**1. Introduction**

Reelin is an extracellular matrix glycoprotein of ~400 kD, expressed in mammals during neurodevelopment by the Cajal-Retzius (CR) neurons, which are located in the marginal zone of the cortex and hippocampus [1], and by the cerebellar granule cells [2]. In adult stages, CR neurons degenerate in both structures [3], limiting Reelin production and se‐ cretion to GABAergic interneurons [4]. Meanwhile, the expression in the cerebellum re‐ mains being exclusive of granule cells [5]. During development, Reelin synthesis also occurs in structures like the hypothalamus, the olfactory bulb, the basal ganglia and the amygdale. In these last two brain regions, Reelin expression continues into adulthood but at low concentrations [1].

Reelin gene encompasses 450 kb of genomic DNA located on human chromosome 7q22 and in murine chromosome 5. Both genes contain 65 exons that encode a protein sharing a 94,2 (%) of identity [6-7]. The transcription initiation region and the exon 1 of the reelin gene is enriched in CG nucleotides, forming a large CpG island [8], which is associated with a meth‐ ylation-dependent negative regulation of reelin transcription [9]. In fact, DNA methyltrans‐ ferases and histone deacetylases inhibitors increase Reelin protein expression, most likely due to decreased reelin promoter methylation [10-11].

In addition to the epigenetic regulation, reelin gene show multiple *cis* elements, which con‐ tain binding sites for transcription factors involved in neurodevelopment such as Sp1, Tbr-1 and Pax6, and elements involved in cytoplasm-to-nucleus signal transduction as CREB and NF-κB [7,12]. Tbr-1 deficient mice show a clear disruption of cortical organization, accompa‐

© 2013 Bórquez et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

nied by decreased Reelin levels [13]. On the other hand, retinoic acid, a known inducer of neuronal growth and differentiation, increase Pax6 and Sp1 levels leading to the activation of the reelin promoter and a subsequent increased Reelin protein synthesis [14].

four successive waves of postmitotic neurons migrate from the ventricular zone, through the sub-plate and neurons already positioned, to reach the marginal zone where the Reelin se‐ creted by the CR neurons acts as a "stop signal" inducing the termination of the neuronal migration. This process is termed as "radial migration", and occurs through an inside-out mechanism, where early migrating neurons are placed at the inner aspects of the cortex [23]. Mechanistically, cortical neurons migrate using two different mechanisms, a glial-depend‐ ent process termed locomotion; and a glial-independent one termed nuclear translocation. During migration across the cortical plate, the neurons adopt morphology characterized by the presence of a cytoplasmic extension oriented toward the most outer aspect of the cortex, the leading process. A secondary cytoplasmic extension emerge orthogonally from the lead‐ ing process and is termed the trailing process. While the leading process will be further de‐

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

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41

The *reeler* mutant shows a clear disruption of the cortical layers, characterized by the ab‐ sence of pre-plate splitting, generating a structure called the superplate [25]. Additionally, migrating neurons fail to establish an inside-out pattern of cortical layers [26]. Thus, in the *reeler* mutant, neurons that migrate earlier during development are placed in the outer as‐

Abnormal neuronal migration is not exclusively for the *reeler* mice cortex. Purkinje neurons in the cerebellum are also aberrantly organized. After birth, the Purkinje cell layer is absent, and a reduction of granule cells number is appreciated, these alterations result in a dramatic reduction of foliation pattern and diminished cerebellar size [30]. At the hippocampal re‐ gion, the *reeler* mutant is characterized by the presence of non-compacted dentate gyrus and

Summarizing, Reeler brain shows smaller size and larger ventricles, the distribution of the dorsal, medial and ventral hippocampus is altered, the cortex display an inverted array of neurons in their layers and the cerebellum shows no foliation and alterations in the organi‐

At the molecular level, the Reelin signaling pathway control several processes required for proper neuronal migration. For example, Reelin stabilize the leading process by inducing cofilin phosphorylation at Ser3, which regulates actin dynamics [33]. Furthermore, Reelin can also induce MAP1B phosphorylation through GSK-3β activation. MAP1B function is in‐ volved in formation of brain laminated areas, therefore, Reelin can modulate neuronal guid‐ ance through post-translational modifications of MAP1B [34]. These two examples show how Reelin can act coordinately to locally regulate the assembly of actin microfilaments and

In addition to its role in neurodevelopment, Reelin controls the formation of neural circuits, promoting the growth and branching of dendrites in hippocampal neurons [35]. Moreover, Reelin can enhance the formation of dendritic spines, supporting a role at the post-synaptic

Most of these cellular functions are dependent on a signaling pathway, triggered by the binding of Reelin to its two main receptors, the very-low density lipoprotein receptor

veloped as the dendritic arbor, the trailing process will become the axon [24].

pect of the cortex, leading to an outside-in pattern of cortical layers [23,27-29].

disorganized pyramidal layer [31].

zation of its layers [32].

microtubules (Figure 1A).

compartment [36].

The full length Reelin protein contains 3461 amino acids, organized from N- to C-terminal by the following domains and motifs: 1.- A signal peptide, 2.- F-spondin-like motif, 3.- 8 re‐ peat domains, composed of a region A and region B spaced by EGF motif, and 4.- A region enriched in basic amino acids [2].

Reelin may undergo proteolytic cleavage at the beginning of the 3rd and 7th A-EGF-B repeat generating many fragments including the N-terminal, the intermediate segment and the Cterminal fragment. Cleavage may be precluded by zinc chelators, known inhibitors of metal‐ loproteinases [15]. Recently a putative protease had been identified as p50 and p70 isoforms of a disintegrin and metalloproteinase with thrombospodin motif 4 (ADAMTS-4). The p50 isoform cleaves at N-terminal only, and p70 cleaves the N- and C-terminal sites [16]. The im‐ portance of the proteolytic processing remains unclear, however; several reports showed that the internalization of Reelin at target cells is independent of its cleavage. In turn, only the central region seems to be sufficient for Reelin functions. Reelin cleavage would be re‐ quired to enable Reelin secretion, allowing the release of a central, active fragment from the extracellular matrix-attached full length protein [17-18]. In contrast to this notion, there are many studies showing that the N-terminal region is important for Reelin secretion (due to the presence of a signal peptide on this region) [19], and to promote the formation of homo‐ polymers, which are essential for proper signal transduction [20]. There is still little evidence about the function of the C-terminal region. The *reeler* Orleans mutation characterized by a deletion of 220 nucleotides at the C-terminal, prevents the secretion of Reelin, suggesting a possible role for this region in normal Reelin functions [21].

### **2. Reelin in neurodevelopment**

As outlined in the previous section, Reelin is a glycoprotein, which is expressed in CR neu‐ rons starting at embryonic day 11 (E11), mainly in the cortex, hippocampus and cerebellum. It expression remains high until day E18, when CR neurons begin to degenerate [1,3]. The importance of Reelin to neurodevelopment had been elucidated through numerous studies using a mice model exhibiting a spontaneous mutation (partial deletion) in the reelin gene, called the *reeler* mice [22]. These mice had pronounced defects in the correct neuronal posi‐ tioning in the laminar structures of the brain. At day E11, postmitotic neurons located in the ventricular zone, migrate toward the pial surface to form the preplate. On E13, a new cohort of migrating neurons originated at the proliferative region separate the pre-plate. Pre-plate splitting originates two regions, the marginal zone and the sub-plate, which are positioned adjacent to the pial surface and near the ventricular zone, respectively. The marginal zone is rich in CR neurons, which are the primary source of Reelin during neurodevelopment. Sev‐ eral waves of postmitotic migrating neurons are positioned between the marginal zone and the sub plate, leading to the formation of the cortical plate. During the E14-E18 time lapse, four successive waves of postmitotic neurons migrate from the ventricular zone, through the sub-plate and neurons already positioned, to reach the marginal zone where the Reelin se‐ creted by the CR neurons acts as a "stop signal" inducing the termination of the neuronal migration. This process is termed as "radial migration", and occurs through an inside-out mechanism, where early migrating neurons are placed at the inner aspects of the cortex [23]. Mechanistically, cortical neurons migrate using two different mechanisms, a glial-depend‐ ent process termed locomotion; and a glial-independent one termed nuclear translocation. During migration across the cortical plate, the neurons adopt morphology characterized by the presence of a cytoplasmic extension oriented toward the most outer aspect of the cortex, the leading process. A secondary cytoplasmic extension emerge orthogonally from the lead‐ ing process and is termed the trailing process. While the leading process will be further de‐ veloped as the dendritic arbor, the trailing process will become the axon [24].

nied by decreased Reelin levels [13]. On the other hand, retinoic acid, a known inducer of neuronal growth and differentiation, increase Pax6 and Sp1 levels leading to the activation

The full length Reelin protein contains 3461 amino acids, organized from N- to C-terminal by the following domains and motifs: 1.- A signal peptide, 2.- F-spondin-like motif, 3.- 8 re‐ peat domains, composed of a region A and region B spaced by EGF motif, and 4.- A region

Reelin may undergo proteolytic cleavage at the beginning of the 3rd and 7th A-EGF-B repeat generating many fragments including the N-terminal, the intermediate segment and the Cterminal fragment. Cleavage may be precluded by zinc chelators, known inhibitors of metal‐ loproteinases [15]. Recently a putative protease had been identified as p50 and p70 isoforms of a disintegrin and metalloproteinase with thrombospodin motif 4 (ADAMTS-4). The p50 isoform cleaves at N-terminal only, and p70 cleaves the N- and C-terminal sites [16]. The im‐ portance of the proteolytic processing remains unclear, however; several reports showed that the internalization of Reelin at target cells is independent of its cleavage. In turn, only the central region seems to be sufficient for Reelin functions. Reelin cleavage would be re‐ quired to enable Reelin secretion, allowing the release of a central, active fragment from the extracellular matrix-attached full length protein [17-18]. In contrast to this notion, there are many studies showing that the N-terminal region is important for Reelin secretion (due to the presence of a signal peptide on this region) [19], and to promote the formation of homo‐ polymers, which are essential for proper signal transduction [20]. There is still little evidence about the function of the C-terminal region. The *reeler* Orleans mutation characterized by a deletion of 220 nucleotides at the C-terminal, prevents the secretion of Reelin, suggesting a

As outlined in the previous section, Reelin is a glycoprotein, which is expressed in CR neu‐ rons starting at embryonic day 11 (E11), mainly in the cortex, hippocampus and cerebellum. It expression remains high until day E18, when CR neurons begin to degenerate [1,3]. The importance of Reelin to neurodevelopment had been elucidated through numerous studies using a mice model exhibiting a spontaneous mutation (partial deletion) in the reelin gene, called the *reeler* mice [22]. These mice had pronounced defects in the correct neuronal posi‐ tioning in the laminar structures of the brain. At day E11, postmitotic neurons located in the ventricular zone, migrate toward the pial surface to form the preplate. On E13, a new cohort of migrating neurons originated at the proliferative region separate the pre-plate. Pre-plate splitting originates two regions, the marginal zone and the sub-plate, which are positioned adjacent to the pial surface and near the ventricular zone, respectively. The marginal zone is rich in CR neurons, which are the primary source of Reelin during neurodevelopment. Sev‐ eral waves of postmitotic migrating neurons are positioned between the marginal zone and the sub plate, leading to the formation of the cortical plate. During the E14-E18 time lapse,

of the reelin promoter and a subsequent increased Reelin protein synthesis [14].

possible role for this region in normal Reelin functions [21].

**2. Reelin in neurodevelopment**

enriched in basic amino acids [2].

40 Understanding Alzheimer's Disease

The *reeler* mutant shows a clear disruption of the cortical layers, characterized by the ab‐ sence of pre-plate splitting, generating a structure called the superplate [25]. Additionally, migrating neurons fail to establish an inside-out pattern of cortical layers [26]. Thus, in the *reeler* mutant, neurons that migrate earlier during development are placed in the outer as‐ pect of the cortex, leading to an outside-in pattern of cortical layers [23,27-29].

Abnormal neuronal migration is not exclusively for the *reeler* mice cortex. Purkinje neurons in the cerebellum are also aberrantly organized. After birth, the Purkinje cell layer is absent, and a reduction of granule cells number is appreciated, these alterations result in a dramatic reduction of foliation pattern and diminished cerebellar size [30]. At the hippocampal re‐ gion, the *reeler* mutant is characterized by the presence of non-compacted dentate gyrus and disorganized pyramidal layer [31].

Summarizing, Reeler brain shows smaller size and larger ventricles, the distribution of the dorsal, medial and ventral hippocampus is altered, the cortex display an inverted array of neurons in their layers and the cerebellum shows no foliation and alterations in the organi‐ zation of its layers [32].

At the molecular level, the Reelin signaling pathway control several processes required for proper neuronal migration. For example, Reelin stabilize the leading process by inducing cofilin phosphorylation at Ser3, which regulates actin dynamics [33]. Furthermore, Reelin can also induce MAP1B phosphorylation through GSK-3β activation. MAP1B function is in‐ volved in formation of brain laminated areas, therefore, Reelin can modulate neuronal guid‐ ance through post-translational modifications of MAP1B [34]. These two examples show how Reelin can act coordinately to locally regulate the assembly of actin microfilaments and microtubules (Figure 1A).

In addition to its role in neurodevelopment, Reelin controls the formation of neural circuits, promoting the growth and branching of dendrites in hippocampal neurons [35]. Moreover, Reelin can enhance the formation of dendritic spines, supporting a role at the post-synaptic compartment [36].

Most of these cellular functions are dependent on a signaling pathway, triggered by the binding of Reelin to its two main receptors, the very-low density lipoprotein receptor (VLDLR) and the ApolipoproteinE receptor 2 (ApoER2) [37]. The binding of Reelin to its re‐ ceptor induce the phosphorylation of the adapter protein mDab1 on tyrosine residues [38]. mDab1 phosphorylation lead eventually to the modulation of cytoskeleton effectors mole‐ cules such as MAP1B and cofilin [23].

**3. Reelin in the adult brain**

Although Reelin function is mainly related to neurodevelopment, several recently studies assign roles in the adult brain, such as the development of dendrites and dendritic spines [36], modulation of synaptogenesis [39], modulation of synaptic plasticity [40-43] and neuro‐ transmitter release [44]. The mechanism by which Reelin can modulate the synaptic trans‐ mission is not fully elucidated. Currently it is strongly suggested that Reelin acting through its canonical signaling pathway facilitates the phosphorylation of NR2A and NR2B subunits

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

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This intracellular calcium increase causes the insertion of the GluR1 subunit of AMPA re‐ ceptors, allowing the phosphorylation and nuclear translocation of CREB [45]. CREB phos‐ phorylation is required to elicit the formation of dendritic spines. In addition, Reelin reduces the number of silent synapses, facilitating the exchange of subunit NR2B by NR2A of NMDA receptor [46]. Therefore, Reelin modulates synaptic plasticity events involved in learning and memory processes in adults. Consistently with a role for Reelin in the control of neurotransmission, *reeler* mice show diminished expression of presynaptic (SNARE, SNAP-25) [44] and postsynaptic (PSD-95, PTEN) markers [43]. These defects cause failures

Owing to the importance that Reelin have in the correct structuration and lamination of the brain during development and in neuronal connectivity and synaptogenesis in the adult brain, its dysfunction has been directly related to the generation or susceptibility to acquire neuropsychiatric conditions such as depression and schizophrenia, or neurodegenerative

The most tangible evidence supporting these putative relationships was obtained through studies of human brains derived from neuropsychiatric and neurodegenerative conditions. Decreased levels of Reelin are shown in postmortem samples from prefrontal cortex of pa‐ tients with schizophrenia and bipolar disorders [47]. This decrease may be explained in schizophrenic patients by an abnormal hypermethylation of the *reelin* promoter, an epige‐ netic modification involved in gene silencing [48]. Furthermore, immunohistochemistry ex‐ periments in depressive and schizophrenic patients show decreased Reelin expression at the hippocampus [49]. On the other hand, diminished Reelin levels in the hippocampus of pa‐ tients with AD had been reported, suggesting a direct correlation between the severity of the disease and the extent of decreased Reelin expression [50]. All of these antecedents provide evidence enough to feature a molecular link between decreased Reelin levels and neurode‐

In order to understand the etiology of neurodegenerative/psychiatric diseases, different ani‐ mal models had been developed. A widely paradigm is the "two hit" model, which suggests

of the NMDA receptor, favoring the calcium influx into the postsynaptic neuron.

in the release of neurotransmitters, impairing synaptic transmission.

**4. Animals models for neuropsychiatric diseases**

diseases such as Alzheimer's disease (AD) [45].

generative/psychiatric diseases.

**Figure 1.** Reelin signaling pathway in normal brain and its impairment in AD brains. The panel A show the intracellular events triggered by the binding of Reelin to its canonical membrane receptors, ApoER2 and VLDLR. mDab1 protein is spe‐ cifically phosphorylated at tyrosine residues, which concomitantly with the activation of PI3K, regulate actin and microtu‐ bule dynamic behavior. LIMK-mediated phosphorylation of cofilin and the Akt inhibitory phosphorylation of GSK-3β are essential to this regulation. On the other hand, in AD brains, diminished Reelin expression and its aggregation in amyloidlike deposits, induce impairment in its signaling pathway (represented by gray lines). Decreased Reelin signaling triggers the dephosphorylation of cofilin, promoting the formation of actin-rods. On the other hand, the activation of GSK-3β and CDK-5, lead to hyperphosphorylation of tau protein inducing its aggregation into PHFs (panel B).
