Phosphorylation of The Tau Protein in Neurodegenerative Disease

Protein phosphorylation is a reversible post-translational modification that involves a series of sequence-specific kinases and occurs on specific residues such as serine, threonine, and tyrosine. The reversible phosphorylation of proteins regulates almost all aspects of the cell’s life cycle and abnormal phosphorylation is the cause or consequence of many diseases. Protein phosphorylation states can mediate protein complex formation and regulate protein function, which is important for cell physiology but can also promote neuropathic events. The tau protein is a very important microtubule-associated protein in the brain, occurring most commonly in neurons and glial cells. Its level of phosphorylation is associated with a variety of diseases of the central nervous system such as Alzheimer’s disease. Under normal circumstances, post-transcriptional tau phosphorylation is conducive to the stability of microtubules. However, hyperphosphorylation can lead to the deformation and aggregation of various types of cytoskeletal components of nerve tissue, causing them to lose normal function. Disease; MTRS: Mercuric Transport; SER: Serine; THR: Threonine; Microtubule; GSK3β: Glycogen Synthase Kinase 3β; ALA: Alanine; Projection Domain; MBD: Microtubule-Binding Domain; Progressive Supranuclear MAP1B: 1B; Synthase 5; Neurofibrillary Helical Filaments; Filaments; Frontotemporal lobar degeneration; Creutzfeldt-Jakob Disease; Proline-Dependent

(AD) is the most extensively studied and significant progress has been made. The function of the microtubule-associated protein tau is to promote microtubule assembly and stabilization in neurons, which is required for axonal transport and neurite outgrowth [2]. Tau is a microtubule-associated phosphoprotein that is abundant in neurons and is regulated by protein kinases and protein phosphatases. Appropriately phosphorylated Tau binds to microtubules, thereby stimulating the assembly of tubulin into microtubules and maintaining microtubule stability [3]. In the brain of Alzheimer's disease, tau is abnormally hyperphosphorylated; it contains three to four times more phosphate than normal tau [4].
In vitro and in vivo, hyperphosphorylation of tau has been shown to reduce the affinity of tau for microtubules, leading to disruption of neuronal cytoskeleton and axonal transport [5]. Abnormal aggregation of hyperphosphorylated tau protein is a common pathological feature of neurodevelopmental disorders commonly referred to as tauopathy, including AD, progressive supranuclear palsy and frontotemporal dementia [6]. Several neurodegenerative diseases, collectively referred to as tauopathy, are characterized by insoluble, highly phosphorylated tau that is a neuronal inclusion of straight or paired helical filaments [7].

Microtubules and the Effects of Tau Phosphorylation on Microtubule Structure
Neuronal development and function are influenced by the cytoskeletal infrastructure of cells, namely microtubules, actin, and intermediate filament networks. Microtubule cytoskeletal networks are organized into stable and dynamic arrays that provide structural support as molecular motion trajectories and serve as signal platforms during neuronal development and plasticity [8][9][10]. Microtubules are composed of alpha-and beta-tubulin heterodimers that assemble into protofilaments and then laterally contact each other to form tubules [11]. β-Tubulin must be in a GTP-bound state to allow the assembly of heterodimers onto the protofilament. Alpha-tubulin binds to β-tubulin but only β-tubulin can hydrolyze GTP. Once the protofilament is assembled, β-tubulin is exposed at the "plus end" and alpha-tubulin is exposed at the "minus end." This structural polarity leads to a difference in the growth rate at each end and it has been observed that end-capping occurs more often [12] and is much faster on the plus end than on the minus end. Microtubules can be modified within cells by switching between assembled and disassembled states in a process called dynamic instability [13]. MAPs have the ability to bind to microtubule lattices, tubulin heterodimers, or both. They can thereby regulate the assembly/disassembly kinetics of microtubules to properly organize and remodel microtubule cytoskeletal structure during neuronal development and activity [14,15].
The α-and β-tubulin heterodimers that assemble into microtubules exist in a state of dynamic equilibrium with non-polymeric tubulin. The filamentous structure of microtubules forms intracellular cytoskeletons in a variety of cells but are particularly enriched in neurons [16][17][18]. The dynamics of microtubule assembly can be regulated by temperature, microtubule protein modifications, small molecules such as paclitaxel, and some mercuric transport Tau protein has been identified as a factor that promotes microtubule assembly and stability. Microtubule assembly is thought to be negatively regulated by tau protein phosphorylation. More than 40 serine (Ser) and Threonine (Thr) residues have been identified as possible phosphorylation sites on the tau protein. Although the biological significance of every single phosphorylation site is not clear, it is known that phosphorylation of tau at Ser-262 of tau (in the 441-residue tau protein) has a profound influence on its interaction with microtubules [25]. Tau Phosphorylation on the Structure of  Threonine 231  cause it separates tau from MTS, which may Interaction with   The six unique isomers are mainly differentiated by the number of N-terminal insertion sequences (0N, 1N, or 2N) and the number of microtubule binding repeats (3R or 4R) that they contain. In the normal adult brain, the ratio of the 3R to 4R isomers is about 1:1.

Structure and Phosphorylation of Tau Protein
The tau protein was identified in 1975 as a protein with the ability to induce microtubule formation [38,39]. It is the most widely occurring MAP in the normal brain and its primary function is to bind tubulin and promote its polymerization into mi- Thr-231is higher in the brains of AD patients and these three phosphorylated forms of tau can therefore be used as biomarkers for AD [50][51][52]. Kinetic analysis showed that pseudophosphorylation increased the tau aggregation rate by increasing the filament nucleation rate. In addition, it increases the tendency to aggregate by stabilizing mature filaments to prevent depolymerization. The covalently bound phosphate is distributed within the tau microtubule-binding domain and adjacent to approximately 40 sites [45,53,54]. The occupancy of these sites may affect the tau aggregation in two ways. First, the occupancy of certain loci regulates the affinity of tau-tubulin [55], promoting an increase in the level of free cytoplasmic tau available for nucleation and supporting aggregation reactions [56][57][58][59]. Second, hyperphosphorylation directly increases the tendency of tau aggregation [60,61]. In addition, tau phosphorylation has been reported to reduce proteasome-mediated tau conversion in neuronal cell models [62]. Thus, the occupancy of certain tau phosphorylation sites can increase the free cytoplasmic tau concentration by a variety of mechanisms.

Tau phosphorylation and Neurological Diseases
Neurodegenerative diseases with abundant filamentous tau protein inclusion bodies are called tauopathies. Some neurodegenerative diseases differ from AD in that they lack the pathology of beta-amyloid plaques [63]. However, the tauopathies other than AD include chromosome 17-linked Parkinson's disease with frontotemporal dementia, chronic traumatic encephalopathy, argicophilia granulosus, Progressive Supranuclear Palsy (PSP), corticobasal degeneration, globular glia tauopathy, and Pick's disease. Due to the abnormal accumulation of phosphorylated tau protein in neuronal and glial cells in these neurodegenerative diseases, synaptic plasticity of hippocampal neurons can be affected, and memory function seriously disrupted [64]. It has been reported that changes in protein phosphorylation affect axonal transport in neurodegenerative disease models. For example, one study showed that as phosphorylation of neurofilament proteins and the microtubule-associated protein MAP1B increased, their respective axonal transport rates decreased [65].
In contrast, another study revealed that the enhanced phosphorylation level of tau increased the overall slow rate of tau protein transport in neurons and that the inhibition of tau phosphorylation by GSK-3 decreased its motility ( Figure 2). Due to these and other similar findings, axonal transport defects have been regarded one of the contributing factors to neurodegenerative disease [66].  , as well as cyclin-dependent kinases (like CDK5) and activator subunit p25, to form highly phosphorylated tau proteins. This highly phosphorylated form of the protein then dissociates into helical filaments that eventually form neurofibrillary tangles (NFTs).
Phosphorylation of tau enhances PHF formation. Phosphorylation can also be a physiologically feasible way to bring tau into a PHF-prone state. Phosphorylation can alter the conformation of tau, making it long and stiff [72]. Negative-stained electron microscopy showed that the core of the PHFs and SFs is composed of a double helix stack of C-shaped subunits [73] and successive steps along the β-strand of the protofilament are linked by helical symmetry. Moreover, the C-terminal region of tau is disordered, and it projects away from the core to form a fuzzy shell [74]. The protofilament cores of the PHFs and SFs are similar, indicating that they are ultrastructural polymorphs. The ultrastructural polymorphism between the PHF and SF is due to the difference in lateral contact between the two protofilaments. In the PHF, the two strands form exactly the same spiral symmetric structure, whereas in the SF, the protofilaments are asymmetric. In AD, tau is highly phosphorylated and many of the major kinases that phosphorylate the tau protein target glycogen synthase kinase-3 (GSK-3)-targeted tau phosphorylation sites [75]. Another of the major kinases responsible for tau hyperphosphorylation is cyclin-dependent kinase 5 (CDK5), a member of the serine/threonine kinase family of cyclin-dependent kinases.
Most AD neurons do not have normal microtubule structure but instead have pathological NFTs that are paired helical filaments of abnormal, hyperphosphorylated tau. Since tau pathology has been shown to be associated with neuronal loss, one of the treatment strategies targeting the molecular basis of AD includes inhibition of tau hyperphosphorylation [76]. To examine whether microtu-  [64]. PSP is a rare, late-onset neurodegenerative disease whose clinical symptoms include early postural instability, vertical gaze palsy, and a later onset of dementia.
From the ultrastructural perspective, the NFT filaments present in PSP are straight and contain only the 4R isoform of the tau protein [78]. Animal models have revealed that mutations in the tau gene led to sprouting in dentate gyrus granule cells of hippocampal mossy fibers, and primary epilepsy is partially caused by mutations in the Tau protein gene. The S169L mutation of the presenilin 1 gene has also been found in patients with epileptic seizures and familial Alzheimer's disease [79]. AD is the most common cause of dementia. It is a degenerative disease of the central nervous system and is mainly characterized by progressive cognitive impairment and memory impairment. The main pathological features of AD are senile plaques and neurofibrillary tangles. The core component of neurofibrillary tangles is the double-helical fibril formed by abnormally modified Tau protein [80]. Creutzfeldt-Jakob disease (CJD) is a rare and fatal human neurodegenerative disease that belongs to family of diseases known as transferable spongiform encephalop-athies or prion diseases. The cerebrospinal fluid level in patients with CJD is significantly higher than that of AD patients and other dementia patients [81] (Table 1). As detailed above, it is clear that tau protein is closely associated with many diseases of the central nervous system and clarifying its mechanism of action can lead to new targets of treatment for tau protein-related diseases. AD Degenerative disorder of the central nervous system Senile plaques and nerve fiber tangles made of double helix fibrils containing abnormally modified tau protein. [80] CJD Neurodegenerative disease that can transmit spongiform encephalopathy Tau protein content in CSF is more than 2131pg/ml, which manifests as mental disability, ataxia, and myoclonus. [81]

Phosphorylation Affects Axonal Transport and Degradation of the Tau protein
The This domain contains either three or four binding repeats (depending on alternative splicing of tenth exons), resulting in a 3R or 4R tau protein isomer, respectively. However, tau also interacts with components of the plasma membrane through its N-terminal projection domain [83]. While we know that phosphorylation of tau reduces its ability to bind and stabilize microtubules, we have recently found that the binding of tau to the plasma membrane is also regulated by phosphorylation [84]. It is well known that increased phosphorylation of tau reduces its affinity for microtubules, leading to instability of the neuronal cytoskeleton [85].  [89], indicating that phosphorylation at other sites is necessary to completely inhibit its biological activity. The 21 phosphorylation sites in PHF-tau have been identified by reactivity with antibody and protein sequencing technologies at various phosphorylation sites. Among them, 10 sites are on the Ser / Thr-Pro motif and 11 are on the non-Ser / Thr-Pro motif [90,91]. Ser / Thr-Pro and non-Ser / Thr-Pro sites may be phosphorylated by proline-dependent protein kinase (PDPK) and non-PDPK, respectively.
In the non-proline-directed phosphorylation site of PHF-tau, both Ser-208 and Ser-210 are in the SR-motif range.
In addition, in addition to the known GSK-3βphosphorylation site on tau, studies have identified a new phosphorylation site Thr-175 and a non-prolineated phosphorylation site Ser-400. TTK is a non-proline-directed Ser / Thr kinase that has been purified from bovine brain [92]. It is the first tau kinase to phosphorylate Ser-208 and Ser-210, both of which are PHE phosphate. Site. Thr-212 is a neighboring residue close to Ser-208 in tau and is known to be the phosphorylation site of GSK-3β [93]. Absorption tests by peptides pS208 and pS210 demonstrated the specificity of anti-pS208.
Therefore, we can confirm that the phosphorylation site Ser-208 is a site separate from Thr-212. In addition to affecting its transport, tau phosphorylation also affects its ability to be degraded [94]. We studied the degradation of tau by the ubiquitin-proteasome system (UPS) and macroautophagy (autophagy) in the context of tau transport. While the UPS eliminates transient proteins by tagging them with chains of ubiquitin, autophagy removes long-lived structural proteins, as well as damaged or misfolded proteins [95]. Autophagy has also been shown to reduce both wild-type and modified tau proteins, including caspase-cleaved and C-terminally truncated species [96].

Tau protein hyperphosphorylation
Aberrant protein phosphorylation can lead to disease-related processes [97]. Accordingly, the abnormal phosphorylation of tau is observed in many neurodegenerative diseases. For example, histopathological investigations of AD showed extra-neuronal accumulation of β-amyloid peptide in plaques, neuronal aggregates of NFTs, and astrogliosis surrounding neurons [98]. Abnormal hyperphosphorylation of tau leads to aggregation, formation of NFTs, microtubule rupture, neuronal dysfunction, and death [99]. NFT

consists of a pair of helical filaments (PHF), which in turn consists
of a microtubule-associated protein tau in a hyperphosphorylated state [100]. In AD, the phosphorylation/dephosphorylation system appears to be greatly affected [101]. It has been shown that brain glucose uptake/metabolism in AD is impaired [102] and this damage has been suggested to be associated with abnormal hyperphosphorylation of tau. This finding implicates astrocytes as a key factor, especially because changes in glucose uptake and/or glutamate uptake (mediated by astrocytes) affect neuronal function and survival. identified in the human brain, PP2A accounts for more than 70% of tau dephosphorylation [110]. In the AD brain, PP2A activity was significantly reduced [111]. PP2A is a multimeric enzyme consisting of a catalytic subunit (C) and two regulatory subunits (A subunit or B subunit). The physiological form of PP2A is considered to be a heterogeneous composition composed of A and C subunits. Trimer. The major natural form of PP2A is a heterotrimer in which the core enzyme binds to one of several regulatory subunits expressed in a cell-and tissue-specific manner [112]. Another potential function of PP2A in the brain is to regulate phosphorylation of microtubule-associated protein (MAPS).  [113]. The hyperphosphorylation of the tau protein and the subsequent formation of NFTs are associated with abnormal activation of protein kinases [114].
In fact, studies have shown that the imbalance of kinase and phosphatase activity may play a causative role in the hyperphosphorylation of tau [102]. The proline-directed protein kinases that catalyze the phosphorylation of tau (such as GSK-3 and CDK5) predominantly do so at Ser-Pro and Thr-Pro sites on the tau protein, whereas the non-proline-directed protein kinases (such as protein kinase A, protein kinase C, calmodulin-dependent kinases, plasmin-dependent kinases, and glucocorticoid-dependent kinases) primarily phosphorylate serine or threonine residues and do not require proline guidance. It has been demonstrated at the cellular, brain, and animal levels that phosphatases play an important role in protein degradation in neurons in diseases such as AD. Studies have reported that inhibiting protein phosphatase activity induced tau hyperphosphorylation and aggregation [115].

Drugs that Affect Tau Phosphorylation Patterns
Nimodipine Attenuates Phosphorylation of Tau at Ser-396: Nimodipine is an L-type calcium channel antagonist that reduces excessive calcium influx in pathological conditions [116] and shows neuroprotective effects. Nimodipine treatment was initially used due to its ability to produce vasodilation in smooth muscle cells lined with blood vessels [117]. Chronic cerebral hypofusion (CCH) has been reported to promote hyperphosphorylation of the tau protein. It showed that nimodipine attenuated CCH-induced tau phosphorylation by up-regulating the expression of miR-132. In addition, nimodipine inhibited CCH-induced activation of GSK-3β and neuronal apoptosis. These findings support the role of nimodipine in inhibiting tau phosphorylation at Ser-396 via miR-132/GSK-3β and points to new potential drug target for the treatment of tauopathy in CCH by regulating the miR-132/GSK3β pathway [116].
Tamoxifen Inhibits CDK5 Kinase Activity and Regulates Tau Phosphorylation: CDK5 is a multifunctional enzyme that plays an important role in brain development. The catalytic subunit of this kinase does not have enzymatic activity as a monomer but is activated by binding to activation subunits p35 or p39. These activation subunits are structurally related to cyclins, activators of cell cycle CDKs, but do not show homology with cyclins at the amino acid level. In contrast to other CDKs, activation of CDK5 does not require phosphorylation of the activation loop.
Studies have shown that neurotoxicity induces proteolytic cleavage of the p35 subunit by calcium-regulated calpains [68]. In vitro experiments have shown that this proteolytic conversion of p35 to p25 does not significantly alter the steady-state kinetics of tau phosphorylation by CDK5 [118]. The binding of CDK5 to p25, the N-terminally truncated proteolytic product, stabilizes CDK5 in the active dimer form and alters its substrate specificity.et al.identified tamoxifen from a large-scale bioluminescent resonance energy transfer (BRET)-based screen of small molecules that inhibit the interaction between CDK5 and p25. They showed that tamoxifen reduced tau phosphorylation by blocking the activation of CDK5 by p25 [118]. This finding paves the way for new therapies for tauopathies by harnessing the drug tamoxifen [118].

Rapamycin Reduces Tau Phosphorylation at Ser-214 by Modulating cAMP-Dependent kinases: Mammalian
target of rapamycin (mTOR) is a highly evolutionarily conserved serine/threonine kinase. mTOR is involved in regulating many cellular processes such as autophagy, protein translation, ribosome biosynthesis, actin organization, mitochondrial oxygen consumption, proliferation, and differentiation [119]. It is worth noting that mTOR acts as a linker to protein kinase signals, receiving inputs from many upstream signaling pathways and delivering various downstream kinases such as cAMP-dependent protein kinases (e.g. PKA), GSK-3β, and mitogen-activated protein kinases [120]. Since all these kinases are tau-associated kinases, whether rapamycin can modulate tau phosphorylation by regulating these kinases remains to be determined. In human neuroblastoma SH-SY5Y cells, a cell model widely used for tau pathology studies, research indicated that rapamycin reduced the PKA-mediated phosphorylation of tau at Ser-214. Similar results were obtained in wild-type human embryonic kidney 293 (HEK293) cells that were stably transfected with the longest isoform of recombinant human tau (tau441; HEK293/tau441). Since Ser-214 is a site that blocks tau hyperphosphorylation [121], the inhibition of mTOR by rapamycin could indirectly prevent or reduce tau hyperphosphorylation.
Research has focused on rapamycin-induced enhancement of autophagy, as autophagy mediates massive degradation of cytoplasmic content and thus enhances the clearance of hyperphosphorylated tau [122]. It is also thought that rapamycin may inhibit the synthesis of the tau protein. However, since autophagyinduced by rapamycin gives priority to the reduction of excessive phosphorylated and insoluble tau and soluble tau is dispersed throughout the cell, it may not be easy to reduce tau levels by autophagic degradation, and showed that rapamycin improved memory deficits in 6-month-old 3xTg AD mice before accumulation of hyperphosphorylated and insoluble tau was observed [123]. Similarly, another study using an AD mouse model showed that the protective effect of rapamycin was apparent only before insoluble tau accumulated in these animals [124]. These studies suggest that the protective effects of rapamycin may not be limited to autophagic clearance of hyperphosphorylated and insoluble tau.

Conclusion
As a major MAP, tau protein plays an important role in neurodegenerative diseases. AD is pathologically identified by the presence