AD is a degenerative brain disease that becomes worse with the age. Symptoms occur because neurons and glial cells in specific regions of the brain involved in thinking, learning, and memory have been damaged. Over time, symptoms tend to increase and start interfering with individuals' ability to perform daily activities, and after nine years of symptom onset, patients die [9, 10]. Therefore, it is imperative to find new drugs that could delay the onset, slow progression, or improve symptoms of AD. Such neurotoxic effects were observed in the present study by the prolonged intake of AlCl3, which significantly prompt neuropathological alterations in HO and PC of rats that are consistent with several reports [43,17,44-46]. After treatment with JM-20, a high level of neuroprotection was observed, confirming our preliminary hypothesis that it provides neuroprotection. Treatment with JM-20 rescued mitochondria from aluminum-induced neurotoxicity, preserved anti-apoptotic proteins and, consequently, restored multiple types of memory and neurobiological status in this AD model. These effects were accompanied by oxidative, AChE and mitochondrial stabilization of level and function, whereas neuronal viability at biochemical and histological level were verified. Overall, JM-20 therapy showed potential to counteract different aspects of AlCl3-induced pathological mechanisms.
Considering that cognitive decline and the lack of visuospatial memory are both clinical symptoms of AD [40], the deficit of learning and spatial memory have been widely reported in studies with transgenic mice and pharmacological model of AD [42,42]. Considering this, we used the AD model induced by AlCl3 neurotoxicity. The deposits of AlCl3 are involved in several signals in the brain, specially related to neuroinflammation [47], mitochondrial disorders [48], cellular death [44], and behavioral signs of dysfunction in working, spatial and emotional-associative memory. Moreover, the hippocampus is one of the most vulnerable cerebral structures; suffering from a decline in synaptic function in this structure can lead to dementia [49].
As previously reported by others, our study indicates that chronic exposure to AlCl3 significantly impaired spatial memory in rats, according to MWM and Y maze tests results, and also the contextual memory as revealed in the passive avoidance test [50-52]. In the Y maze test, AlCl3-treated rats had impaired working memory, failed to make correct alternations in a new environment. In the MWM, AlCl3 over loaded rats also showed less capacity to retrieve and retain the location of hidden platform reflecting the inability to encode and remember the spatial information even after several days of training. In the passive avoidance test, AlCl3-treated rats do not remember the aversive stimulus past 24 h and enter the dark chamber associated with electric shock earlier in comparison to control rats that could remember and avoid the dark chamber. JM-20 co-administration after 15 days of AlCl3 treatments, until day 30, reversed aluminum induced memory deficits, which indicate its memory protecting effect. Our previous work denotes that JM-20 reversed the memory deficits induced by scopolamine, an indication of cognitive protection mediated by this molecule, but in a transient model of dementia, useful to detect JM-20 activity against a very fast cholinergic dysfunction (thirty minutes after injection)[16]. In the present study, JM-20 also showed a protective effect but against chronic aluminum exposure, more similar to clinical conditions.
AChE, a cholinesterase that hydrolyses acetylcholine (ACh), is considered one of the biomarkers of cholinergic function in the brain. AChE has high sensitivity to exogenous factors including aluminum [53-55]. Increased AChE activity stimulates the breakdown of ACh, leading to cholinergic system abnormal function. As cholinergic transmission contributes to learning, memory and cognition and it is closely related to short- and long-term memory [56], the degree of impairment in these synapses correlates with the severity of dementia in patients [57]. Aluminum is a well-known potent cholinotoxin [58], which can alter the blood brain barrier to trigger changes in the cholinergic transmission [59]. In addition, it significantly elevates AChE activity directly [60], through the interaction between aluminum and AChE peripheral sites, modifying the secondary structure of the enzyme [61,62]. Here, in line with these previous studies, our results showed considerably elevated AChE activity in AlCl3 group when compared to control groups [52,63,64]. However, co-administration of JM-20 to AlCl3-intoxicated rats restored physiological AChE activity. Thus, JM-20 can reduce the negative effects of AlC3 in the brain mediated by AChE hyperactivity, as well as ameliorated the learning and memory impairment by AlCl3 exposure. It is noteworthy that JM-20 has inherent anti-AChE activity properties. Previously we have demonstrated using in vitro and in silico studies that JM-20 has a strong AChE inhibitory activity (IC50 ≈ 200 nM) [16,65]. Therefore, JM-20 administration can directly inhibit AChE activity in the Hippocampus, restoring memory dysfunction because cholinergic neurons densely innervate the hippocampus, mediating the formation of episodic memory [66,56].
The cognitive impairment found in AD has been also associated with oxidative damage and the subsequent unbalance on the endogen antioxidant system in the brain, one of the major causes of brain aging. Previous studies have reported elevation of brain oxidative stress markers due to the impairment of the antioxidant enzyme system in neurodegenerative diseases, a feature that can be induced by the presence of aluminum [68, 69]. Regarding this experimental evidence, our study revealed that AlCl3-treated rats showed significant decrease of CAT activity (antioxidant enzymes activities), while increasing MDA production in the HO and PC, corroborating with the hypothesis of impaired antioxidant system in this AD model. Treatment with JM-20 was able to counteract both the decrease in antioxidant activity and the accumulation of oxidative stress markers, maintaining levels similar to healthy-control rats, while SOD activity was unaltered. Despite these indirect antioxidant effects, JM-20 has intrinsic antioxidant potential. Its reduction potential of -0.72 V makes it an excellent electron acceptor, capable of preventing the generation of ROS at the mitochondrial level [15]. Recent results confirmed these effects on scopolamine-induced cholinergic and memory impairment and in rotenone-induced neurotoxicity in experimental model of Parkinson’s disease [16]. Collectively, these data suggests that the effects of JM-20 in decreasing oxidative stress may involve not only its direct antioxidant effects, but also an indirect antioxidant action favoring a cellular antioxidant environment.
Considerable evidence suggests there are beneficial effects of antioxidants on brain degeneration and dementia. A large body of evidence demonstrates a real oxidative environment on an AD brain over a long progression period [70-72]. In fact, recent evidence states that oxidation products could act as biomarkers in some neurodegenerative diseases, such as lipid peroxidation markers 4-hydroxynonenal and MDA, that were are identified in the cortex and hippocampus in AD patients [73]. The oxidative damage may be related to a decrease in antioxidants and repair systems. In this sense, enzymes such as SOD, CAT, and GPx (glutathione peroxidase), can determine the clearance of free radicals [74]. Brains with decreased SOD and the CAT activities observed in hippocampal neurons and glial cells may stimulate an overproduction of superoxide and H2O2 molecules, alongside with excessive NO production (via iNOS activity) cause severe cellular injury [75,76,74]. Moreover, SOD released from astrocytes is hypothesized to protect GSH by reducing the production of superoxide [77]. In addition, the increase in GPx activity may induce decreased levels of GSH, the main antioxidant defense of the brain, which may contribute to excitotoxicity because it may increase the vulnerability to oxidation [78] and may induce an excessive inflammatory response in age-related neurodegenerative diseases [79].
Several investigations indicate that the mitochondrion is a target for AD. It has been hypothesized that the dysfunction in this organelle may be at the heart of the progression of AD itself and is primarily involved in ROS production in the CNS [80,81]. In addition, brains with decreased hippocampal SOD and the CAT activities may stimulate an overproduction of superoxide and H2O2 molecules, causing severe cellular injury [74-76]. After 38 days of treatment, isolated brain mitochondria of AlCl3 treated rats showed high membrane potential dissipation, increased ROS production and significant swelling. In this sense, our work verified the role of JM-20 on mitochondrial dysfunction rescue, after a period of AlCl3 exposure. JM-20 was able to reverse mitochondrial malfunction and preserve this organelle by maintaining membrane potential, normalizing H2O2 production and partially preventing mitochondrial swelling. We did not detect signs of damage at this level on ex vivo preparations, and normal rates of O2 production were measured. These result are in correspondence with our previous reports to evaluating JM-20 effects on ischemic, Parkinson and in AD transient’s model, were detected even protection of mitochondrial anatomy [15,16]. Importantly, the structural characteristics of JM-20: small molecular weight (404.14 g/mol), cationic, and lipophilic molecule with a log P of 3.46 (partition coefficient of a molecule to predict solubility and permeability), allow it to reach the mitochondrial compartment promptly making this organelle a potential pharmacological target for it actions. Taking all together, we hypothesized that mitochondrial preservation is the major mechanism of JM-20 neuroprotective actions, which resulted in better performances in memory tests by treated animals.
Having higher metabolic rates and energy demands, neuronal cells depends a lot on mitochondrial function. Mitochondria supply ATP (adenosine triphosphate) to the cells (via oxidative phosphorylation), synthesize key molecules, and respond to oxidative stress as well as in apoptosis/survival signals. Mitochondrial production of ATP supports synapse assembly, generation of action potentials, and synaptic transmission, all of this essential for cognitive function [82,83,84]. Numerous works describe the central role of damaged mitochondria on the neurodegenerative pathophysiology, as a background of oxidative stress is perpetuated by the installation of mitochondrial dysfunction. Damaged mitochondria produce excess superoxide and hydroxyls radicals, H2O2, and protein-lipid peroxidation, which disrupts mitochondrial DNA and cause an imbalance in mitochondrial respiratory chain, Ca2+ homeostasis, excitotoxicity, membrane permeability, apoptosis and mitochondrial defense systems [80,85]. Those are notable causes of the propagation of neuronal dysfunction, triggering neurodegeneration with the consequent loss of cerebral functions [80,85]. In this sense, a mitoprotective drug is a potential candidate for treating dementia signs and its progression, in order to avoid the ordinary course of neurodegeneration [83,86].
Neurodegenerative processes are associated with induction of neuroinflammation and release of cytokines, its propagation and its consequences, ending in dysfunction and cell death [87]. Patients suffering with AD exhibit increased levels of cytokines in serum, brain, and cerebrospinal fluid [89,90]. In addition, oxidative/nitrosative stress can also induce an inflammatory response by increasing TNF-α levels [88]. These and other sings of neuroinflammation are the basis of neuroinflammatory hypothesis of AD late onset [87]. TNF-α induces astrocyte and microglial activation and is closely associated with increases in another proinflammatory cytokines, such as IL-1β. In the present study, we investigated AlCl3 effect on brain homogenates and detected high levels of TNF-α on HO and PC of AlCl3 treated rat, but we did not observe abnormal levels of IL-1β. In these conditions, JM-20 avoided the increase of TNF-α and exerted a strong protection, both in the HO and PC, in rats treated during 38 days with AlCl3.
The early release of TNF-α is essential in priming microglial cells and innate immunity to effectively resolve initial damage, and its absence delays activation of microglial cells which leads to an exaggerated and nonspecific activation, amplifying secondary damages [91]. However, TNF-α is considered a pro-inflammatory factor that is synthesized by microglia, astrocytes, and some populations of neurons [92,93], with receptors in the same cellular population, exhibiting direct and indirect actions on neurons [94-96]
TNF-𝛼 can bind to two specific receptors: TNFR1 (tumor necrosis factor receptor 1), with an intracellular death domain, and TNFR2 (tumor necrosis factor receptor 2), with a higher affinity and mostly involved in neuroprotection but also contributes to cell injury mediated by NF-ƙB and caspase-3 activation leading neuronal death [97]. Such activities amplify the oxidative stress that is deleterious to biochemical and cellular processes, in cyclic events of damage. The result is chronic neuroinflammation, dysregulation of cellular functions, and further neuronal death. In memory function, it was described that TNF-𝛼 overproduction also regulates synaptic plasticity region dependently, for example, inhibition of long-term potentiation in the hippocampus [98,99]. Therefore, we propose that JM-20 mitigates neuroinflammation through the attenuation of cytokine synthesis and release, potentially increasing neuronal survival and preserving mnemonic process. This mechanism may be an indirect result of JM-20 mitoprotective effects.
In both AD pathology and in AlCl3-induced toxicity, we have several signs of injury: the mitochondria, which are important sensors and executioners in the cell’s decision to live or die by intrinsic pathways, the expression of TNF-𝛼 that lead to neuronal death in long-lasting conditions mediated by extrinsic signals and a continuous, not resolved, oxidative stress. In this sense, we quantify levels of essential key mediators of survival and death signals (Akt, GSK-3β, Bax/Bcl-2 ratio, caspase 8), as well, as the executer of apoptosis cellular death (caspase 3). Our results pointed out a significant increase in the inactivation of GSK-3β against AlCl3-treated rats, both in the hippocampus and prefrontal cortex. In the brain, GSK-3β regulates many crucial cellular processes, acting as a key switch that controls numerous signaling pathways [reference]. Different evidences support the role of GSK-3β on beta amyloid (Aβ) and tau neurotoxicity, mediated by mechanism of direct production and regulation (e.g. inhibiting ADAM activity, or in tau protein hyperphosphorylation (Ptau) together CDK-5 kinase action) [reference]. Increased GSK-3β activity has been used to model events occurring in AD, interventions that exacerbate cognitive impairments, and neuropathology in rodent models of AD, like in aluminum rat model of AD [102]. Even when we do not evaluate Aβ and Ptau formation, several reports mention the putative capacity of aluminum to produce these reactive species, keys on AD neuropathology [103]. Then, we suppose that the increased levels of GSK-3β on AlCl3 group is the beginning of a neurotoxic pathway that it is possibly acting in the APP processing and multiple tau phosphorylation. In addition, concerning with synaptic transmissions is well documented that neuronal GSK-3β overexpression causes a decrease in postsynaptic density number and volume in hippocampal granule neurons [104], a phenomenon that maybe related to cognitive impairment and altered LTP generation [105]. At that point, JM-20 possibly stopped these slowly by consistent process that lead damage, and prevent cognitive decline.
Apoptosis might also trigger an adaptive immune response, associated to the release of cell death associated danger signals (cDAMPs) including (but not limited to): ATP, nucleic acids the non-histone nuclear DNA-binding protein high mobility group box 1 (HMGB1), cytokines like type I interferon (IFN) chaperones like calreticulin (CALR), heat shock protein family and reactive oxygen species (ROS) [100].
Hippocampal and prefrontal cortex homogenates under AlCl3 effects revealed the subsequent impact in different subsystems leading to apoptotic signaling. Values of Bax/Bcl-2 ratio, and levels of caspase 3 and caspase 8 reflect the relative cells status in the hippocampus and prefrontal cortex and indicate irrevocable death of brain cell precedents of AlCl3-treated rats control group. In contrast, treatment with JM-20 preserves at least a few target pathways as oxidation/reduction balance, energetic provision mediated by the mitochondria, physiological non-toxic activity levels of AChE for memory and cognitive processes contributing to brain restoration and non-deleterious process resulting on degeneration and cellular death. After 1 month of JM-20 daily doses, pro-apoptotic mediators of intrinsic and extrinsic pathways and executers markers were similar to healthy group: stable and balanced. At this point, aware that an entire cascade of intermediaries should be quantified, we were led to believe that JM-20 is a molecule able to prevent neuronal death under neuropathological alterations similar to that in AD in humans. Nevertheless, we do not discard the occurrence of other immunogenic and programmed cell death modalities like necroptosis in AlCl3 treated group, which might be modulated by JM-20. Indeed, a previous report provided the first in vivo evidence for a role of RIP3 protein in TNF-𝛼-induced toxicity of hippocampal neurons, demonstrating that TNF-𝛼 promotes RIP3-MLKL-mediated necroptosis of hippocampal neurons bypassing ROS accumulation and calcium influx supporting this hypothesis [105]. Evaluation of these markers could contribute to expand the molecular effector mechanisms elicited by JM-20 in the context of AlCl3-induced alterations. To confirm the irreversibility of the apoptosis induced by the AlCl3-treated group, and the protective role of JM-20, it would be relevant to evaluate some markers of late apoptosis, like loss of cell membrane integrity, DNA fragmentation or cytomorphological alterations [39].
To associate JM-20 neuroprotective effects with histological analysis, we evaluated neuronal population by H&E. In the AlCl3 group, a large number of signs of neuronal death and cell loss were detected, whereas JM-20 8 mg/kg protects more than 95% of neurons on HO and PC regions. The results supported that the previous mechanism described for JM-20, mediated memory protection also stopped the degenerative process leading to dysfunction and death, reported in final states of dementia.
In conclusion, based in this and our previous reports, JM-20 has potential as a novel candidate for treating AD condition, acting in key pathological mechanisms of neurodegeneration and preserving structural architecture of brain structures that are essential for cognitive functions. The current results do not cover the full spectrum of possible mechanisms of memory dysfunction and neuronal death; thus, other studies should be performed.