Interplay Between Hippocampal Glutathione Depletion and pH Increment in Alzheimer’s Disease

Proposed schematic model for GSH and pH interplay in AD

OS is attributed to an imbalance between pro-oxidant and antioxidant levels [18]. To understand how OS relates with antioxidant regulation, energy metabolism processes, and altered pH levels, it is important to consider the cascade of molecular events. Excessive OS leads to the production of free radicals, which are further neutralized by the active sulfhydryl (-SH) group of the Cys moiety of GSH molecule. Thus, increased OS becomes a major driving factor for GSH depletion. From the experimental studies, a significant GSH depletion in the left hippocampus of MCI patients as compared to healthy old was observed from our previous studies [17, 19]. Here, GSH quantitation was performed through ¹H magnetic resonance spectroscopy (MRS) using MEGA-PRESS pulse sequence, and pH was obtained using 2D ³¹P-MRS with dual tuned (¹H/³¹P) transmit/receive volume head coil [17]. These findings are in concurrence with post-mortem reports confirming GSH depletion [20].

OS also plays a critical role in the generation of cytotoxic products. Due to increasing oxidative damage, ROS reacts with several macromolecules, including poly unsaturated fatty acids in the cell membrane. This reaction results in lipid peroxidation and generation of cytotoxic 4-hydroxynenal (4-HNE) as a by-product [21]. Peroxidized lipids then react with many mitochondrial enzymes, which in turn escalate OS through increased free radical release into the cytoplasm [22]. 4-HNE-induced neurotoxicity has been reported in AD [23, 24] and supported by postmortem studies [25–28]. 4-HNE influences the phospholipid asymmetry of the membrane lipid bilayer. In this event cascade, it induces apoptotic neuronal cell death by affecting the activity of various enzymes, which induces membrane damage and causes GSH depletion [29, 30]. It also interacts with the (-SH) group of the cysteine residue of GSH and forms GSH–4-HNE adduct, significant amount of which was found in the hippocampal region of AD-affected brains in some postmortem studies [24, 31]. These studies further raise the possibility of GSH–4-HNE interaction being a probable reason for GSH decline in AD.

Brain creatine kinase

CK is a cytosolic dimeric enzyme and exists in three major isoforms: muscle CK, brain CK, and muscle–brain CK [32]. Brain creatine kinase (BBCK) plays a central role in neurons due to their high energy requirement. BBCK transfers a phosphate group from adenosine triphosphate (ATP) to adenosine triphosphate (ADP) in a reversible reaction (as shown in Eq 1). The reaction generates phosphocreatine (PCr) and H⁺ ions, thereby maintaining PCr/Cr shuttle and cellular energy homeostasis [33–37]. ATP + Cr ⇌ BBCK ADP + PCr + H +(1)

Highly reactive and essential Cys-283 residue of BBCK is involved in substrate-binding synergy [38, 39], further making it susceptible to modifications by ROS [40] and 4-HNE [41]. This oxidative modification of BBCK via ROS takes place through post-translational modification [42].

Therefore, the combined effect of post-translational modifications and 4-HNE interaction with the active site potentially hinders enzyme activity, subsequently causing its impairment. BBCK is crucial for energy metabolism as well as for contributing to the net cytosolic H⁺ ion concentration maintenance. Thus, changes in its kinetics can potentially alter pH. Moreover, postmortem studies in AD report that the significant depletion of BBCK in the hippocampus [43] causes deficits in optimal energy levels and reduces energy supply to glia, neurons, and synapses [44]. Ultimately, BBCK should be further studied in detail considering its criticality in CK reactions affecting certain cellular microenvironmental factors, such as pH.

Brain creatinine kinase, mitochondrial respiration, and interplay with glutathione

The differential micro compartmentation of CK isoenzymes is involved in maintaining high local [ATP]/[ADP] ratios in the vicinity of cellular ATPases for optimal energy required for ATP hydrolysis. On the other hand, a relatively low [ATP]/[ADP] ratio in the mitochondrial matrix is required to stimulate oxidative phosphorylation [45]. Due to the specific differential localization of mitochondrial and cytosolic isoenzymes and the slightly faster diffusion rate of PCr as compared to ATP, the CK/PCr system provide a temporo-spatial “energy shuttle” [46].

Mitochondrial creatine kinase together with its cytosolic counterpart BBCK adds to the build-up of a large intracellular pool of PCr. This provides a buffer to maintain a rapid fall in global ATP concentrations both temporally and spatially. PCr is an important metabolite present ubiquitously throughout the body and acts as a buffer by donating its phosphate (P_i) group to ADP for ATP regeneration. Under conditions of elevated OS, there is a profound effect on metabolism, including inactivation of the enzymes of glycolysis which can lead to metabolic failure and hence anaerobic conditions [47].

The CK/PCr system also aids in buffering of protons, thus preventing acidification of cells which are actively involved in ATP hydrolysis [34]. Another major function of the CK/PCr system is to prevent the rise of intracellular free ADP concentrations, which may lead to the inactivation of cellular ATPases [34]. Due to the reversible nature of CK, PCr can donate a P_i to ADP, generating ATP and creatine. Thus, in low energy conditions, more amount of ADP is required for ATP production which maybe the reason for the mitochondria’s lowered sensitivity to ADP when ATP is needed. In the reverse scenario, creatine produces ADP by accepting a P_i group from ATP. Levels of both ATP and ADP have to be regulated to maintain proper scheme of biochemical reactions.

Basal membrane proton conductance is a facilitator of metabolic homeostasis but also causes mild uncoupling from oxidative phosphorylation [48]. The mitochondrial permeability transition pore (MPTP) complex is an inner mitochondrial membrane resident protein. Large amounts of proton leak through MPTP can cause necrotic cell death as well as ATP depletion [49]. Age-related oxidative stress could be an important factor in increasing the sensitivity of brain mitochondria to MPTP opening [50]. Due to ATP depletion, PCr comes into play by donating its P_i moiety to ADP, leading to replenished levels of ATP. The antioxidant GSH is an important scavenger of ROS produced due to OS in the brain; and hence, its depletion can be acquitted as a factor leading to proton leakage through MPTP. However, specifically for hippocampal GSH, no reports on GSH depletion affecting membrane proton conductance are available to the best of our knowledge.

Brain creatine kinase, glutathione depletion, and AD

CK is involved in the catalysis of PCr and ADP from creatine using ATP as a cofactor. Due to the reversible nature of CK, ATP can be generated from PCr. Thus, in cells that require high amount of energy for functioning, PCr serves as an energy reservoir for the regeneration of ATP [34] and is hence important for maintaining bioenergetic balance. BBCK, the major isoform of CK present in brain tissues, is responsible for maintaining ATP levels in neurons [42]. Decrease in BBCK activity is considered as a biochemical marker for AD [8]. BBCK is also sensitive to OS. Due to increased ROS formation, oxidation of BBCK occurs in the brains of AD patients, leading to reduction in enzymatic activity [42]. Another study reported an aberrant cytosol/membrane partitioning of CK as well as CK inactivation in AD [51].

Glutathione level and memory performance in AD

GSH is the master antioxidant in brain tissues that is involved in the critical maintenance of redox homeostasis by scavenging ROS. Memory formation is a complex neurobiological process involving the hippocampus and cortex [52]. In AD, the left hippocampal region has been observed to be affected differently than the right hippocampal region, thus affecting the formation of episodic and semantic memory [53].

A ¹H-MRS study followed that the reduction in GSH was correlated with a decline in the global cognitive status of patients as assessed by different neuropsychological tests, such as Mini-Mental Status Examination (MMSE), Clinical Dementia Ratio (CDR), and Trail Making Tests Parts A and B (TMT B-A) [54]. For both MMSE and CDR examination, significant correlation was observed between the neuropsychological test scores and GSH depletion in both hippocampal regions and the frontal cortex. A significant negative correlation was also observed between TMT B-A and GSH depletion for the hippocampus and frontal cortex, parts of the brain primarily involved in memory formation and executive functioning [54].

In a combined ³¹P-MRS and MEGA-PRESS ¹H MRS study, significant GSH depletion has been observed in the left hippocampus in patients with MCI as compared to healthy controls, leading to microenvironment changes like pH increment, thus disturbing the neurochemical integrity of brain [17]. In both MCI and AD, pH increases with the decrease in GSH. However, in healthy control subjects, both pH and GSH have a linear dependence. Thus, increased OS and GSH depletion have been well associated with AD [55], and these studies reflect the importance of uncovering the underlying mechanism between hippocampal GSH depletion and pH increase leading to memory deficits in AD patients.

All the aforementioned evidence collectively explains how OS plays an important role in the mutual interplay of antioxidant depletion and enzyme inefficiency central to changes in energy metabolism, thereby contributing to microenvironmental changes, such as change in brain tissue pH as depicted in Fig. 1. Apart from the proposed mechanistic interplay, factors most likely to be involved in the hippocampal pH increment are presynaptic Ca²⁺ /H⁺-ATPase [14], extracellular carbonic anhydrase [15], and GABA-A receptor-mediated bicarbonate efflux [16].

OPEN IN VIEWER

Fig. 1

Schematic model of GSH–pH interplay illustrates the collective evidence of GSH depletion due to oxidative stress and its association with GSH–4-HNE adduct formation. Subsequent oxidative stress and 4-HNE-induced impact on BBCK is also depicted.