**5. C-terminal truncation of tau protein in AD**

#### **5.1. PHF-core concept**

It is technically difficult to determine the half life of the different tau isoforms and several factors may regulate tau degradation such as, for example, the extent of phosphorylation and acetylation of tau (see below). The half life of tau decreases in rats by neonatal period

Two main mechanisms for tau protein degradation have been documented: 1) the proteaso‐ mal ubiquitin pathway and 2) the lysosomal autophagic pathway. Proteasomal degradation of tau protein has been described by 20S proteasomal processing [51], although there have also been reports suggesting that tau is not normally degraded by the proteasome [52]. Tau, modified by phosphorylation, can be ubiquitinated by the CHIP-hsc70 complex and degrad‐ ed by the proteasome [53]. Furthermore, acetylation of tau can regulate its proteasomal deg‐ radation by modifying those lysine residues needed for ubiquitination. In this way, acetylation of tau inhibits its degradation through a competition between ubiquitination and

On the other hand, tau may get processed through a lysosomal autophagic mechanism. It has been reported that tau can be degraded by lysosomal proteases [55] and, more recently, it was shown that tau fragmentation and clearance can occur by lysosomal processing [56]. Tau protein is a microtubule-associated protein. It's mostly abundant in neurons in the Cen‐ tral Nervous System (CNS). The main function of Tau protein is to interact with tubulin to stabilize microtubules and promote tubulin assembly into microtubules. Tau protein con‐

Normally, the tau protein is very important, as it manages the transport of materials within soma and other cellular regions through the myelin sheaths. Once it spotted something sus‐ picious or irrelevant, it stops the information sending process automatically. However, in Alzheimer's disease, the tau proteins started to perform uncommon reaction, where it trans‐

Once the above problem happening, it causes the brain overloading with information and might lead to inflammation, clumps or tangles, which kill most of the brain cells (Fig. 5).

Protein phosphorylation is the addition of a phosphate group, by esterification, to one of three different amino acids: serine, threonine and tyrosine. Phosphorylation is the most common post-translational modification of tau described and increased tau phosphorylation reduces its affinity for microtubules leading to cytoskeletal destabilization.  The formation of fibrillar aggregates of post-translationally modified tau protein in the brain are characteristic of AD and other tauopathies. The phosphorylation of tau protein affects its solubility, localization, function, interaction with partners and susceptibility to other posttranslational modifications. However, the role of specific sites of tau phosphorylation in ear‐ ly neurodegenerative mechanisms is unknown. The molecular mechanisms of aggregation

trols microtubule stability in two different ways : isoforms and phosphorylation.

mitting the information to the brain simultaneously, regardless of its validity.

**4. Phosphporylation of tau protein**

P20 and there is less demand for tau in non-dividing, mature neurons [50].

acetylation [54].

94 Understanding Alzheimer's Disease

In 1988, Wischik et al, [7, 22] identified tau protein as the major constituent of Pronase-resist‐ ant PHFs and tau was characterized by a specific C-terminal truncation of the protein at Glu-391. This truncation is identified by the monoclonal antibody (mAb) 423 [23, 31], and the acid-reversible occlusion of the intact core tau domain. PHFs are labeled by the fluores‐ cent dye, thiazin-red, a dye which can be used to differentiate between amorphous and fi‐ brillar states of tau and amyloid proteins in AD. The minimum tau fragment in the PHF [20, 24] corresponds to the tandem repeat region in the C-terminal domain of tau protein, a spe‐ cies having a molecular weight of 12.5 kDa. Characteristically, this fragment is highly stable to proteolysis, insoluble and toxic and is referred to as PHF-core tau [22, 57, 58]. PHF-core tau and mAb 423 immunoreactivity of NFTs, have a close clinical-pathological relationship; the density of NFTs immunolabelled with mAb 423 is correlated with the progression of neurofibrilary pathology, as determined by Braak staging criteria (Fig. 2). Most significantly, there is a correlation between mAb 423 immunoreactivity and both clinical severity and pro‐ gression to dementia [3]. On the other hand, over-expression of PHF-core tau, in cell culture, favors a programmed cell death or apoptosis, which shows that it is highly toxic[59]. Re‐ combinant tau protein truncated at Glu-391 also shows increased rates of polymerization compared with recombinant full-length tau. From confocal microscopy studies, it has been shown that this fragment of tau is hidden within the PHF-core and exposed by formic acid treatment [57]. In the cytoplasm of susceptible neurons, this truncated tau triggers an auto‐ catalytic process in which the fragment has a high affinity for full length tau and, once bound, leads to cycles of proteolysis and further tau binding to form a proto-filament [60]. In this scenario, the initiating tau species that gave rise to the filament is hidden within a large number of covering tau molecules, some of which become hyperphosphorylated. This would correspond to the early aggregation of tau protein associated with PHF in small NFT. Tau molecules of the NFT would become exposed on death of the neuron to reveal the ex‐ tracellular NFT, or "ghost tangle" (Fig. 1D, small arrow) which shares the properties of being stable, insoluble and immunoreactive with mAb 423 [57, 61]. The proteases responsible for truncation at Glu-391 are not known.

**6. Truncation of tau protein at Asp-421**

terminus of tau protein has not yet been generated.

**of tau protein in AD**

Alz-50 immunoreactive and in the absence of PHFs [64, 67].

In 2003, a second truncation of tau protein was found to be associated with PHFs [62-65]. This truncation is found at position Asp-421 in the C-terminus of the tau molecule and its presence can be detected specifically by using mAb TauC-3[25, 63]. Unlike truncation at Glu-391, for which the protease responsible is unknown, caspase-3 (an enzyme involved in the apoptotic pathway) is responsible for the truncation at Asp-421 *in vitro* [59, 62, 63]. This suggests that cleavage of the carboxyl terminus of tau protein, could result as a neuronal re‐ sponse to prevent or control the polymerization of tau in PHF [58]. In 2005, Binder and col‐ leagues discovered a truncation at the amino terminus of the tau protein associated with PHFs. This cut corresponds to Asp-13, which is produced by caspase-6, another enzyme in‐ volved in the apoptotic pathway [66]. An antibody to detect this cleavage site of the amino

Phosphorylation of Tau Protein Associated as a Protective Mechanism in the Presence of Toxic…

http://dx.doi.org/10.5772/54228

97

Tau-C3 has an affinity for NFTs, NDs and neuropil threads in AD brains. Immunohisto‐ chemical studies indicated that truncation at Asp-421 occurs after conformational change; the antibody binds with greatest affinity when the amino terminus of tau molecule contacts the third microtubule binding repeat (MTBR), as recognized by mAb Alz-50 [26]. However, other studies have shown Tau-C3 immunoreactivity in pre-tangle cells before they become

**7. Impact of phosphorylation and truncation on the abnormal processing**

**7.1. A neuroprotective mechanism for the phosphorylation of tau protein in the AD brain** During neurodegeneration in AD, tau protein is abnormally phosphorylated in the prolinerich region at Ser and Thr residues [68], and such phosphorylation sites can be identified us‐ ing highly specific antibodies such as: AT8 (Ser-202/Thr-205) AT100 (Ser-212 and Ser-214), TG3 (Thr-231/Ser-235) and PHF-1 (Ser-396/Ser-404), among others (Fig. 6). However, NFTs are found in viable neurons at late stages of the disease, and they persist in neuronal cells for decades with a significant number of NFTs being found in the cognitively intact elderly [69, 70]. Such NFT-bearing neurons contain normal content and structure of microtubules [68]. The findings from studies in transgenic mice and human data, suggest that tau accu‐ mulation in the somatodendritic compartment may represent the manifestation of a protec‐ tive mechanism or a cellular adaptation that arises with advancing age. An increase in tau phosphorylation in AD brain has been associated with a protective mechanism against oxi‐ dative stress [71]. In another study, intact microtubules were found in NFT-bearing neurons [8], calling into question whether accumulation of phosphorylated tau and destabilization of microtubules are necessarily linked. Although microtubules are depolymerized in neurons with fibrillary degeneration, one study found evidence that the reduction of microtubules in AD is marked and specifically limited to vulnerable pyramidal neurons, and that even these alterations were observed in the absence of PHF [72]. This finding is also consistent with

**Figure 6.** Location of tau phosphorylation sites and epitopes for tau antibodies.  Putative phosphorylation sites that have not yet been demonstrated *in vitro* or *in vivo* (black). Localization of antibody epitopes are indicated arrows. Residues are numbered according to the longest tau isoform.


**5. Future research**

**a**

As mg/kg

0

As mg/kg

0.00

**4. Discussion**

**Figure 11.** Comparison of (b) soil extractable of As with (a) total soil concentrations in Libya

0.01

0.02

0.03

0.04

0.05

0.06

**b**

2

4

6

8

10

868 Environmental Risk Assessment of Soil Contamination

East South West

East South West

Total As mean concentrations in ESR are nearly 4.8 and 4.1 times higher than in SSR and WSR, respectively. Total As loads in ESR may be related to the soil parent material, which is comprised of subsequently covered Lower Palaeozoic rocks. This may be the reason why the clay agricultural soils of ESR have the highest background levels of As. This is in agreement with [8], who reported higher concentrations of trace elements in Eastern Libyan soil than in other areas. In the present study, the trace elements present in Eastern Libyan soil (Co, Zn, Cu,


**2.1 Antimicrobial activity**

There are several commercial products with organic acids on the market, all with their own specific chemical and functional properties. As shown in Table 1, the inclusion of organic acids can reduce pH and the feed's buffering capacity, while their antimicrobial effect can prevent the growth of bacteria (especially Gram negative bacterial species, like *Salmonella spp.* and *E. coli*), yeasts and moulds. In the stomach, the pH is decreased, reducing the concentration of all the types of bacteria. In the small intestine, only the organic acids with antibacterial activity are able to inhibit bacteria growth. This is the main reason that the use of these acids has been proposed as a way of preventing or reducing the incidence of diarrhea in young pigs (Jensen et al. 2001; Tsiloyiannis et al. 2001a, 2001b: Piva et al. 2002a; Papatsiros et al. 2011). Thus, the organic acids are divided into two large groups. In the first group are included those with indirect effect on the decrease of the bacterial population by pH reduction and acting mainly on the stomach because the animal organism has the capability of preventing the decline in the acidity in the small intestine by buffering the medium with bicarbonate (fumaric, citric, malic and lactic acids). In the other group, are involved those organic acids (formic, acetic, propionic and sorbic acid), that have the ability to reduce the pH and affect directly Gram- bacteria by interfering in the bacterial cell with complex enzymes. These enzymes destroy the cell membrane and influence the mechanism of DNA duplication which prevents bacterial reproduction (Castro, 2005).

Many studies with dietary acidifiers have shown positive effects in improving growth rate, feed efficiency and acting against bacteria, yeast, fungi, moulds (Table 2), but others have found a negligible and even negative negative response (Radecki et al. 1988; Eidelsburger et al. 1992a; Manzanilla et al. 2004; Štukelj et al. 2010). It is likely that the antimicrobial effects of the organic acid ions, which act by controlling bacterial populations in the upper gastrointestinal tract, are responsible for the beneficial effects of these acids (Roth & Kirchgessner, 1998). Moreover, organic acids can also enhance the effects of antibiotics by improving their absorption (Radecki et al. 1988; Eidelsburger et al. 1992b). In addition, acidifiers can have an initial

Dietary buffering capacity varies substantially between different feedstuffs (Bolduan et al. 1988a, 1988b). The acid-buffering capacity is lowest in cereals and cereal by-products, intermediate or high in protein feedstuffs and very high in mineral sources (Jasaitis et al. 1987). Addition of organic acids reduces dietary pH curvili nearly depending on the acid pKa value and buffering capacity (Bolduan et al. 1988a, 1988b) of the diet. The pH-lowering effect of different organic acids is reduced in the following order: tartaric acid>citricacid>malic acid> fumaric acid>lactic and formic acids>acetic acid> propionic acid. Salts of organic acids have only a small influence on dietary pH, but the addition of protein and mineral sources to the diet weakens the pH-lowering effect of the acid (Roth & Kirchgessner, 1989). It seems reasonable to assume that the buffering capacity of feed can be considerably influenced by the selection of feed ingredients, and it may in part reflect the differences in the effectiveness of acidifiers. In general, organic acids lower dietary buffering capacity,

The greatest acidification benefits have been observed in diets formulated from cereals and plant proteins, while the growth-promoting effect in diets containing milk products is small (Giesting et al. 1991). The latter presumably holds true when lactose in milk products is converted to lactic acid by lactobacilli in the stomach, creating the desired reduction in pH

There are several commercial products with organic acids on the market, all with their own specific chemical and functional properties. As shown in Table 1, the inclusion of organic acids can reduce pH and the feed's buffering capacity, while their antimicrobial effect can prevent the growth of bacteria (especially Gram negative bacterial species, like *Salmonella spp.* and *E. coli*), yeasts and moulds. In the stomach, the pH is decreased, reducing the concentration of all the types of bacteria. In the small intestine, only the organic acids with antibacterial activity are able to inhibit bacteria growth. This is the main reason that the use of these acids has been proposed as a way of preventing or reducing the incidence of diarrhea in young pigs (Jensen et al. 2001; Tsiloyiannis et al. 2001a, 2001b: Piva et al. 2002a; Papatsiros et al. 2011). Thus, the organic acids are divided into two large groups. In the first group are included those with indirect effect on the decrease of the bacterial population by pH reduction and acting mainly on the stomach because the animal organism has the capability of preventing the decline in the acidity in the small intestine by buffering the medium with bicarbonate (fumaric, citric, malic and lactic acids). In the other group, are involved those organic acids (formic, acetic, propionic and sorbic acid), that have the ability to reduce the pH and affect directly Gram- bacteria by interfering in the bacterial cell with complex enzymes. These enzymes destroy the cell membrane and influence the mechanism

whereas certain salts of organic acids can increase it.

**2.1 Antimicrobial activity**

and thus reducing the need for diet acidification (Easter, 1988).

of DNA duplication which prevents bacterial reproduction (Castro, 2005).

Many studies with dietary acidifiers have shown positive effects in improving growth rate, feed efficiency and acting against bacteria, yeast, fungi, moulds (Table 2), but others have found a negligible and even negative negative response (Radecki et al. 1988; Eidelsburger et al. 1992a; Manzanilla et al. 2004; Štukelj et al. 2010). It is likely that the antimicrobial effects of the organic acid ions, which act by controlling bacterial populations in the upper gastrointestinal tract, are responsible for the beneficial effects of these acids (Roth & Kirchgessner, 1998). Moreover, organic acids can also enhance the effects of antibiotics by improving their absorption (Radecki et al. 1988; Eidelsburger et al. 1992b). In addition, acidifiers can have an initial eradicating effect on bacteria in the feed (Lueck, 1980) and remain there as a first barrier, preventing re-contamination. Even *under good conditions*, all compound feeds have a certain content of germs (bacteria, viruses, fungi and protozoa), which may be proliferate under unfavourable harvest and storage conditions (Schöner, 2001). Preservatives reduce the incidence of germs in the feed and thus the quantity of germs consumed by the animals. The hygienic quality of feed is significantly improved. The addition of organic acid lowers the pH value of the feed and also provides acid-binding capacity.

In fact, organic acids associated with specific antimicrobial activity are short-chain acids (SCFA, C1–C7) and are either simple monocarboxylic acids such as formic, acetic, propionic and butyric acids, or carboxylic acids, bearing a hydroxyl group (usually on the carbon) such as lactic, malic, tartaric, and citric acids. Four organic acids commonly used in feed formic, acetic, propionic and lactic acid - have a specific ability to penetrate the bacterial cell wall and kill bacteria by interfering with their metabolism. These acids only pass the membrane in non dissociated form. Their primary antimicrobial action (strain-selective growth inhibition or delay) is through pH depression of the diet. However, the ability of organic acids to change from undissociated to dissociated form, depending on the environmental pH, makes them effective antimicrobial agents. When acid is in the undissociated form it can freely diffuse through the semi permeable membrane of microorganisms into their cell cytoplasm. Once inside the cell, where the pH is maintained near 7, the acid dissociates and suppress cell enzymes (decarboxylases and catalases) and nutrient transport systems (Lueck, 1980). The efficacy of an acid in inhibiting microbes is dependent on its pKa value which is the pH at which 50% of the acid is dissociated. Organic acids with higher pKa values are more effective preservatives and their antimicrobial efficacy is generally improved with increasing chain length and degree of unsaturation (Foegeding & Busta, 1991). In practice this means that the stomach pH has to be lower than 5 for optimal results. Without these specific antimicrobial acids, the pH needs to be very low to destroy bacteria. Some of the above acids' salts, have also shown to have benefits on growth performance. Other acids, such as sorbic and fumaric acid, have some antifungal activity and are short chain-carboxylic acids, containing double bonds. Organic acids are weak acids and are only partly dissociated; most of them, with antimicrobial activity, have a pKa 3 - 5.

In addition, each acid has its own spectrum of antimicrobial activity. Their antimicrobial effects vary from one acid to another, depending on concentration and pH (Chaveerach et al. 2002). For example, lactic acid is more effective in reducing gastric pH and coliforms (Jensen et al. 2001; Tsiloyiannis et al. 2001a; Øverland et al. 2007), whereas other acids, such as formic, propionic have broader antimicrobial activities and they can be effective against bacteria (e.g. coliforms, clostridia, Salmonella), fungi and yeast (Partanen & Mroz, 1999; Bosi et al. 2005; Creus et al. 2007; Øverland et al. 2007). Several reports have shown that the use of organic acids may reduce the coliform burden along the gastrointestinal tract (Bolduan et al. 1988b) and reduce scouring and piglet mortality or control postweaning diarrhea and edema disease in piglets (Tsiloyiannis et al. 2001a, 2001b; Piva et al. 2002a, Papatsiros et al. 2011). The following order of killing potency of coliform bacteria in the gastric digesta at pH 3, 4, and 5, are: propionic<formic<butyric<lactic<fumaric<benzoic were established (Naughton & Jensen, 2001; Knarreborg et al. 2002). Jensen et al. (2001) demonstrated that the potency of these acids against *Salmonella typhiumurium* in gastric digesta at pH4 was in the following order: acetic <formic < propionic < lactic < sorbic < benzoic. Inconsistent results may be due to the variety of diets with different buffering capacities that were used in these

The Prophylactic Use of Acidifiers as Antibacterial Agents in Swine 303

The use of acidifiers appears to be most beneficial in the early period after weaning. Studies demonstrating the improved feed conversion ratio, weight gain and growth-promoting effects of acidifiers indicated that the effect was greater in young pigs than older pigs (Radcliffe et al. 1998; Øverland et al. 2000; Partanen et al. 2007), but there is some evidence that they may be beneficial for improvement of daily gain and feed efficiency in growing-finishing pigs

The results of trials including the addition of inorganic acids in pig diets has indicated positive responses on growth performance (Walsh et al. 2007; Stein, 2007), especially during the period after weaning (Mahan et al. 1996, 1999). However, the use of other inorganic acids, such as sulfuric acid, has not shown positive effects on growth performance (Ravindran & Kornegay, 1993). In addition, salts of organic acids, such as formates and diformates can be used to significantly improve growth rate and feed conversion in pigs (Table 3). However, there are also studies with no responses (Biagi et al. 2007) or involving risk factors (Pallauf & Huter, 1993; Øverland et al. 2000). For example, calcium formate

a. Acidifiers may have a negative effect on diet palatability, when they are added at excessive levels, resulting in lower feed intake or feed refusal (Partanen & Mroz, 1999). Certain acids, e.g. tartaric and formic acids have a strong odour and flavour, and an increasing dietary acid level, which is generally associated with a dramatic decrease in feed intake, as reflected by lower daily gains (Eckel et al. 1992; Kirchgessner et al. 1993). Addition of excessive amounts of formates to the diet may also disturb the acid-base status of pigs leading to metabolic acidosis, which results in decreased feed intake and slower growth (Giesting et al. 1991; Eckel et al. 1992; Eidelsburger et al. 1992e). Organic acids metabolized via the citric-acid cycle, e.g. fumaric and citric-acids, do not seem to

cause acidosis, irrespective of their dietary inclusion (Eidelsburger et al. 1992c). b. Acids at high levels in feed are corrosive to cement and galvanized steel in pig housing, resulting to pose handling and equipment issues to the feed manufacturer. For example, formic acid is the most corrosive for the equipment and it is dangerous to handle, while fumuric acid is easy to handle (Mateos et al. 1999). Salts of organic acids are generally odorless and less corrosive than their acid forms, making them easier to handle in the

c. The use of organic acids in their free form, at levels that have been proven to be efficacious, can cause palatability problems (Partanen, 2001), damage the stomachal and duodenal mucosas (Argenzio & Eisemann, 1996), as well as cause bone demineralization (Partanen & Mroz, 1999) and an acidic stress, inducing a resistance

In order to minimize these effects, the natural buffering capacity of feeds (related to mineral and protein content) should be evaluated to determine the minimum effective amount of acid to use (Best, 2000). Another strategy to extend the effectiveness of acid supplements and reduce corrosion damage to housing materials is the use of a slow-release form of acids. It consists on the use of organic acids with fatty acids and mono- and diglycerides mixed to form microgranules. A study by Cerchiari (2000) showed that use of these granules, as

mechanism towards organic acids in certain bacteria (Bearson et al. 1997).

compared to use of free acids, results in greater feed intake and growth.

(Øverland et al. 2000; Partanen et al. 2001a; Gauthier 2002; Canibe et al. 2005).

decreased feed intake and daily gain (Pallauf & Huter, 1993; Øverland et al. 2000).

The use of organic acids in feed appears two main problems:

feed manufacturing process (Jacela et al. 2009).

**3. Risk factors of acidifier use**

experiments. Bacteria are known to develop acid-resistance when exposed to acidic environments for some time (Mroz, 2005).

#### **2.2 Antibacterial activity and growth promoting effects**

The beneficial effects of organic acids and their salts on growth performance have been confirmed in several studies. Acidifiers added to pig diets may potentially help improve growth performance (Table 2 & 3) by improving digestive processes through several mechanisms. It is believed that acidifiers can enhance the growth performance by:

The use of some organic acids has been found to reduce the formation of biogenic amines (such as cadaverine and putrescin) that are produced particularly in high protein feeds and in feeds, containing added synthetic amino acids. Biogenic amines have unfavourable effects on growth and feed conversion. The growth stimulation effects of formic, acetic and propionic acids are partly caused by their inhibitory effect on biogenic amines (Eckel et al. 1992). However, a clear mode of action has not been fully described yet and the magnitude and consistency of the response may vary, depending on inclusion rate and other dietary factors. The use of acidifiers appears to be most beneficial in the early period after weaning. Studies demonstrating the improved feed conversion ratio, weight gain and growth-promoting effects of acidifiers indicated that the effect was greater in young pigs than older pigs (Radcliffe et al. 1998; Øverland et al. 2000; Partanen et al. 2007), but there is some evidence that they may be beneficial for improvement of daily gain and feed efficiency in growing-finishing pigs (Øverland et al. 2000; Partanen et al. 2001a; Gauthier 2002; Canibe et al. 2005).

The results of trials including the addition of inorganic acids in pig diets has indicated positive responses on growth performance (Walsh et al. 2007; Stein, 2007), especially during the period after weaning (Mahan et al. 1996, 1999). However, the use of other inorganic acids, such as sulfuric acid, has not shown positive effects on growth performance (Ravindran & Kornegay, 1993). In addition, salts of organic acids, such as formates and diformates can be used to significantly improve growth rate and feed conversion in pigs (Table 3). However, there are also studies with no responses (Biagi et al. 2007) or involving risk factors (Pallauf & Huter, 1993; Øverland et al. 2000). For example, calcium formate decreased feed intake and daily gain (Pallauf & Huter, 1993; Øverland et al. 2000).