Dihydrolipoic acid is a hydrolyzed form of α-LA. Several studies indicated DHLA as a metal chelator, antioxidant, radical scavenger and anti-inflammatory agent [20–22]. Both α-LA and DHLA have also been proposed to have neuroprotective effects and can be an option in treating AD [23–26]. However, very little data were published about the ability of this α-LA to reduce possible damage caused by aluminum, whereas no data is available for DHLA [27, 28]
In the present study, we report that Al exposure causes a significant increase in oxidative stress parameters and ROS levels. Al's toxicity is primarily due to its pro-oxidant activity, which promotes oxidative stress, free radical attack, and oxidation of cellular proteins and lipids. When Al ions interact with protein polypeptides via oxygen-containing amino acids, side chains, and protein backbone, secondary structure alteration occurs, culminating in eventual denaturation or conformational or structural change, such as forming A plaques [29]. Al appears to enhance oxidative stress and damage by affecting iron homeostasis. This particular effect results in iron overload. Elevated cellular iron concentrations can exacerbate oxidative damage and are associated with etiology of certain neurodegenerative diseases. Iron overload caused by Al has been linked to enhanced lipid peroxidation, DNA damage, and ROS-induced apoptosis [30]. In the present study, Al-exposed cells treated by DHLA showed a significant decline in ROS and oxidative stress due to this compound's ability to neutralize the free radicals and metal chelation. In one study, it is stated that DHLA lowers the redox activity of transition metal ions but does not remove them from the active site of enzymes[31]. In another study, it is suggested that relative to the Fe3+/Fe2+reduction with ascorbate, DHLA can fully inhibit the formation of hydroxyl (•OH) radicals but not with superoxide (O2−) [32].
Moreover, DHLA protects against iron-induced lipid peroxidation [1]. We can state that the phosphorylated protein Tau is deposited by Fe3+, and its induced aggregation can be reversed when Fe3+ is converted to Fe2+. These findings suggest that iron may play a role in improperly phosphorylated Tau aggregation, which culminates in neurofibrillary tangles. According to the latest theory, Tau is primarily involved in the interactions of iron ions in neuronal cells throughout the pathogenic framework of AD [33]. In the present work, when treated with DHLA, AD and Al-exposed groups showed a significant decrease in tau hyperphosphorylation, which can be assigned to the metal chelation effect of DHLA. However, this effect should be proven by further investigations.
Tau is a phosphoprotein whose principal roles are stabilizing microtubules' structure and enhancing axon transit. Tau hyperphosphorylation, primarily produced by aberrant protein kinase and phosphatase activity, is one of the initial stages in the pathophysiology of AD and is regulated by Tau protein kinase activation and phosphatase inhibition. Oxidative stress may directly interact with GSK-3β and cause Tau phosphorylation and the development of neurofibropathy [34]. Under pathological conditions, ROS disrupts Tau's normal function, causing it to engage with cyclin-dependent protein kinase-5 and GSK-3β kinases, resulting in hyperphosphorylation. As a result, it loses its capacity to connect to microtubules and accelerates microtubule depolymerization, disrupting neuronal signal conduction. Su et al. (2010) demonstrated that persistent oxidative stress increases Tau phosphorylation in M17 neuroblastoma cells and plays an important role in nerve fiber disease in vivo [35]. Additionally, ROS inhibits PP2A, allowing GSK-3β to be activated [36]. As a result, increased activation of GSK-3β may result in tau hyperphosphorylation and the formation of neurofibrillary tangles in AD. Thus, as we showed in our experiments, increased Tau hyperphosphorylation has been evident in the presence of ROS and oxidative stress in AD and Al groups. However, treatment with DHLA reduced Tau phosphorylation by decreasing oxidative stress and downregulating the GSK-3β pathway.
Deposition of peptide carbonyls has previously been noted in the progression of several neurodegenerative disorders, including Alzheimer's disease, as oxidative stress arises to play a key role in the progression of Alzheimer's disease through ROS generation and increases the expression of carbonylated proteins. Patients with Alzheimer's disease reported higher levels of protein carbonyls in the inferior parietal lobe and the hippocampus than controls. Furthermore, protein carbonyls have been linked to forming Aβ and neurofibrillary tangles in AD. The increase in carbonylated proteins in regions with persistent pathophysiological changes suggests that carbonylation may play a role in the pathogenesis of AD. Protein carbonyl formation could be caused by oxidative stress and oxidative proteasomal deactivation as the organism ages [37]. α-LA considerably reduces oxidative stress by limiting lipid and protein oxidation and significantly inhibits free radical production. Two nuclear factors mediate the antioxidant activity of α-LA: (i). nuclear factor erythroid-related factor-2 (Nrf2) and (ii). nuclear factor kappa light chain (NF-κB) re B-cell activators [38]. In DHLA-treated study groups, we observed substantial decreases in protein oxidation, possibly due to the potent ability of this compound to scavenge carbonyls. In addition, DHLA decreased lipid peroxidation and increased GSH levels possible because of its potent reductant capacity.
The Akt pathway supports neuronal survival, whereas its blockage causes neuronal death. Akt has a wide range of biological effects, mostly by boosting the phosphorylation of Bax (one of the Bcl-2 family's apoptotic promoters), mammalian target of rapamycin (mTOR), GSK-3β and other downstream substrates. Downregulation of Akt with increased GSK-3β activity is linked to brain dysfunction and neuropathophysiological alterations. GSK-3β, in its active state, translocates from the cytoplasm to the nucleus and causes apoptotic effects. In the present study, Akt activity was impaired in the AD model and Al exposure. The overactivity of GSK-3β in AD and AD-Al groups was due to lower Akt levels. DHLA treatment regulated the Akt levels by downregulating GSK-3β and decreasing Tau hyperphosphorylation. A study by Huang et al. [39] reported that aluminum maltolate reduced Akt phosphorylation and enhanced GSK-3β activity. Hyperforin showed neuroprotection against Al nontoxicity by regulating Akt/GSK3 pathway in the PC21 cell line.
Wnt signaling is important in the nervous system, from early patterning to higher functions such as synaptic plasticity and memory in the adult brain. Several Wnt pathway components, like Wnt ligands (Wnt2, Wnt4, and Wnt9a), as well as transcription factors lymphoid enhancer-binding factor 1 (Lef1) and transcription factor 3 (Tcf3), are down-regulated in the elderly rodent brain. Wnt3 and Wnt3a expression in the dentate gyrus area of the hippocampus decreases between 1 and 22 months of age. Furthermore, there is a distinct decrease in canonical Wnt signaling in the hippocampus of elderly rats where disheveled segment polarity protein 2 (Dvl2), Axin2, and nuclear β-catenin are downregulated by age [40]. Reduced Wnt signaling increases GSK-3β activity, contributing to Tu hyperphosphorylation, a fundamental pathogenic AD feature. GSK-3β overexpression in the brain induces neurodegeneration and learning impairments [40, 41]. The Wnt signaling pathway was impaired in our AD groups, indicating decreased Wnt activity. In addition, the reported increased activity of GSK-3β in our study is evidence to conclude that Wnt signaling has been suppressed in AD. Along with AD groups, a decrease in the Wnt signaling pathway was also observed in cells exposed to Al. This may indicate that an impaired Wnt signaling pathway might be an underlying mechanism behind the neurotoxicity of Al. Treatment with DHLA, especially in the AD and Al groups, improved Wnt signaling. As a result, DHLA reduced the activity of GSK-3β and Wnt signaling improved in all DHLA-treated cells.
In conclusion, dysregulation of the GSK-3β pathway and Wnt signaling are important mechanisms behind AD. These pathways are also impaired by Al exposure. DHLA, a potent antioxidant, can decrease ROS levels and oxidative parameters. Moreover, DHLA treatment can decrease GSK-3β activity and increase Wnt signaling in Al exposure and/or AD model. DHLA is responsible for most of the pharmacological activities of α-LA. Thus, we propose that DHLA be added to AD patients' treatment regimens to improve their overall health. However, further in vivo experiments and clinical studies are needed to decide the proper dosage of DHLA and possible indications.