**6. Is the AD-associated Reelin reduction a major factor involved in the neuronal cytoskeleton pathology?**

### **6.1. Reelin reduction in AD brains**

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‐

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

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

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

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‐

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].

phatases functions [88]. (Figure 1B)

structures could be precursors of Hirano's bodies.

**5.2. Cofilin and actin-rods**

46 Understanding Alzheimer's Disease

dynamic assembly.

end of filaments.

creases the activity of PAK [92].

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

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‐ nile plaques [98].

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‐ paired at early stages of AD.

### **6.2. Cytoskeletal pathologies and Reelin signaling**

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 VLDLR and ApoER2.

As VLDLR/ApoER2 or mDab1 deficient mice exhibit a phenotype indistinguishable from *re‐ eler* mice, it is suggested that both receptors and the adapter protein can be linearly placed on the same signal transduction pathway [37,101].

**Protein Functions in Reelin signaling Association with Alzheimer´s**

ApoER2 Reelin receptor VLDLR and ApoER2 dKO mice

VLDLR Reelin receptor VLDLR and ApoER2 dKO mice

mDab1 Intracellular adapter for Reelin receptors mDab1-deficient mice show

Akt Phosphorylates and inhibits GSK-3β Impairment in PI3K-Akt pathway was

GSK-3β Major tau kinase Phosphorylates tau at AD-associated

PI3K Lipid kinase essential for membrane recruiting of Akt

tau Microtubule-associated protein. Promotes

Cdc42 Small GTPase associated with actin

LIMK1 Major effector of Rho-family GTPases.

Cofilin Actin binding protein with F-actin depolymerizing activity

dynamics

microtubule assembly and stabilization

Phosphorylates and inactivates cofilin

**Table 1.** Association of Reelin signaling pathway with Alzheimer´s disease

Reelin Extracellular matrix glycoprotein Diminished levels in restricted areas

**disease**

of AD brain. Reelin-deficient mice show increased tau phosphorylation

present elevated levels of phosphorylated tau

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

present elevated levels of phosphorylated tau

early death.

mice.

mice.

epitopes

neurons

The Reelin signaling pathway can target not only microtubule cytoskeleton, but also the ac‐ tin microfilament formation. Acting through its canonical signaling pathway that involve re‐ ceptors VLDLR and ApoER2, the adapter protein Dab1 and activation of PI3K, Reelin is able to activate the small GTPases Rac1 and Cdc42, increasing actin polymerization. These

the core of NFTs

increased tau phosphorylation and

Impairment in PI3K-Akt pathway was observed in aged APP-PS1 transgenic

observed in aged APP-PS1 transgenic

Hyperphosphorylated tau constitutes

A lesser Aβ-induced actin-rod formation is observed in cdc42 null

The expression of constitutively active LIMK1 reduces Aβ-induced actin-rods in hippocampal slices

Ser3 dephosphorylation triggers its aggregation into actin-rods

**References**

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

49

[50, 98]

[38]

[38]

[106]

[112]

[112]

[85]

[77]

[96]

[90]

[89]

The binding of Reelin to its receptors induces mDab1 tyrosine phosphorylation, mediat‐ ed by non-receptor tyrosine kinases from the Src family [102]. The mutation of these ty‐ rosines residues by phenylalanines in a *knockin* mouse recapitulates several features of the *reeler* mouse, supporting that these phosphorylation events are required for proper Reelin signaling [103].

Several genetic models suggest that canonical Reelin signaling plays an essential role in con‐ trolling the phosphorylation state of tau and, therefore, modulating a critical event in the progression of AD (Table 1).

Mice deficient in various components of the Reelin signaling pathway, including Reelin it‐ self, VLDLR, ApoER2 and Dab1 show increased tau phosphorylation in several AD-associ‐ ated epitopes, such as those recognized by the antibodies AT8 (pSer202/205) and PHF1 (pSer396/404) [38,104-106].

The increase in tau phosphorylation is caused by increased activity of two main kinases, Cdk5 and GSK-3β [105], suggesting that Reelin is playing a negative control over the activi‐ ties of these kinases.

GSK-3β is normally inhibited by phosphorylation at its N-terminal region by the protein kinase Akt, mainly at the Ser9. Reelin signaling in turn, activates Akt through its recruit‐ ment to membrane domains rich in phosphatidylinositol 3-phosphate (PIP3), whose forma‐ tion is involved the activity of the phosphatidilinositol 3-kinase (PI3K). Reelin activates PI3K by potentiating the interaction between tyrosine phosphorylated-mDab1 and the p85α subu‐ nit of PI3K [107-108].

Moreover, it has been proposed that the increased activity of Cdk5 in Dab1 or Reelin defi‐ cient mice may be due to a remarkable increase of the proteolyzed form of a Cdk5 activa‐ tor, called p25 [105]. This fragment induces a non-physiological activation of Cdk5, which is present mainly in pathological conditions, including brains of patients with AD [109]. Since the proteolysis of the Cdk5 activator is due to the activity of calpain, it may be hy‐ pothesized that the Reelin signaling pathway could regulate calpain-dependent proteoly‐ sis of p35.

It has been proposed that Cdk5 could not be directly regulated by the Reelin signaling cas‐ cade, because cortical neurons treated with Reelin do not exhibit any significant change in the Cdk5 activity [107] or a diminished phosphorylation state of Cdk5-dependent substrates [110]. However, it may not be ruled out that a subset of substrates still not analyzed can be phosphorylated by Cdk5 due to impairment in Reelin signaling.

The Amyloidogenic Pathway Meets the Reelin Signaling Cascade: A Cytoskeleton Bridge Between... http://dx.doi.org/10.5772/54038 49


**Table 1.** Association of Reelin signaling pathway with Alzheimer´s disease

As VLDLR/ApoER2 or mDab1 deficient mice exhibit a phenotype indistinguishable from *re‐ eler* mice, it is suggested that both receptors and the adapter protein can be linearly placed

The binding of Reelin to its receptors induces mDab1 tyrosine phosphorylation, mediat‐ ed by non-receptor tyrosine kinases from the Src family [102]. The mutation of these ty‐ rosines residues by phenylalanines in a *knockin* mouse recapitulates several features of the *reeler* mouse, supporting that these phosphorylation events are required for proper

Several genetic models suggest that canonical Reelin signaling plays an essential role in con‐ trolling the phosphorylation state of tau and, therefore, modulating a critical event in the

Mice deficient in various components of the Reelin signaling pathway, including Reelin it‐ self, VLDLR, ApoER2 and Dab1 show increased tau phosphorylation in several AD-associ‐ ated epitopes, such as those recognized by the antibodies AT8 (pSer202/205) and PHF1

The increase in tau phosphorylation is caused by increased activity of two main kinases, Cdk5 and GSK-3β [105], suggesting that Reelin is playing a negative control over the activi‐

GSK-3β is normally inhibited by phosphorylation at its N-terminal region by the protein kinase Akt, mainly at the Ser9. Reelin signaling in turn, activates Akt through its recruit‐ ment to membrane domains rich in phosphatidylinositol 3-phosphate (PIP3), whose forma‐ tion is involved the activity of the phosphatidilinositol 3-kinase (PI3K). Reelin activates PI3K by potentiating the interaction between tyrosine phosphorylated-mDab1 and the p85α subu‐

Moreover, it has been proposed that the increased activity of Cdk5 in Dab1 or Reelin defi‐ cient mice may be due to a remarkable increase of the proteolyzed form of a Cdk5 activa‐ tor, called p25 [105]. This fragment induces a non-physiological activation of Cdk5, which is present mainly in pathological conditions, including brains of patients with AD [109]. Since the proteolysis of the Cdk5 activator is due to the activity of calpain, it may be hy‐ pothesized that the Reelin signaling pathway could regulate calpain-dependent proteoly‐

It has been proposed that Cdk5 could not be directly regulated by the Reelin signaling cas‐ cade, because cortical neurons treated with Reelin do not exhibit any significant change in the Cdk5 activity [107] or a diminished phosphorylation state of Cdk5-dependent substrates [110]. However, it may not be ruled out that a subset of substrates still not analyzed can be

phosphorylated by Cdk5 due to impairment in Reelin signaling.

on the same signal transduction pathway [37,101].

Reelin signaling [103].

48 Understanding Alzheimer's Disease

progression of AD (Table 1).

(pSer396/404) [38,104-106].

ties of these kinases.

nit of PI3K [107-108].

sis of p35.

The Reelin signaling pathway can target not only microtubule cytoskeleton, but also the ac‐ tin microfilament formation. Acting through its canonical signaling pathway that involve re‐ ceptors VLDLR and ApoER2, the adapter protein Dab1 and activation of PI3K, Reelin is able to activate the small GTPases Rac1 and Cdc42, increasing actin polymerization. These changes in small Rho GTPases are responsible of increased mobility of growth cones and promote the appearance of filopodia in the axon of cortical neurons in culture [111]. The sta‐ bilization of actin filaments is mediated directly by an increase in activation of LIMK and phosphorylation of cofilin Ser3 [33]. LIMK and cofilin phosphorylation are two key events that regulate actin microfilament turnover in a Rac-dependent manner. Currently, there are no studies showing a causal relationship between impaired Reelin signaling and molecular changes affecting cofilin phosphorylation that could regulate the formation of actin-rods. However, it is tempting to speculate that further studies may solve a linkage between the decreased Reelin signaling observed in AD brains and abnormal actin dynamics.

Proceedings of the National Academy of Sciences of the United States of America

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

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

51

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