*3.1.2. Tauopathy animal models*

Pathogenic tau mutations typically result in either RNA splicing variation causing the ratio change of 3R/4R or a structure change, which will further affect its binding affinity with microtubule and other proteins or promote its self-assembly [66, 67, 69]. The mutation sites cover the whole protein and could also be in the introns affecting the RNA splicing [67]. Some much more common mutations have been selected in generating transgenic models for studying tauopathies [70]. To date, 28 tauopathy mouse models have been reported according to Alzhforum.org and many of which are overexpressing models [67]. Other tauopathy animal models in *Drosophila*, zebrafish, and *C. elegans* also help the field to untangle the molecular and cellular complexity of this clinical condition [71–74].

the pathogenesis and treatment methods for tauopathy with a mouse. Another major concern is related to the modulation of endogenous enzymes at the time of human tau expression, which is an issue difficult to control. Some key features related to general pathogenesis process were missed from the transgenic mice models, including robust tau propagation and significant cell apoptosis [48, 81]. In one study, by injecting insoluble tau to mouse hippocampus, it was found that the seeding and propagation ability of the synthesized insoluble tau are much weaker than the insoluble fraction of isolated tau from the AD in vivo [48]. Together, it shows there is still a long way to go before using these animal models to find a strategy for detour tauopathy.

Tauopathy

41

http://dx.doi.org/10.5772/intechopen.73198

Recent advances in human stem cell research may provide a solution for tauopathy and related research. By grafting human stem cell-derived neuron to express amyloid-beta in mouse brain, a study revealed that the human neurons are more susceptible to the toxicity of amyloid-beta than mouse neurons [81]. This observation proved the discrepancy of cells from the two organisms. Therefore, if the quality of the grafted cells and the surrounding microenvironment represent the physiological conditions in the human brain, shall we also expect human neurons

Most patients who suffer from tauopathies carry wild-type MAPT. Therefore, posttranslational modifications of tau are believed to be the key of tau pathogenesis and have been the major area of tauopathy research for years. Understanding the posttranslational modifications of tau not only helps us to pin down the pathogenic mechanism but also offers a viable path for drug screen by targeting certain enzymes that modifying tau. As mentioned in the above sections, altered posttranslational modifications of tau could render the protein to lose its native unfolded structure and by which to promote aggregate formation. In this process, the modifications also changed the interactions between tau and other proteins in addition to tubulins. So far, 10 types of tau posttranslational modifications have been documented [82], among which phosphorylation and truncation are most found, representing the majority of the modifications, while other modifications (ubiquitylation, oxidation, glycosylation, glycation, nitration, acetylation, and sumoylation) are either not often discussed in this setting or just recently founded. In total, over 100 sites on a tau protein have been proposed that could

In one of the pioneer studies aimed to prove that neurofibrillary tangles are made of tau, the researchers found treating the tissue section with phosphatase could dramatically increase the antibody labeling of tau on the tangles [9], and the researchers coined the staining as "atypical phosphorylated" tau. Since then, the "hyperphosphorylated" tau under pathological conditions received a great deal of attention. Many protein kinases have been proposed to play roles in tau phosphorylation, and some of them have been confirmed by in vivo studies [9, 84]. Recent developments have proposed to try out some kinase inhibitors as potential tauopathy therapeutics [85]. For a 2N4R tau protein, it consists of 45 serine residues, 35 threonine

are more susceptible to the toxicity of tau?

**3.2. Posttranslational modifications of tau**

be modified if not considering the truncation [83].

*3.2.1. Phosphorylation*

Transgenic expression of normal human tau in mouse models with either 3R or 4R forms were unable to induce significant pathological changes [75]. While with strong pan-neuronal promoters could induce more pathological features, this approach also raises a concern of overwhelmed tau expression, which may lead to possible artificial effects by causing heavy burdens on protein degradation systems, which obviously deviated from the progressive pathogenesis that is responsible for the sporadic tauopathy [75–77]. Notably, a recent study created transgenic mice expressing an N-terminal truncated tau under the control of human tau promoter so to mimic the normal expression level and by which recapitulated some major pathological features of tauopathies [76]. The authors claimed that a similarly truncated tau could be found in postmortem progressive supranuclear palsy brains, which makes this finding quite interesting.

In comparison, expressing disease-linked mutant tau can induce more pronounced pathological effects, and some of the models are widely used in basic or pre-clinical research settings if not in combined with other tauopathy-related protein expression [67, 70, 75, 77]. The most commonly adopted tau mutations in transgenic animal models are P301L, P301S, R406W, and V337 M [59, 77]. All these mutants were found in FTD patients, and their expression showed reduced binding affinity to the microtubule. Importantly, all of them could efficiently induce tau filaments formations in mouse models, although the composition of the filaments may be different for different tau mutations [66, 70]. Among them, three mouse models stand out in terms of their wide usage in basic research, as well as in the pre-clinical tests of drug development. These are P301L, PS19, and rTg4510 (r for regulatable), all of which were developed in the mid-2000s [78–80]. P301L mice overexpress 2N4R tau with P301L mutation under the pan-neuronal driver Thy1 [78]. PS19 mice overexpress 1N4R tau with P301S mutation driven by mouse prion protein promoter [79], and rTg4510 adopts the tet-off system to overexpress 0N4R tau bearing P301L mutation only in the absence of tetracycline that controlled by Ca2+/ calmodulin-dependent protein kinase II (CAMKII) promoter [80]. In general, rTg4510 and PS19 mice show more massive pathology burdens in comparison with to P301L. Although overt tau aggregations were observed in all three types of mice and showed cognitive defects, only rTg4510 and PS19 were reported to induce significant neuronal loss [79, 80]. PS19 mice showed severe hippocampus shrinkage at the age of 9 months, while rTg4510 mice showed gross forebrain atrophy at the age of 10 months [79, 80]. It is noteworthy that human tau expression levels were several times higher than the endogenous mouse tau levels for all three models [78–80].

These data collectively show that while significant tau aggregates can be induced by expressing mutant tau, the models are different from sporadic tauopathy, especially in terms of studying the pathogenesis and treatment methods for tauopathy with a mouse. Another major concern is related to the modulation of endogenous enzymes at the time of human tau expression, which is an issue difficult to control. Some key features related to general pathogenesis process were missed from the transgenic mice models, including robust tau propagation and significant cell apoptosis [48, 81]. In one study, by injecting insoluble tau to mouse hippocampus, it was found that the seeding and propagation ability of the synthesized insoluble tau are much weaker than the insoluble fraction of isolated tau from the AD in vivo [48]. Together, it shows there is still a long way to go before using these animal models to find a strategy for detour tauopathy.

Recent advances in human stem cell research may provide a solution for tauopathy and related research. By grafting human stem cell-derived neuron to express amyloid-beta in mouse brain, a study revealed that the human neurons are more susceptible to the toxicity of amyloid-beta than mouse neurons [81]. This observation proved the discrepancy of cells from the two organisms. Therefore, if the quality of the grafted cells and the surrounding microenvironment represent the physiological conditions in the human brain, shall we also expect human neurons are more susceptible to the toxicity of tau?

#### **3.2. Posttranslational modifications of tau**

Most patients who suffer from tauopathies carry wild-type MAPT. Therefore, posttranslational modifications of tau are believed to be the key of tau pathogenesis and have been the major area of tauopathy research for years. Understanding the posttranslational modifications of tau not only helps us to pin down the pathogenic mechanism but also offers a viable path for drug screen by targeting certain enzymes that modifying tau. As mentioned in the above sections, altered posttranslational modifications of tau could render the protein to lose its native unfolded structure and by which to promote aggregate formation. In this process, the modifications also changed the interactions between tau and other proteins in addition to tubulins. So far, 10 types of tau posttranslational modifications have been documented [82], among which phosphorylation and truncation are most found, representing the majority of the modifications, while other modifications (ubiquitylation, oxidation, glycosylation, glycation, nitration, acetylation, and sumoylation) are either not often discussed in this setting or just recently founded. In total, over 100 sites on a tau protein have been proposed that could be modified if not considering the truncation [83].

#### *3.2.1. Phosphorylation*

microtubule and other proteins or promote its self-assembly [66, 67, 69]. The mutation sites cover the whole protein and could also be in the introns affecting the RNA splicing [67]. Some much more common mutations have been selected in generating transgenic models for studying tauopathies [70]. To date, 28 tauopathy mouse models have been reported according to Alzhforum.org and many of which are overexpressing models [67]. Other tauopathy animal models in *Drosophila*, zebrafish, and *C. elegans* also help the field to untangle the molecular

Transgenic expression of normal human tau in mouse models with either 3R or 4R forms were unable to induce significant pathological changes [75]. While with strong pan-neuronal promoters could induce more pathological features, this approach also raises a concern of overwhelmed tau expression, which may lead to possible artificial effects by causing heavy burdens on protein degradation systems, which obviously deviated from the progressive pathogenesis that is responsible for the sporadic tauopathy [75–77]. Notably, a recent study created transgenic mice expressing an N-terminal truncated tau under the control of human tau promoter so to mimic the normal expression level and by which recapitulated some major pathological features of tauopathies [76]. The authors claimed that a similarly truncated tau could be found in postmortem progressive supranuclear palsy brains, which makes this find-

In comparison, expressing disease-linked mutant tau can induce more pronounced pathological effects, and some of the models are widely used in basic or pre-clinical research settings if not in combined with other tauopathy-related protein expression [67, 70, 75, 77]. The most commonly adopted tau mutations in transgenic animal models are P301L, P301S, R406W, and V337 M [59, 77]. All these mutants were found in FTD patients, and their expression showed reduced binding affinity to the microtubule. Importantly, all of them could efficiently induce tau filaments formations in mouse models, although the composition of the filaments may be different for different tau mutations [66, 70]. Among them, three mouse models stand out in terms of their wide usage in basic research, as well as in the pre-clinical tests of drug development. These are P301L, PS19, and rTg4510 (r for regulatable), all of which were developed in the mid-2000s [78–80]. P301L mice overexpress 2N4R tau with P301L mutation under the pan-neuronal driver Thy1 [78]. PS19 mice overexpress 1N4R tau with P301S mutation driven by mouse prion protein promoter [79], and rTg4510 adopts the tet-off system to overexpress 0N4R tau bearing P301L mutation only in the absence of tetracycline that controlled by Ca2+/ calmodulin-dependent protein kinase II (CAMKII) promoter [80]. In general, rTg4510 and PS19 mice show more massive pathology burdens in comparison with to P301L. Although overt tau aggregations were observed in all three types of mice and showed cognitive defects, only rTg4510 and PS19 were reported to induce significant neuronal loss [79, 80]. PS19 mice showed severe hippocampus shrinkage at the age of 9 months, while rTg4510 mice showed gross forebrain atrophy at the age of 10 months [79, 80]. It is noteworthy that human tau expression levels were several times higher than the endogenous mouse tau levels for all three

These data collectively show that while significant tau aggregates can be induced by expressing mutant tau, the models are different from sporadic tauopathy, especially in terms of studying

and cellular complexity of this clinical condition [71–74].

ing quite interesting.

40 Cognitive Disorders

models [78–80].

In one of the pioneer studies aimed to prove that neurofibrillary tangles are made of tau, the researchers found treating the tissue section with phosphatase could dramatically increase the antibody labeling of tau on the tangles [9], and the researchers coined the staining as "atypical phosphorylated" tau. Since then, the "hyperphosphorylated" tau under pathological conditions received a great deal of attention. Many protein kinases have been proposed to play roles in tau phosphorylation, and some of them have been confirmed by in vivo studies [9, 84]. Recent developments have proposed to try out some kinase inhibitors as potential tauopathy therapeutics [85]. For a 2N4R tau protein, it consists of 45 serine residues, 35 threonine residues, 12 histidine residues, and 5 tyrosine residues. Current postulated phosphorylation sites have essentially covered most of the available sites, and indeed many of these residues are found being phosphorylated under physiological conditions. Therefore, the widespread, and likely dynamic, tau phosphorylation appears to serve for certain uncovered functions. Nevertheless, it also indicates that the differences in general phosphorylation and changes of phosphorylation state in certain residues may play a critical role in tauopathies [83, 84]. Moreover, hyperphosphorylated tau could also be detected in other pathological conditions aside from tauopathy. It was found that phosphorylated tau proteins are co-aggregated with alpha-synuclein in Parkinson disease and Lewy bodies dementia [86]. In some reports, phosphorylated tau aggregation can be found in Huntington disease and amyotrophic lateral sclerosis brains [87, 88]. In traumatic brain injury patients, the levels of phosphorylated tau, but not total tau, are significantly increased [89]. Recently, it is also suspected that tau phosphorylation may play a role in type 2 diabetes, rendering the patients incline to have cognitive defects [90]. These data collectively indicate hyperphosphorylation of tau has a strong correlation with a variety of brain pathologies, not just tauopathies.

study found active GSK3β could efficiently facilitate tau tangles formation after tau are initially polymerized in the presence of arachidonic acid [100]. It should be noticed that effects of GSK3β in tau-mediated toxicity are unsettled; a report found that overexpression of GSK3β in tauopathy models may not necessarily lead to shortened lifespan or accelerate pathological burden in the animal model [101]. In contrast, results from some studies hinted that activation of GSK3β is critical for exacerbating tauopathy [102, 103]. These observations should be interpreted carefully as the tau models used or compared are not in the same background.

Tauopathy

43

http://dx.doi.org/10.5772/intechopen.73198

GSK3β is a constitutively active protein that can autophosphorylate its tyrosine residues like Tyr 216 to increase the enzyme stability [104]. The activity of GSK3β is mainly regulated by insulin and Wnt signaling pathways [105]. When insulin pathway is activated, protein kinase B/Akt will be activated, which in turns phosphorylate serine 9 on GSK3β and causes its inactivation. In the case of activated Wnt signaling pathway, the inhibition of GSK3β activity would alleviate the degradation of β-catenin, whose nuclear translocation is responsible for the downstream genes activation of the pathway, but the precise mechanism regarding tauopathy modulation is still unknown [106, 107]. Nevertheless, a report showed both path-

It was postulated that the phosphorylation by GSK3β requires the priming of adjacent proline residue as GSK3β is a member of proline-directed kinase family [84]. So far, more than 40 sites in tau, either serine or threonine, have been reported could be phosphorylated by GSK3β, and some of them are exclusively found in pathological conditions [84]. Among these sites, in vitro study first identified tau could be phosphorylated by GSK3β at the sites S202, S396, and S404 [108]. Since then, different studies reported many different phosphorylation sites with the availability of the corresponded phospho- site-specific antibodies. Some in vitro/in vivo studies later confirmed that frequent phosphorylation sites include S262, S396, and S404 [101, 109]. The phosphorylation of some residues may play important roles in affecting the binding affinity between tau and tubulins or regulating synaptic plasticity [4, 110, 111]. However, it is still unknown which residues are most frequently phosphorylated by GSK3β under different pathological states in contrast to normal condition, and if there is any protective effect by GSK3β phosphorylation against tau from forming the aggregates. Probing these questions is a major challenge but will help our understanding of the role of GSK3β in tau-associated

Besides GSK3β, other kinases such as p38, cyclin-dependent kinase 5 (CDK5), c-Jun N-terminal kinase (JNK), extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), casein kinase (CK), protein kinase A (PKA), and Ca2+ /calmodulin-dependent protein kinase II (CAMKII) have been reported to involve in directing tau phosphorylation [4, 84, 112–114]. Although the possible phosphorylation sites mediated by these kinases, especially in tauopathy conditions, may be less than GSK3β, it is fair to say that their roles in modulating tau toxicity are less well studied, thus whether the modulations by these kinases are less critical than GSK3β in tauopathy remain to be addressed. Indeed, a recent study found P38γ overexpression could ameliorate excitotoxicity

ways are being downregulated in AD [96].

disease conditions.

*3.2.1.2. Other kinases*

However, the scenario of pathogenesis in tau hyperphosphorylation is more than merely the activation of some kinases or down-regulation of some phosphatase. As a matter of fact, different kinases are interacting, regulating, and even competing with each other for acting or interfering on same sites [91, 92], it is conceivable that the dynamics of transferring/removing phosphate groups on tau protein could be complex. Besides, kinases have multiple substrates, and some of them have important roles in normal cell functions [91]. The activities change of a kinase could lead to a domino effect toward the change of cellular activities. Last but not least, not all phosphorylations are toxic as some of them are required for normal tau functions, and certain sites phosphorylation may even serve as a protective effect in tauopathies [4, 5]. Therefore, a systemic dissection of disease-prone tau phosphorylations and their regulation is a pre-requisite before aiming such complex regulation for a therapeutic exploration.

To study the phosphorylations, many antibodies recognizing specific phosphorylated residues on tau have been generated, and a list can be found on Alzforum.org [93]. For analyzing the effect of phosphorylation, recombinant MAPT constructs bearing site-specific mutations to mimic potential phosphorylation status of tau are regularly utilized in tauopathy research, which provides some insights regarding the genotoxic and structural impacts upon modifications [94]. However, even with the recombinant tau with or without pseudo-phosphorylations, it is hard to generate significant polymerization in vitro postulated due to a lack of "nucleation" process, although pseudo-phosphorylated tau may be prone to aggregate [95].

#### *3.2.1.1. GSK3β*

It is well acknowledged that glycogen synthase kinase 3 beta (GSK3β) plays a pivotal role in tau hyperphosphorylation [96, 97]. An early study showed that recombinant tau and microtubule-associated GSK3β that were harvested from bacterial lysates could be co-eluted in immunochromatography with anti-GSK3β and co-immunoprecipitated [98]. A subsequent study found active GSK3β co-localized with tau inclusions in tauopathy brain tissues, and the amount of active GSK3β was significantly increased in the patients [99]. Moreover, in vitro study found active GSK3β could efficiently facilitate tau tangles formation after tau are initially polymerized in the presence of arachidonic acid [100]. It should be noticed that effects of GSK3β in tau-mediated toxicity are unsettled; a report found that overexpression of GSK3β in tauopathy models may not necessarily lead to shortened lifespan or accelerate pathological burden in the animal model [101]. In contrast, results from some studies hinted that activation of GSK3β is critical for exacerbating tauopathy [102, 103]. These observations should be interpreted carefully as the tau models used or compared are not in the same background.

GSK3β is a constitutively active protein that can autophosphorylate its tyrosine residues like Tyr 216 to increase the enzyme stability [104]. The activity of GSK3β is mainly regulated by insulin and Wnt signaling pathways [105]. When insulin pathway is activated, protein kinase B/Akt will be activated, which in turns phosphorylate serine 9 on GSK3β and causes its inactivation. In the case of activated Wnt signaling pathway, the inhibition of GSK3β activity would alleviate the degradation of β-catenin, whose nuclear translocation is responsible for the downstream genes activation of the pathway, but the precise mechanism regarding tauopathy modulation is still unknown [106, 107]. Nevertheless, a report showed both pathways are being downregulated in AD [96].

It was postulated that the phosphorylation by GSK3β requires the priming of adjacent proline residue as GSK3β is a member of proline-directed kinase family [84]. So far, more than 40 sites in tau, either serine or threonine, have been reported could be phosphorylated by GSK3β, and some of them are exclusively found in pathological conditions [84]. Among these sites, in vitro study first identified tau could be phosphorylated by GSK3β at the sites S202, S396, and S404 [108]. Since then, different studies reported many different phosphorylation sites with the availability of the corresponded phospho- site-specific antibodies. Some in vitro/in vivo studies later confirmed that frequent phosphorylation sites include S262, S396, and S404 [101, 109]. The phosphorylation of some residues may play important roles in affecting the binding affinity between tau and tubulins or regulating synaptic plasticity [4, 110, 111]. However, it is still unknown which residues are most frequently phosphorylated by GSK3β under different pathological states in contrast to normal condition, and if there is any protective effect by GSK3β phosphorylation against tau from forming the aggregates. Probing these questions is a major challenge but will help our understanding of the role of GSK3β in tau-associated disease conditions.

#### *3.2.1.2. Other kinases*

residues, 12 histidine residues, and 5 tyrosine residues. Current postulated phosphorylation sites have essentially covered most of the available sites, and indeed many of these residues are found being phosphorylated under physiological conditions. Therefore, the widespread, and likely dynamic, tau phosphorylation appears to serve for certain uncovered functions. Nevertheless, it also indicates that the differences in general phosphorylation and changes of phosphorylation state in certain residues may play a critical role in tauopathies [83, 84]. Moreover, hyperphosphorylated tau could also be detected in other pathological conditions aside from tauopathy. It was found that phosphorylated tau proteins are co-aggregated with alpha-synuclein in Parkinson disease and Lewy bodies dementia [86]. In some reports, phosphorylated tau aggregation can be found in Huntington disease and amyotrophic lateral sclerosis brains [87, 88]. In traumatic brain injury patients, the levels of phosphorylated tau, but not total tau, are significantly increased [89]. Recently, it is also suspected that tau phosphorylation may play a role in type 2 diabetes, rendering the patients incline to have cognitive defects [90]. These data collectively indicate hyperphosphorylation of tau has a strong correla-

However, the scenario of pathogenesis in tau hyperphosphorylation is more than merely the activation of some kinases or down-regulation of some phosphatase. As a matter of fact, different kinases are interacting, regulating, and even competing with each other for acting or interfering on same sites [91, 92], it is conceivable that the dynamics of transferring/removing phosphate groups on tau protein could be complex. Besides, kinases have multiple substrates, and some of them have important roles in normal cell functions [91]. The activities change of a kinase could lead to a domino effect toward the change of cellular activities. Last but not least, not all phosphorylations are toxic as some of them are required for normal tau functions, and certain sites phosphorylation may even serve as a protective effect in tauopathies [4, 5]. Therefore, a systemic dissection of disease-prone tau phosphorylations and their regulation is

a pre-requisite before aiming such complex regulation for a therapeutic exploration.

To study the phosphorylations, many antibodies recognizing specific phosphorylated residues on tau have been generated, and a list can be found on Alzforum.org [93]. For analyzing the effect of phosphorylation, recombinant MAPT constructs bearing site-specific mutations to mimic potential phosphorylation status of tau are regularly utilized in tauopathy research, which provides some insights regarding the genotoxic and structural impacts upon modifications [94]. However, even with the recombinant tau with or without pseudo-phosphorylations, it is hard to generate significant polymerization in vitro postulated due to a lack of "nucleation" process, although pseudo-phosphorylated tau may be prone to aggregate [95].

It is well acknowledged that glycogen synthase kinase 3 beta (GSK3β) plays a pivotal role in tau hyperphosphorylation [96, 97]. An early study showed that recombinant tau and microtubule-associated GSK3β that were harvested from bacterial lysates could be co-eluted in immunochromatography with anti-GSK3β and co-immunoprecipitated [98]. A subsequent study found active GSK3β co-localized with tau inclusions in tauopathy brain tissues, and the amount of active GSK3β was significantly increased in the patients [99]. Moreover, in vitro

tion with a variety of brain pathologies, not just tauopathies.

*3.2.1.1. GSK3β*

42 Cognitive Disorders

Besides GSK3β, other kinases such as p38, cyclin-dependent kinase 5 (CDK5), c-Jun N-terminal kinase (JNK), extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), casein kinase (CK), protein kinase A (PKA), and Ca2+ /calmodulin-dependent protein kinase II (CAMKII) have been reported to involve in directing tau phosphorylation [4, 84, 112–114]. Although the possible phosphorylation sites mediated by these kinases, especially in tauopathy conditions, may be less than GSK3β, it is fair to say that their roles in modulating tau toxicity are less well studied, thus whether the modulations by these kinases are less critical than GSK3β in tauopathy remain to be addressed. Indeed, a recent study found P38γ overexpression could ameliorate excitotoxicity induced by amyloid-beta in a tau-dependent manner. Further experiments showed that the effect was mediated by the phosphorylation of tau S205 through the action of P38γ, and the phosphorylation of this residue could abolish the interaction between tau and Fyn and PSD95, which otherwise could form a complex interacting with NMDA receptor to induce excitotoxicity [4]. This result strongly suggests different kinases may have different roles in tauopathy, and not every up-regulated tau phosphorylations under pathological conditions are for enhancing the toxicity. Given such complex modifications, more studies shall emphasize the difference of activity among these kinases under physiological and pathological conditions, as another recent study showed that ERK1/2 does not phosphorylate tau under physiological condition [115]. Furthermore, since the pathology development in tauopathy usually takes years, whether there is any sequential activation of the kinases appears to be another intriguing issue. Indeed, a recent study examining tau-staining in the postmortem AD brains of different stages showed that N-terminal side of tau is preferentially phosphorylated at early stages [116].

most attention by researchers concerning neurodegeneration, and calpain 2, on the other hand, is mostly expressed by glial cells. Studies have shown that calpain 1 can cleave p35 to generate p25, which could further induce prolonged activation of CDK5 [120]. Calpain can also cleave GSK3β to generate a C-terminal truncated form, which makes its inhibitory site less likely to be phosphorylated and thus produces a dominant-active GSK3β [121, 131]. Also, calpain can directly process tau and generate small fragments. However, the physiological or pathological impacts of those cleaved calpain products in regarding tauopathy are unclear,

Tauopathy

45

http://dx.doi.org/10.5772/intechopen.73198

Among the caspases, executive caspases, especially caspase-3, play a critical role in the direct processing of tau at the site D421. As mentioned above, D421-truncated tau can be found in AD brains, and this cleaved form is suspected to facilitate tau aggregate formation and thus enhancing the toxicity [124, 129, 133, 134]. Phosphorylation at S422 could prevent the truncation, which could be mediated by JNK and TTBK-1 [114, 135, 136]. Nevertheless, JNK and TTBK-1 could also phosphorylate tau at the sites other than S422, which complexes the protective scenario [114, 136]. Caspase 3 can also regulate the phosphorylation of tau through

Besides caspase-3, other caspases may involve in tauopathy as well. A recent study showed that caspase-2 could cleave tau at the site D314. This truncated tau could not participate in tau aggregation but is existing in the brains of P301L mice by a significant amount. Pseudophoshorylation of this site prevented caspase-2 cleavage and consequently caused memory and cognitive

Tau lysine residues could be acetylated by certain endogenous acetyltransferase, and such modulations were first demonstrated by p300 and Creb-binding protein (CBP) [138, 139]. Importantly, the insoluble tau protein fraction isolated from postmortem brains of the AD patients could be recognized by an anti-acetylated tau antibody [138]. Since then, tau acetylation studies start to emerge, and up to date, four tau acetylation sites, K174, K274, K280, and K281, have been confirmed in pathological conditions [140]. Acetylation of K280 and K281 reduced tau binding affinity to microtubules in vivo and facilitated tau aggregation in vitro [140, 141]. Moreover, acetylation of K280 exacerbated tau toxicity in a *Drosophila* model, and acetylation of K174 worsened neurodegeneration and behavior defects in PS19 mice [142, 143]. An overall tau acetylation effect is likely to aggravate tau toxicity. A study showed the administration of salsalate, a drug that could inhibit p300, ameliorated the tau pathology and memory defects in PS19 mice [143]. Interestingly, a recent study reported acetylation of K321 could impede S324 phosphorylation, a frequent modification in postmortem AD brains. This observation leads to an intriguing prospect that some switches from acetylation to phosphorylation might affect disease progression [144]. Altogether, these studies bring up questions including to what extent tau acetylation could affect tau toxicity and whether there are interactions

and the calpain-mediated tau cleavage site(s) remains elusive [120, 127, 132].

cleaving and activates protein kinase B and thereby activates GSK3β [119].

between tau acetylation and other posttranslational modifications.

*3.2.2.2. Caspases*

defects of the mice [137].

*3.2.3. Acetylation*

#### *3.2.2. Truncation*

Proteases including calpain and caspases are involved in tauopathy pathogenesis [117, 118]. They are activated in tauopathies, either directly cleave tau or indirectly cleaving its associated kinases, affecting the structure/function of tau [118–121]. While less commonly reported, other proteases are suspected to play roles in tauopathy-related protein truncations [122].

Two truncated tau proteins have caught attention in tauopathy, as they are abundant in the postmortem AD brains [123]. In fact, truncated tau could also found in other tauopathies beyond AD. Importantly, researchers used live multiphoton imaging combined with thioflavin S administration and a dye for activated caspases and observed that the tangle formation was preceded by caspases activation in a classic P301L tauopathy mice (tg4510) [124], indicating a close tight between tau cleavage and toxicity. Currently, two truncation sites, E391 and D421, have been characterized and both are on the C-terminus. We also learned that the truncation on D421 is mediated by caspases, mainly by Caspase-3. D421-truncated tau is associated with lysosome in AD brain, indicating that the truncated tau may be favored to be degraded through autophagy or maybe impairing the autophagy system [124]. However, it is still unclear what kind of proteases are responsible for the cleavage at E391, albeit the site was the first-identified cleavage site in tau, and its C-terminal cleavage product appears in PHF core [118, 123, 125–128]. While various reports have suggested that caspase and calpain are capable of cleaving tau and both present as an early event in pathogenesis and could aggravate tau toxicity [124, 129, 130], an important issue should be solved in studying tau truncation that is whether the aberrant increase of tau truncation is a consequence of tau aggregation or actually the cause that induces tau aggregate formation [124].

#### *3.2.2.1. Calpains*

Calpains are cytosolic calcium-dependent cysteine proteases. In an analysis of a postmortem brain lysates, calpain 1 was found to activate at early stages of AD, close to the stage when GSK-3β and CDK5 were activated [130]. The human genome has two identified calpain family members, calpain 1 and calpain 2. Calpain 1 is mainly expressed by neurons and thus received most attention by researchers concerning neurodegeneration, and calpain 2, on the other hand, is mostly expressed by glial cells. Studies have shown that calpain 1 can cleave p35 to generate p25, which could further induce prolonged activation of CDK5 [120]. Calpain can also cleave GSK3β to generate a C-terminal truncated form, which makes its inhibitory site less likely to be phosphorylated and thus produces a dominant-active GSK3β [121, 131]. Also, calpain can directly process tau and generate small fragments. However, the physiological or pathological impacts of those cleaved calpain products in regarding tauopathy are unclear, and the calpain-mediated tau cleavage site(s) remains elusive [120, 127, 132].

#### *3.2.2.2. Caspases*

induced by amyloid-beta in a tau-dependent manner. Further experiments showed that the effect was mediated by the phosphorylation of tau S205 through the action of P38γ, and the phosphorylation of this residue could abolish the interaction between tau and Fyn and PSD95, which otherwise could form a complex interacting with NMDA receptor to induce excitotoxicity [4]. This result strongly suggests different kinases may have different roles in tauopathy, and not every up-regulated tau phosphorylations under pathological conditions are for enhancing the toxicity. Given such complex modifications, more studies shall emphasize the difference of activity among these kinases under physiological and pathological conditions, as another recent study showed that ERK1/2 does not phosphorylate tau under physiological condition [115]. Furthermore, since the pathology development in tauopathy usually takes years, whether there is any sequential activation of the kinases appears to be another intriguing issue. Indeed, a recent study examining tau-staining in the postmortem AD brains of different stages showed that N-terminal side of tau is preferentially phosphorylated at early stages [116].

Proteases including calpain and caspases are involved in tauopathy pathogenesis [117, 118]. They are activated in tauopathies, either directly cleave tau or indirectly cleaving its associated kinases, affecting the structure/function of tau [118–121]. While less commonly reported, other proteases are suspected to play roles in tauopathy-related protein truncations [122].

Two truncated tau proteins have caught attention in tauopathy, as they are abundant in the postmortem AD brains [123]. In fact, truncated tau could also found in other tauopathies beyond AD. Importantly, researchers used live multiphoton imaging combined with thioflavin S administration and a dye for activated caspases and observed that the tangle formation was preceded by caspases activation in a classic P301L tauopathy mice (tg4510) [124], indicating a close tight between tau cleavage and toxicity. Currently, two truncation sites, E391 and D421, have been characterized and both are on the C-terminus. We also learned that the truncation on D421 is mediated by caspases, mainly by Caspase-3. D421-truncated tau is associated with lysosome in AD brain, indicating that the truncated tau may be favored to be degraded through autophagy or maybe impairing the autophagy system [124]. However, it is still unclear what kind of proteases are responsible for the cleavage at E391, albeit the site was the first-identified cleavage site in tau, and its C-terminal cleavage product appears in PHF core [118, 123, 125–128]. While various reports have suggested that caspase and calpain are capable of cleaving tau and both present as an early event in pathogenesis and could aggravate tau toxicity [124, 129, 130], an important issue should be solved in studying tau truncation that is whether the aberrant increase of tau truncation is a consequence of tau aggregation

Calpains are cytosolic calcium-dependent cysteine proteases. In an analysis of a postmortem brain lysates, calpain 1 was found to activate at early stages of AD, close to the stage when GSK-3β and CDK5 were activated [130]. The human genome has two identified calpain family members, calpain 1 and calpain 2. Calpain 1 is mainly expressed by neurons and thus received

or actually the cause that induces tau aggregate formation [124].

*3.2.2. Truncation*

44 Cognitive Disorders

*3.2.2.1. Calpains*

Among the caspases, executive caspases, especially caspase-3, play a critical role in the direct processing of tau at the site D421. As mentioned above, D421-truncated tau can be found in AD brains, and this cleaved form is suspected to facilitate tau aggregate formation and thus enhancing the toxicity [124, 129, 133, 134]. Phosphorylation at S422 could prevent the truncation, which could be mediated by JNK and TTBK-1 [114, 135, 136]. Nevertheless, JNK and TTBK-1 could also phosphorylate tau at the sites other than S422, which complexes the protective scenario [114, 136]. Caspase 3 can also regulate the phosphorylation of tau through cleaving and activates protein kinase B and thereby activates GSK3β [119].

Besides caspase-3, other caspases may involve in tauopathy as well. A recent study showed that caspase-2 could cleave tau at the site D314. This truncated tau could not participate in tau aggregation but is existing in the brains of P301L mice by a significant amount. Pseudophoshorylation of this site prevented caspase-2 cleavage and consequently caused memory and cognitive defects of the mice [137].
