TAU HYPOTHESIS

The tau hypothesis suggests that hyperphosphorylation of tau protein leads to the conversion of normal tau into the paired helical filament (PHF-tau) and NFTs23,24,25,26,27,28,29. Previously, Aβ had been the focus of AD research. Only recently have researchers begun to shift focus to tau protein due to the fact that a variety of reports have shown tau proteins being among the key elements contained in the NFTs. Tau protein, a member of the microtubule-associated protein, is also a functional monomeric and unfolded cell membrane protein, located within the cytosol of a neuron and very crucial in tubulin stabilization30. In addition, tau is known to control neurite growth and have a role in axonal guidance, thus enhancing the normal function of neurons31. In humans, tau protein is only found in trace levels in non-neuronal cells32. Tau goes through various post-translational changes, especially hyperphosphorylation, a process which acts as a significant factor in influencing the stability of microtubules, thereby leading to tau protein accumulation in AD30. Previous findings have shown that hyperphosphorylation of tau protein occurs via conformational changes, which are followed by the transformation of tau monomer to tau oligomer, leading to the paired helical filament and NFT formation33,34,35,36,37. Recent studies have revealed that NFT itself does not appear to be implicated in causing neurotoxicity leading to the onset of neurodegeneration38, though the hyperphosphorylated tau that disintegrates to form oligomers (the toxic form of tau) is implicated in neuronal damage39. In addition, recent and findings in rats have highlighted that tau pathology may have far-reaching effects in distinct brain regions40,41,39. Tau, a microtubule-associated protein, acts as a scaffolding component to assist in stabilizing microtubules by influencing tubulin stability in order to control the normal neuronal function42,43.

The position of the tau gene, microtubule-associated protein tau (MAPT), is essential and located on 100 kb of the long arm of human chromosome 17 at locus 17q21, and has 16 exons44. Moreover, it has been shown that in the brain of humans, the tau proteins encode six isoforms that have different sizes, with their length range being between 352 and 441 amino acids 45. These isoforms have some variations, three repeats (3R) or four repeats (4R) in the C terminal, and also the presence or absence of one (29 amino acids) or two inserts (58 amino acids) in the N-terminal part, which bind to actin proteins and differentiate them. Findings have revealed that the repeat areas (244-268 amino acids) located in the C-terminal are the main domain, which causes the clinging of tau to microtubule46,47. Furthermore, findings have suggested that tau phosphorylation at specific epitopes could adjust the capability of tau to cling to microtubule48. Many post-translational changes, mostly tau hyperphosphorylation, were considered to be involved as essential elements that influence the assembly of microtubule which, in turn, cause tau accumulation in AD49. Thus, tau protein goes through conformational alterations whereas the transformation of tau monomer to tau oligomer causes the accumulation of tau, thereby causing it to pair with a helical filament and produce NFTs (due to hyperphosphorylation of tau). Emerging studies have revealed that NFTs are not associated with causing neurotoxicity. Nonetheless, the intermediary tau oligomer was reported to be a toxic form of tau that is implicated in synaptic destruction in AD50. Tau pathophysiology is accompanied by abnormality of amyloid precursor protein (APP) and is eminently seen in the brain regions implicated in the memory-hippocampus as well as parts of the cerebral cortex51.

Formation of Aβ

Aβ peptide 52,58 is a derivative of Amyloid β-precursor protein (AβPP), also an intrinsic type I glycoprotein59 which has a broad ectodomain. The position of the chromosome for AβPP is found on 21q21.2. There are 18 exons in the APP gene that exceeds 170 kb, thereby creating 10 isoforms through discrete splicing. These isoforms measure between 563 and 770 amino acids APP is 695 amino acids in length and is one of the isoforms that has been previously reported to be found in neurons of the central nervous system 60. The specific area that codes Aβ-strings is made up of 16 and 17 exons, of which therein are (40 and 43) amino acid residues. Specifically, this area continues from the ectodomain to the protein’s transmembrane domain53. APP’s central domain possesses strong affinity; hence, it can bind to ions such as copper (Cu) and Zinc (Zn), as well as heparin and collagen, within the extracellular matrix, thereby mediating the interplay of APP with the extracellular matrix. The sphere implicated in the neuritic process is defined by a precise string which is seen following the inclusion of exon 7 product 61. APP isoforms have been known to play vital roles in enhancing coordination between cells and, thus, a few of those roles are hereby outlined62. APP isoform (of 695 amino acid) has been shown to be involved in aiding coordination between cells and enhancing the connection to the extraneuronal matrix, thereby leading to stability. APP within the intracellular domain may be associated with a cytoskeletal system that transports constituents within a particular cell. APP1-671 (βAPPs) and APP1-687 (αAPPs) are the other two APP isoforms produced in segments; they offer a protective role to the neurons and regulate events at the synapse 44.

Processing of APP

Proteolytic series of events taking place around and in the APP transmembrane sphere yield a better expression for the production of toxic Aβ42 protein, and eventually for AD pathogenesis. Three different divisions release the APP ectodomain from the membrane. These divisions have been confirmed to be α-secretase split (which separates Aβ-domain and inhibits Aβ-development- by generating amyloidogenic substance- due to the incapability of developing pathogenic Aβ) 63, β-secretase cleavage (the segment produced from α-secretase, while the β-secretase segments remains connected to the membrane; following further conversion by γ-secretase it becomes weakened), and Aβ-protein (Aβ42) breakage (whose aggregation is thought to cause AD due to the presence of toxic and fibril aggregates) 52,54,58.

Biochemistry of Senile Plaques

Blocq and Marnesco, in 1892, were the first to demonstrate that senile plaques (SP) could be described as densely packed structures called amyloid bodies. The process by which amyloid bodies develop and are implicated in AD development is known as amyloidosis. Fibrillogenic proteins (about 10 nm in diameter; amyloid fibrils that are smooth and straight) and non-fibril components (ApoE and serum Aβ components) are the contents of the amyloid bodies. The SPs, which are also called amyloid plaques, are the product of an extracellular accumulation of Aβ protein60.

Aluminum as one of the frequently used heavy metals for induction of cognitive impairment

Environmental heavy metals are agents that have been well-established to impact the development of the brain through neurotoxicity100. Among the commonly used Alzheimerogenic chemicals, heavy metals have been identified to induce high levels of toxicity that is linked with several diseases, including neurodegenerative disorders, following long-term exposure in humans or chronic administration in rodents. A growing body of support has reported the relation between heavy metals and neurodegenerative diseases, including AD and Parkinson’s disease 100. Heavy metals are generally known to damage the nervous system. Of all the heavy metals, the effect of aluminum on biological systems has been well-reported101,45,102. Al is an ubiquitously scattered environmental and industrial toxicant associated with anemia103, osteomalacia, and hepatic and neurological disorders103. The high amount of Al has been identified in the brain of subjects suffering from AD and causes toxicological effects, including encephalopathy, bone disease, and anemia. Very recently, orally administered Al (300 mg/kg body weight) was reported to induce oxidative stress, cholinergic deficit, and accumulation of Aβ & NFTs in the brain of rats101.

Moreover, in the Al-treated group, there are marked histopathological changes and diffuse gliosis accompanied by pericellular edema in the cerebral region, in addition to neuronophagia and loss of neurons. A number of studies have shown that Al neurotoxicity is highly linked with cognitive impairment of AD, via oxidative stress and cholinergic dysfunction104. The findings have indicated an upregulation in the expression and activity of acetylcholinesterase (AChE) and malondialdehyde (MDA), but a significant decrease in expression and activity of glutathione-s-transferase (GST), glutathione peroxides (GPX), and glutathione reductase (GR).

Scopolamine-induced AD-like dementia

A growing body of knowledge has established that scopolamine is an anticholinergic drug mostly employed as an approved chemical in pilot studies to cause memory deficit105. Administration of scopolamine leads to deficits in visual recognition memory, verbal recall, visuospatial praxis, visuospatial recall psychomotor speed, and visuoperceptual processes106. It is one of the most potent and commonly used drugs to prevent motion sickness107. Furthermore, researchers have documented that scopolamine non-selectively occludes the adhesion site of ACh muscarinic receptors in the cerebral cortex and results in the unequal discharge of ACh, which annihilates hippocampal neurons and induces learning and memory impairment in mice in a dose-related manner108,109. However, the main effects of scopolamine likely result from blockage of some receptors (M1 and M5) due to their distinct distribution in the brain110. Previous studies have also suggested that it may lead to memory and learning deficit in a dose-dependent way108,109. In some instances, ordinary doses of scopolamine led to agitation, confusion, hallucination, paranoid behaviors, delusions, and incoherent speech111. Previous studies have indicated there is potential participation of N-methyl-D-aspartate (NMDA) receptor mechanisms of the dorsal hippocampus in memory loss due to scopolamine induction112. Currently, histone deacetylase-2 and DNA methyl transferase-1 are important for synaptic plasticity induced by scopolamine after a decrease in memory113. Hippocampal administration of scopolamine occludes long-term potentiation114 and impedes spatial encoding115.

Furthermore, the injection of scopolamine into the medial septum impedes learning and decreases ACh discharge into the hippocampus116, injection of scopolamine in the CA3 area of the hippocampus induces encoding impairments selectively, but memory retrieval in Hebb-William maze was not affected117. Indeed, scopolamine has been identified to cause spatial learning and memory deficiencies. Consequently, it leads to the excitation of glycogen synthase kinase-3 beta (GSK-3β), inadequate spine maturation, and the arborization of dendrites connected with alterations in CREC, Homer1, and amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPA)118.

Colchicine-induced AD-like symptoms

Advances in chemicals used for modelling AD have suggested that colchicines are potential drugs that induce dementia through dysfunctional cholinergic neurons by either inhibiting cholinergic reversal or eroding cholinergic passage cascades119,120. Colchicine induces hippocampal lesions resulting in cognitive impairments and ChAT reduction, suggesting that it can be utilized as a candidate for modelling AD. Colchicine could cause neurotoxicity and memory decline by inhibiting cholinergic pathways, thereby leading to a reduction in the number of cholinergic neurons and subsequently decreasing cholinergic renewal primarily within the hippocampal area of the brain121. Memory impairment seen induced by colchicine may be a result of a decrease in serotonin, dopamine, and norepinephrine within the caudate nucleus, hippocampus, and entire cerebral cortex122.

Furthermore, colchicine was shown to cause the production of protein carbonyls following lipid peroxidation123. Colchicine has also been found to raise cyclooxygenase-1, and 2 (COX-1 and COX-2) expression levels124 and ROS generation 125. Colchicine raises glutamate/GABA ratio in the cortex126 and triggers excitation of MDA receptors that lead to a sharp rise in the influx of Ca, therefore resulting in the triggering of enzymes that depend on Ca (. phospholipases A2, xanthine oxidase, proteases, cyclooxygenases, and protein kinases)127. Intracerebroventricular (ICV) administration of colchicine (7.5 g in 10 L) was found to recapitulate cognitive memory decline in rats128 and similarly in mice119. A significant memory deficit was observed two weeks after cognitive induction impairment using colchicine119. Furthermore, 3 g/mice (ICV injection) of colchicine induces spatial memory impairment129. The main advantage of this model is the fact that it causes some symptoms of sporadic Alzheimer's type of dementia, similar to those found seen in human subjects, like time-variant changes in onset, behavioral, and biological patterns123.

Streptozotocin-induced AD-like cognitive decline

Streptozotocin (STZ), a compound derived from glucosamine nitrosourea and found in the strain of , when administered ICV in rodents130, is known to exert a severe and long-lasting effect on the brain’s cytoarchitecture, biochemistry, metabolism, and functions (such as decreased glucose uptake and energy consumption, oxidative tissue stress, cholinergic differential, and cognitive abilities). STZ is another alkylating agent used in cancer treatment. It has also been found to mimic certain properties of nitrosourea, an anti-cancer agent that has been reported to have hypoglycemic effects in addition to being involved in memory damage110. Induction of neuronal damage and hyperphosphorylation of tau is caused by STZ, resulting in the release of ROS and reactive nitrogen species (RNS) 131. Findings revealed that STZ destructs the glycolytic enzyme activity within the brain which eventually results in the lowering of ATP and creatine phosphate concentrations. This destructed energy system and lowered acetyl CoA synthesis can inhibit cholinergic conductance130. Rats induced by STZ have been reported to demonstrate raised activities of AChE in their brains and decreased ACh. In other studies, STZ has been found to activate Aβ peptide-like aggregates by modifying the GSK alpha/beta132.

Furthermore, there is evidence that gene expression associated with the development of glial-derived NF, brain-derived neurotrophic factors (BDNF) and integrin –alpha-M becomes increased by STZ, while expression of genes for NGE-IB and metallothionein 1/2 is down-regulated and eventually results in the modification in apoptosis and cell survival process133. Administration of ICV-STZ resulted in a substantial decrease in memory and learning skills of rats, leading to oxidative stress134. Additionally, ICV administration of STZ (3 mg/kg) in a model of cognitive impairment in rats mimicked the sporadic dementia of AD135. Memory impairment was observed in the rats during the last phase of the experiment using the Morris water maze (MWM) task in order to assess AD-like symptoms. In addition, impaired glucose metabolism, a decrease in cholinergic markers, and oxidative stress were seen in the rats’ brains. Altogether, these long-lasting effects mimicked those seen in AD patients.

Ethanol/Alcohol induced AD-like symptoms

Chronic intake of alcohol is associated with many problems including attention deficits, impairment in language and social skills, hyperactivity, motor dysfunction, and learning deficits171. Previous studies have shown that ethanol consumption enhances the generation of ROS and results in a decrease of the antioxidants in the brain172. Another finding has suggested that ethanol could damage hippocampal and cholinergic neurons with a resultant effect on the sensory-motor system as well as disruption of learning and memory173. High ethanol treatment resulted in excessive nitric oxide (NO), which has been found to destruct memory and learning, while higher doses of ethanol disrupted the glutamatergic system and increased GABAergic conveyance in the brain areas that are associated with memory173. Ethanol also elevates the level of adenosine, which may, in turn, lead to memory damage174 and can result in a shorter route by accelerating memory impairment and promoting active, spontaneous motion.

Furthermore, results obtained following the MWM test in mice showed a notable increase in escape latency and total swimming distance, with a sharp drop in cross time. Thus, chronic alcohol administration can significantly affect spatial learning ability and cognition by mice175. However, memory impairment caused by prolonged alcohol intake can be observed from the anti-inflammatory activity and from the control of the equilibrium of Glu and GABA176. The particular molecular mechanisms still need further investigations, but overall, alcohol can significantly disrupt learning and memory.

Memory deficit induced by excitotoxin

Ibotenic acid is a strong neurotoxin that aggravates signs and pathophysiology analogous to AD177,178. It is a useful model to appreciate drug efficiency in evading AD pathology. It has also been found that intrahippocampal administration of ibotenic acid (5 µg/µl PBS) produces memory impairment and results in increased AChE activity as well as elevated MDA levels, thereby inducing neuronal toxicity179. Bilateral ICV injection of ibotenic acid may elicit AD-like pathology and symptoms. Indeed, a particular study found that ibotenic acid decreased the activity of cholinergic neurons in rats180. Many other cholinotoxins and neurotoxins that present AD-like symptoms are kainic acid, quinolinic acid, anti-NGF, NMDA antagonist dizocilpine, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Previous studies have reported that intrahippocampal kainic acid (0.4 µg/2µl) administration led to oxidative damage (revealed by the increase of hippocampal lipid peroxidation, nitrite level, and diminished superoxide dismutase (SOD)). Thus, it is clear that kainic acid activates oxidative damage and memory loss in rats 181. Stimulation of glutamate receptors, particularly NMDA receptors, in a diseased condition can lead to disrupted ion homeostasis, decrease in energy, neuronal loss, and cell death (excitotoxicity) by an abnormally high concentration of glutamate 182,183.

Sodium azide-induced AD-like memory impairment

Sodium azide (NaN3) is a white crystalline solid that has been reported to be a mitochondrial toxin implicated in the production of lead azide explosive184. NaN3 administration has been known to induce mitochondrial dysfunction and inhibit cytochrome oxidase, a critical mitochondrial enzyme169. Cytochrome oxidase is critical in the mitochondrial respiratory chain; its interference impedes with mitochondrial complex-IV and decreases ATP levels, which add to metabolic impairment and ROS generation170, ultimately producing some sequelae of AD. Neuronal loss in the CA3 area of the hippocampus is one of the essential features used to confirm the induction of AD using minipumps to deliver NaN.

However, due to high cost, one-time usability and long treatment time, minipumps are unlikely to be useful tools for screening. A new technique was developed to provide an AD-like dementia model by selective cytochrome C complex inhibition using an intraperitoneal injection of NaN in different doses (10-15 mg/kg/day) in rats. The intraperitoneal injection dose-effect-interdependence study employed mitochondrial poison and developed a complex test system to research changes in cognitive functions induced by treatment with NaN. It has been shown that NaN administration impairs learning and memory, and increases AChE levels in the brain169. NaN was reported to cause oxidative damage that resulted in neuronal cell death. The progressive loss of neurons and necrosis were observed in the cortical and hippocampal areas of treated rats, thereby further indicating the potency of NaN. These researchers confirmed that intraperitoneal injection of NaN for 15 days resulted in a comparable level of dementia using implanted osmotic minipumps in rats, ultimately suggesting the involvement of NaN in inducing neurodegenerative disorder185.

A growing body of knowledge has reported that oxidative stress (Figure 1) is a trigger, as well as one of the critical molecular changes for AD commencement 186. Oxidative stress may be caused by inhibition of mitochondrial function. The mitochondrial enzyme, Cytochrome oxidase (COX), has long been established to be predominantly implicated in aerobic energy metabolism and mitochondrial tasks in AD patients 187. Brains of AD patients have shown some mitochondrial abnormalities, particularly of CoX type. The primary toxic effect of NaN has been identified as decreasing the action of CoX in the mitochondrial electron transport chain 185.

LPS-induced AD-like symptoms

LPS has been known to be used in various experiments, both and models of amyloidosis and neuroinflammation 188,189. A variety of neurodegenerative diseases, namely AD 189, Parkinson’s disease190, amyotrophic lateral sclerosis191, and multiple sclerosis192, have been modeled using LPS-induced systemic inflammation (Figure 2). LPS is extracted from the external membrane of gram-negative bacteria. It has been reported as a strong endotoxin with resistivity to degradation by mammalian enzymes, therefore resulting in continual inflammatory stimuli193, which produce proinflammatory cytokines. These proinflammatory cytokines stimulate both neuroimmune and neuroendocrine systems194 that lead to virtually similar feedback formed by behavioral stress195. Inflammation has been reported to be crucial in AD pathogenesis. However, the exact mechanisms through which it participates remains to be elucidated. Inflammatory proteins in the blood, two of which are C reactive protein (CRP) and IL6, have been reported to increase several years before the onset of clinical dementia in different studies12,196. Inflammation is one of the factors that cause AD, although the link between infections and AD etiology has been an issue of debate for more than three decades197. Previous studies have indicated that the expression levels of TNF-α, IL-1β, and IL-6 within the hippocampus are upregulated in comparison with those of control following three days of LPS administration198. Proinflammatory cytokines have already been found as the critical molecules which modulate immune responses; inability to reverse them during chronic inflammation would increase dyshomeostasis198. Moreover, long-standing microglia activation which facilitates inflammatory mediators release, resulting in enhanced oxidative stress and nitrosative stress, is characterized by chronic inflammation 199.

Okadaic acid-induced memory impairment

Okadaic acid (OKA), a major polyether toxin, is a marine microalgae product that causes diarrhetic shellfish toxicity200. IC injection of OKA has been reported to induce memory decline in rats, therefore making it a valuable model agent to study for anti-dementia drug screening201. In terms of mechanism of action, OKA has been found to be a restrictive and modest antagonist of serine/threonine phosphatases 1 and 2A202,203, is associated with short- and long-term memory disruption in rats 204, and triggers tau hyperphosphorylation (Figure 2) and neuronal cell death both 205 and 206. Previous findings have identified that OKA decreases basal synaptic transmission and inhibits the commencement of synaptic plasticity207. Also, OKA has been found to augment Ca in a cultured hippocampal neuronal cells by ionotropic excitatory amino acid receptors, thereby leading to loss of neurons208.

Furthermore, research has demonstrated that OKA triggers the production of ROS in the hippocampus and reduces mitochondrial activity and mitochondrial potency, ultimately resulting in mitochondrial abnormalities in the brain of rats201. Additionally, OKA has been known to inhibit phosphatases and cause hyperphosphorylation of proteins which subsequently leads to neuronal stress and ultimately to neurodegeneration201. Previous findings revealed that OKA-mediated memory decline in rats is linked with exacerbated proinflammatory cytokines, such as TNF-α and IL-1, and with iNOS expression and total nitrite in the hippocampus and cortex 209. Furthermore, infusion (bilateral) of OKA in the hippocampus produced spatial cognitive deficit as a result of raised GFAP expression, decreased GSH, and enhanced protein carbonylation and mitogen-activated protein kinase 38 (p38MAPK)210. Several kinases, including MAPK, GSK-3β and cyclin-dependent kinase 5 (Cdk5), have been identified to be implicated in the phosphorylation of tau protein at different positions seen in AD hyper-phosphorylated tau211,212. Meanwhile, tau dephosphorylation is catalyzed through phosphatase PP2A and other phosphatases (PP1, 2B, and 5)213,214. The precise blocking of PP2A by OKA could result in Alzheimer-like hyperphosphorylation and aggregation of tau 215 and 216,217. Memory loss due to OKA (intra-hippocampal) injection has been documented to be linked with apparent neuropathological changes, such as the formation of Aβ peptide-like structures, helical filament-like phosphorylated tau, and neurodegeneration in the hippocampus210. OKA is an essential means for analyzing218 the cellular mechanisms involved by reversible protein phosphorylation during cell division, signal transduction, and formation of memory217. At present, drugs that act by blocking tau phosphorylation are not available. Thus, this suggests that OKA could be a useful replacement tool towards unravelling therapeutic approaches for AD pathology198.