**5. Cytoskeletal abnormalities in Alzheimer´s disease**

#### **5.1. Tau protein and neurofibrillary tangles**

that genetic and environmental factors may affect the development of central nervous sys‐ tem, acting as "the first hit". These early disorders are linked to long-term vulnerability, which after a "second hit" could cause the symptoms for a disease [51-52]. For diseases such as depression, autism and schizophrenia, the heterozygous *reeler* mice had been used as the genetic "first hit", while stress events after the birth or in adulthood are used as the environ‐ mental "second hit". The results indicate that heterozygous *reeler* mice, after a stressful event, such as maternal deprivation or corticosterone injection, exhibit significantly in‐ creased depressive or schizophrenic behaviors as compared with wild type littermates [53-54]. Indeed, *reeler* heterozygous animals in the absence of a stressful event, display a

The "two hit" model has also been used to study the molecular mechanisms leading to the AD [56]. It is proposed that both oxidative stress and failures in mitotic signaling can inde‐ pendently triggers the onset of the disease; however both are necessary for their progression [57]. In addition, a correspondence had been established between the Reelin expression in the entorhinal cortex of aged rats with their cognitive abilities. A study revealed that aged "cognitively disabled" rats show a significant decreased of Reelin in neurons on layer II of the entorhinal cortex. Such a reduction in Reelin expression was not observed in juvenile or

Since Reelin is expressed from development to adult stages, is conceivable that alterations in Reelin expression, induced by genetic or environmental factors generate a vulnerable stage, and a secondary factor, present in normal aging, may trigger the onset and progression of a

The Reelin-activated signaling pathways, which may be involved in the generation and de‐ velopment of AD are still unclear and will be discussed in next sections. In the last part of this section, we present some of the evidences that correlate altered levels of Reelin and AD. Pyramidal neurons placed in layer II of the entorhinal cortex and the hippocampus derived from AD patients brains exhibit decreased Reelin expression [50]. On the other hand, an in‐ crease in the full length and 180 kD proteolytic fragment of Reelin had been observed in the frontal cortex of AD derived samples [59]. The increase of this proteolytic fragment is attrib‐ uted to problems with the proteolysis of Reelin, associated with decreased Rab11-endocyto‐ sis of full length Reelin [60]. In the other hand, an increase of Reelin is also observed in the frontal cortex of AD patients, which may involve a compensatory mechanism in response to the lower expression in disease-related most vulnerable areas like the entorhinal cortex and

The CR neurons participation in AD is a controversial issue. While electronic microscopy analysis suggested that CR neurons of the temporal cortex were dramatically reduced in AD patients [61], another study showed no difference between AD patients and normal, healthy subjects [62]. On the other hand, there are some polymorphisms in the Reelin gene which had been associated with AD. Seripa and colleagues reported significant differences in two analyzed polymorphisms in the Reelin gene, in a group of 223 Caucasians AD patients.

These differences were exacerbated in female patients [63].

phenotype indistinguishable from control animals [55].

elderly "cognitively able" rats [58].

pathological condition.

44 Understanding Alzheimer's Disease

hippocampus [50].

Neurofibrillary tangles are amongst the standard characteristics of AD brains. These struc‐ tures were firstly described by Alois Alzheimer more than a century ago and are com‐ posed of a densely packed array of fibers of 20 nm in diameter, called paired helical filaments (PHF), which at the core are mainly composed by the microtubule-associated protein, tau [69-70]. Tau protein stabilizes and enhances microtubule polymerization. It is a heterogeneous protein giving rise to 6 isoforms derived from alternative splicing [71]. It contains 3 or 4 imperfect repeats of 31 or 32 amino acids each in tandem which confers the microtubule-binding properties of the protein. These repeats are enriched in basic aminoacids that interact electrostatically with the mostly acidic C-terminal of β-tubulin subunit [72]. Tau protein is highly phosphorylated in fetal brain [73], but minimally phos‐ phorylated in normal adult brain [74]. The abnormal phosphorylation state of several res‐ idues in tau protein plays an important role modulating the affinity to microtubules and promoting its aggregation [75] forming the core of PHFs [69,76-77]. Tau protein can be phosphorylated by many protein kinases such as calcium-calmodulin dependent kinase [78]; PKA [79-81] and PKC [82-83]. Interestingly, many of these residues are hyperphos‐ phorylated in AD brains mainly due to an imbalance in the activity of kinases belongs to the family of proline-directed Ser/Thr protein kinases (PDPKs), such as mitogen-activated protein kinases (MAPK) [84], the glycogen synthase kinase (GSK)-3β [85], JNK [84], p38 [86] and Cyclin-dependent kinase (Cdk)-5 [87]. The abnormal phosphorylation state of tau protein is not only contributed by protein kinases, but also by deregulated protein phos‐ phatases functions [88]. (Figure 1B)

Similarly, cofilin dephosphorylation and the subsequent formation of actin-rods seem to be also a spatial-restricted phenomenon. In example, actin-rods occur in a subpopulation of

The Amyloidogenic Pathway Meets the Reelin Signaling Cascade: A Cytoskeleton Bridge Between...

http://dx.doi.org/10.5772/54038

47

The mechanisms involved in the Aβ-mediated cofilin dephosphorylation are dependent on changes in the activity of its upstream kinase, LIMK [90], and the activity of two known cofi‐

Interestingly, ATP depletion induces chronophin activation in a mechanism involving the dissociation of chronophin-HSP90 complex. This mechanism would be responsible for the

**6. Is the AD-associated Reelin reduction a major factor involved in the**

There is an increasing body of evidence indicating that a deficiency in Reelin signaling may play a major role in the progression of AD. First, decreased Reelin expression is early ob‐ served in brains of AD transgenic mice model, even before Aβ deposition. Accordingly, Reelin expression is also decreased in brains of patients at the presymptomatic stages of AD. The progression of the disease causes in both cases, potentiate the Reelin deficiency from the hippocampus to the entorhinal cortex in mice and from the frontal cortex to the hippocam‐ pus and entorhinal cortex in humans [50,98]. The decrease in Reelin expression is linked to a

Reelin itself can form amyloid deposits in advanced stages of AD, which can or cannot be associated with Aβ senile plaques [64-66]. However, Aβ pathology seems to be a pre‐ requisite for the formation of Reelin aggregates, as these only occur after formation of se‐

On the other hand, the proteolytic fragments of Reelin showing aberrant glycosylation pattern are increased in the cerebrospinal fluid of patients with AD [59,99]. Altogether these antecedents support the hypothesis that the Reelin intracellular signaling is im‐

Reelin signaling is triggered by the binding of Reelin to two members of the lipoprotein re‐ ceptor family, the very low density lipoprotein receptor (VLDLR) and the ApoE receptor 2 (ApoER2)[100]. The signal is then transduced by a cytoplasmic adapter protein, the mamma‐ lian homologue for the *Drosophila* protein *disabled* (mDab)-1, which interacts with the NPXY motifs of the intracellular domain of several members of the LDL receptor family, including

neurons in organotypic slices treated with Aβ [96]. (Figure 1B)

formation of actin-rods under energy deprivation conditions [97].

reduction in CR cells at the cortical layer I in AD brains [61].

**6.2. Cytoskeletal pathologies and Reelin signaling**

lin phosphatases, chronophin [97] and slingshot [94].

**neuronal cytoskeleton pathology?**

**6.1. Reelin reduction in AD brains**

nile plaques [98].

paired at early stages of AD.

VLDLR and ApoER2.

#### **5.2. Cofilin and actin-rods**

NFTs are not the only intraneuronal cytoskeletal protein aggregates found in the brains of patients affected by AD. Hirano´s bodies and actin-rods are two closely related aggre‐ gates primarily composed of actin and the actin binding protein, cofilin. Cofilin concerted‐ ly with the actin depolymerizing factor (ADF) constitutes the major modulators of actin dynamic assembly.

Hirano's bodies were originally described in 1965 and are defined as paracrystalline struc‐ tures, eosinophilic intracellular arrangements resembling rod-shaped filaments of 7 nm. The actin-rods differ from Hirano´s bodies by it smaller size, so it is hypothesized that these structures could be precursors of Hirano's bodies.

The formation of actin-rods in neurons seems to be the result of several neurodegenerative insults, such as ATP depletion, excitotoxic levels of glutamate, oxidative stress [89], and Aβ1-42 oligomers [90]. A common event to all these stimuli triggers the formation of rods is the dephosphorylation (activation) of cofilin [89]. Cofilin/ADF is inactivated by phosphory‐ lation of a highly conserved serine (Ser3), which precludes its binding to actin filaments and, therefore, its role as promoters of filament severing and actin subunits turnover at the minus end of filaments.

The Ser3 of ADF/cofilin is the only known substrate for the two isoforms of LIM domain kinases (LIM, an acronym for three *Caenorhabditis elegans* genes, *lin-11*, *isl-1* and *mec-3*). LIMKs is activated by phosphorylation at the Thr508, mediated by PAK or ROCK, two kinases that act as effectors for small GTPases Rac1 and RhoA respectively [91]. The regu‐ lation of signaling cascades, which target the functions of small GTPases, connect the dy‐ namic control of the actin cytoskeleton with extracellular signals. In AD, different components of the signaling cascade involved in cofilin phosphorylation are altered, in‐ cluding decreased phosphorylation of PAK at Ser141, which is necessary for activation. Although a decrease in phosphorylation and activity of PAK is observed in large areas of cortex and hippocampus of AD brains, neurons located near to amyloid plaques exhibit strong staining for pSer141 PAK, suggesting that while the dephosphorylation is predom‐ inant in the brain of patients with AD, the amyloid fibrils present in amyloid plaques in‐ creases the activity of PAK [92].

Consistently, hippocampal neurons treated with fibrillar Aβ1-42 show increased activity of PAK and its downstream substrate LIMK1 [93-94], most likely through a Rac1 and Cdc42 dependent mechanism [95]. Moreover, the treatment with oligomers of Aβ1-40 has the oppo‐ site effect, decreasing the phosphorylation of PAK, indicating that oligomeric forms may be responsible for the overall reduction in PAK phosphorylation [92].

Similarly, cofilin dephosphorylation and the subsequent formation of actin-rods seem to be also a spatial-restricted phenomenon. In example, actin-rods occur in a subpopulation of neurons in organotypic slices treated with Aβ [96]. (Figure 1B)

The mechanisms involved in the Aβ-mediated cofilin dephosphorylation are dependent on changes in the activity of its upstream kinase, LIMK [90], and the activity of two known cofi‐ lin phosphatases, chronophin [97] and slingshot [94].

Interestingly, ATP depletion induces chronophin activation in a mechanism involving the dissociation of chronophin-HSP90 complex. This mechanism would be responsible for the formation of actin-rods under energy deprivation conditions [97].
