reduction in the antioxidant GSH [329], thereby implicating oxidative stress in the pathogenesis of PD.
Initially, ROS probably originate from the ETC, external factors, and dopamine auto-oxidation [330], potentiated later by monoamine oxidase B metabolism of dopamine, inflammatory responses, or the contribution of heavy metals [331].
ROS have been shown to induce unorthodox activation of Iron Regulatory protein 1, thereby contributing to iron accumulation in dopaminergic cells [332]. Hydrogen peroxide can easily diffuse to adjacent neurons, where it can generate hydroxyl radicals through its interaction with iron [55].
PINK1 mutations in the kinase domain of the mitochondrially located molecule also increase the cells' susceptibility to oxidative stress [55,333].
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