**5. The role of AICD**

### **5.1. Signaling functions of AICD**

As mentioned above, the observations that the common enzyme, γ-secretase, modulates proteolysis and the turnover of possible signaling molecules led to the signaling hypothesis. This suggests that mechanisms similar to the Notch signaling pathway may contribute widely to γ-secretase–regulated signaling mechanisms, including APP signaling. If APP sig‐ naling exists, it may be closely related to AD.

we concluded that AICD could induce neuron-specific apoptosis [29]. The effects of AICD

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Thus, although further studies are required, these results strongly suggest that AICD plays a role in APP signaling and induces neuronal cell death, which may closely relate to the onset

**Figure 4.** Expression of AICD in P19 cells induced neuronal cell death. After aggregation culture with RA, AICD-ex‐ pressing P19 and control P19 cells carrying vector alone were replated and cultured for the indicated periods on dishes and allowed to differentiate. Undifferentiated AICD-expressing P19 cells retained epithelial cell-like morpholo‐ gy similar to control cells, while the differentiated cells became round and showed a bipolar morphology with neurite extension. Two days after replating (Day 2), all cell lines grew well and neurons with long neurites appeared. Four days after replating (Day 4), control cells still grew well as clusters and many neurons had differentiated from these cells. However, many AICD-expressing P19 cells showed severe degeneration, becoming spherical with numerous vacuoles

If APP signaling exists, AICD should change the expression of certain genes. To examine this possibility and identify the genes involved in this neuron-specific apoptosis, we performed DNA microarray analyses to evaluate the changes in the expression of more than 20,000 inde‐ pendent genes induced by AICD through the process of neuron-specific apoptosis [30]. Gene expression levels were deduced by hybridization signal intensity on the DNA microarrays, and the data from AICD-overexpressing cells were compared to data from control cells at the same 3 points during culture: 1) the undifferentiated state; 2) after 4 days of aggregation with RA (ag‐ gregated state); and 3) 2 days after replating (differentiated state). According to our expecta‐ tions, AICD was shown to alter the expression of a great many genes; in the presence of AICD, the expression levels of 277 genes were upregulated by more than 10-fold, while those of 341

were restricted to neurons, with no effects observed in non-neural cells.

and progression of AD.

and detached from the culture dishes.

**5.2. AICD changes the gene expression profile**

genes were downregulated to less than 10% of the original level [30].

Actually, there is accumulating evidence for the existence of APP signaling and its contribu‐ tion to the onset and progression of AD. As mentioned above, the highest degree of se‐ quence conservation within the APP homologues is found in the ICD [9, 29]. This sequence conservation suggests the functional importance of AICD, which may reflect the existence of APP signaling. In addition, several AICD-interacting proteins, which may regulate AICD stability, cellular localization, and transcriptional activity, have been identified. Based on this, several models of APP signaling have also been proposed. As mentioned above, it has been suggested that AICD recruits Fe65 proteins and translocates into the nucleus where the AICD-Fe65-Tip60 ternary complex may control transcription of target genes [27]. Further‐ more, *NEP* gene expression requires binding of the AICD to its promoter [94].

Transgenic mice overexpressing both AICD and Fe65 showed abnormal activity of glycogen synthase kinase 3 beta (Gsk3b protein) [95], leading to hyperphosphorylation and aggrega‐ tion of tau. This results in microtubule destabilization and the reduction of nuclear β-catenin levels causing a loss of cell-cell contact that may contribute to neurotoxicity in AD. Subse‐ quent neurodegeneration and working memory deficits were also observed in these trans‐ genic mice [96]. In other experiments, similar transgenic mice exhibited abnormal spiking events in their electroencephalograms and susceptibility to kainic acid-induced seizures in‐ dependent of Aβ [97]. Furthermore, the function of c-Abl kinase in the transcriptional regu‐ lation of AICD was reported and c-Abl was shown to modulate AICD-dependent cellular responses, transcriptional induction, as well as apoptotic responses [98]. Interestingly, ele‐ vated AICD levels have also been reported in AD brains [96]. In addition, AICD was detect‐ ed in the nucleus in injured brains [99]. Taken together, it is likely that APP signaling changes the expression of certain genes, which may cause AD pathology.

To explore APP signaling, we established several AICD-overexpressing embryonic carcinoma P19 cell lines [29]. Undifferentiated AICD-overexpressing cells retained epithelial cell-like mor‐ phology and healthy as well as control cells. Although neurons were differentiated from these cell lines after aggregation culture with all-*trans*-retinoic acid (RA) treatment, AICD expression induced neuron-specific cell death. Indeed, as shown in Fig.4, while neurons from control cells that carried the vector alone were healthy, almost all neurons differentiated from AICD-overex‐ pressing P19 cells showed severe degeneration, becoming spherical with numerous vacuoles and detaching from the culture dishes 4 days after the induction of differentiation.

Since DNA fragmentation was detected, these cells died by apoptosis. In addition, all termi‐ nal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick end-labeling (TUNEL)-positive cells were also Tuj1-positive neurons. Taken together, we concluded that AICD could induce neuron-specific apoptosis [29]. The effects of AICD were restricted to neurons, with no effects observed in non-neural cells.

Thus, although further studies are required, these results strongly suggest that AICD plays a role in APP signaling and induces neuronal cell death, which may closely relate to the onset and progression of AD.

**Figure 4.** Expression of AICD in P19 cells induced neuronal cell death. After aggregation culture with RA, AICD-ex‐ pressing P19 and control P19 cells carrying vector alone were replated and cultured for the indicated periods on dishes and allowed to differentiate. Undifferentiated AICD-expressing P19 cells retained epithelial cell-like morpholo‐ gy similar to control cells, while the differentiated cells became round and showed a bipolar morphology with neurite extension. Two days after replating (Day 2), all cell lines grew well and neurons with long neurites appeared. Four days after replating (Day 4), control cells still grew well as clusters and many neurons had differentiated from these cells. However, many AICD-expressing P19 cells showed severe degeneration, becoming spherical with numerous vacuoles and detached from the culture dishes.

#### **5.2. AICD changes the gene expression profile**

**5. The role of AICD**

72 Understanding Alzheimer's Disease

**5.1. Signaling functions of AICD**

naling exists, it may be closely related to AD.

As mentioned above, the observations that the common enzyme, γ-secretase, modulates proteolysis and the turnover of possible signaling molecules led to the signaling hypothesis. This suggests that mechanisms similar to the Notch signaling pathway may contribute widely to γ-secretase–regulated signaling mechanisms, including APP signaling. If APP sig‐

Actually, there is accumulating evidence for the existence of APP signaling and its contribu‐ tion to the onset and progression of AD. As mentioned above, the highest degree of se‐ quence conservation within the APP homologues is found in the ICD [9, 29]. This sequence conservation suggests the functional importance of AICD, which may reflect the existence of APP signaling. In addition, several AICD-interacting proteins, which may regulate AICD stability, cellular localization, and transcriptional activity, have been identified. Based on this, several models of APP signaling have also been proposed. As mentioned above, it has been suggested that AICD recruits Fe65 proteins and translocates into the nucleus where the AICD-Fe65-Tip60 ternary complex may control transcription of target genes [27]. Further‐

Transgenic mice overexpressing both AICD and Fe65 showed abnormal activity of glycogen synthase kinase 3 beta (Gsk3b protein) [95], leading to hyperphosphorylation and aggrega‐ tion of tau. This results in microtubule destabilization and the reduction of nuclear β-catenin levels causing a loss of cell-cell contact that may contribute to neurotoxicity in AD. Subse‐ quent neurodegeneration and working memory deficits were also observed in these trans‐ genic mice [96]. In other experiments, similar transgenic mice exhibited abnormal spiking events in their electroencephalograms and susceptibility to kainic acid-induced seizures in‐ dependent of Aβ [97]. Furthermore, the function of c-Abl kinase in the transcriptional regu‐ lation of AICD was reported and c-Abl was shown to modulate AICD-dependent cellular responses, transcriptional induction, as well as apoptotic responses [98]. Interestingly, ele‐ vated AICD levels have also been reported in AD brains [96]. In addition, AICD was detect‐ ed in the nucleus in injured brains [99]. Taken together, it is likely that APP signaling

To explore APP signaling, we established several AICD-overexpressing embryonic carcinoma P19 cell lines [29]. Undifferentiated AICD-overexpressing cells retained epithelial cell-like mor‐ phology and healthy as well as control cells. Although neurons were differentiated from these cell lines after aggregation culture with all-*trans*-retinoic acid (RA) treatment, AICD expression induced neuron-specific cell death. Indeed, as shown in Fig.4, while neurons from control cells that carried the vector alone were healthy, almost all neurons differentiated from AICD-overex‐ pressing P19 cells showed severe degeneration, becoming spherical with numerous vacuoles

Since DNA fragmentation was detected, these cells died by apoptosis. In addition, all termi‐ nal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick end-labeling (TUNEL)-positive cells were also Tuj1-positive neurons. Taken together,

more, *NEP* gene expression requires binding of the AICD to its promoter [94].

changes the expression of certain genes, which may cause AD pathology.

and detaching from the culture dishes 4 days after the induction of differentiation.

If APP signaling exists, AICD should change the expression of certain genes. To examine this possibility and identify the genes involved in this neuron-specific apoptosis, we performed DNA microarray analyses to evaluate the changes in the expression of more than 20,000 inde‐ pendent genes induced by AICD through the process of neuron-specific apoptosis [30]. Gene expression levels were deduced by hybridization signal intensity on the DNA microarrays, and the data from AICD-overexpressing cells were compared to data from control cells at the same 3 points during culture: 1) the undifferentiated state; 2) after 4 days of aggregation with RA (ag‐ gregated state); and 3) 2 days after replating (differentiated state). According to our expecta‐ tions, AICD was shown to alter the expression of a great many genes; in the presence of AICD, the expression levels of 277 genes were upregulated by more than 10-fold, while those of 341 genes were downregulated to less than 10% of the original level [30].


AICD strongly induced expression of several genes, representative examples of which are listed in Table 2. For example, AICD-overexpressing P19 cells showed strong expression of the protein tyrosine phosphatase receptor T (*Ptprt*) gene at all sampling points: 906-fold, 204-fold, and 116-fold upregulation, in undifferentiated, aggregated, and differentiated states, respectively, compared with control cells. In contrast to these upregulated genes, the expression of several genes was strongly inhibited by AICD. Although *Hes5* expression was markedly increased through the process of neural differentiation, with an increase of almost 300-fold in control cells, AICD inhibited this induction. As shown in Fig.5, these results were confirmed by RT-PCR. Thus, AICD may induce both upregulation and downregulation of

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**Figure 5.** RT-PCR analysis of representative 7 upregulated genes and 7 downregulated genes, as well as 3 housekeep‐ ing genes, in P19 cells overexpressing AICD. The RNA samples same as applied to DNA microarray analysis was used in

this RT-PCR analysis.

certain genes, suggesting that AICD plays an important role in APP signaling.

Relative expression levels (fold) were estimated from the intensities of hybridization signals. Housekeeping gene ex‐ pression was unaltered in AICD-overexpressing P19 and control P19 cells in all states, suggesting that these genes are not affected by AICD. These results also indicated that the observed differences in expression were not due to techni‐ cal problems, such as uneven hybridization or poor RNA quality.

**Table 2.** Expression levels of 7 upregulated and 7 downregulated genes, as well as 3 housekeeping genes

AICD strongly induced expression of several genes, representative examples of which are listed in Table 2. For example, AICD-overexpressing P19 cells showed strong expression of the protein tyrosine phosphatase receptor T (*Ptprt*) gene at all sampling points: 906-fold, 204-fold, and 116-fold upregulation, in undifferentiated, aggregated, and differentiated states, respectively, compared with control cells. In contrast to these upregulated genes, the expression of several genes was strongly inhibited by AICD. Although *Hes5* expression was markedly increased through the process of neural differentiation, with an increase of almost 300-fold in control cells, AICD inhibited this induction. As shown in Fig.5, these results were confirmed by RT-PCR. Thus, AICD may induce both upregulation and downregulation of certain genes, suggesting that AICD plays an important role in APP signaling.

**Gene Symbol**

*Sdha*

*Eef1a1*

*Ptprt*

*Dnahc7c*

*Ctgf*

*Hes5*

*Slc10a6*

*LOC213332*

*Downregulated genes*

*Upregulated genes*

*Non-regulated genes (housekeeping genes)*

74 Understanding Alzheimer's Disease

succinate dehydrogenase subunit A

eukaryotic translation elongation factor-1 alpha 1

protein tyrosine phosphatase receptor T

*Cpb1* carboxypeptidase B1

*Nr2e1* tailless homolog

*Myh1* myosin heavy chain 1

*Alkbh3* alkylation repair homolog 3

axonemal dynein heavy chain

connective tissue growth factor

hairy and enhancer of split 5

sodium-dependent organic anion transporter

analog of Na<sup>+</sup>-dependent glucose transporter 1

cal problems, such as uneven hybridization or poor RNA quality.

*Rbp4* retinol-binding protein 4

**Gene Name Function**

*Actb* β-actin cytoskeleton protein -1.2 1.2 1

electron transporter in the TCA cycle and respiratory chain

essential component for the elongation phase during protein translation

protein tyrosine phosphatase that regulates STAT3 activity

hydrolysis of C-terminal end of basic amino acid peptide bond

transcription factor that is essential for neural stem cell proliferation and selfrenewal

one of the components of the motor protein myosin

AlkB enzyme that repairs methylation damage in DNA and RNA

skeletogenesis/vasculogenesis by modulating BMP, Wnt, and IGF-I signals

transcription factor that inhibits neurogenesis

transport of sulfoconjugated steroid hormones and bile acids

*Nid1* nidogen-1 extracellular matrix linker protein -304 -165 -507

*Dtx1* Deltex1 regulator of Notch signaling pathway -30 -85 -691

*Col3a1* collagen type III alpha 1 extracellular matrix protein 4.1 -29 -234 Relative expression levels (fold) were estimated from the intensities of hybridization signals. Housekeeping gene ex‐ pression was unaltered in AICD-overexpressing P19 and control P19 cells in all states, suggesting that these genes are not affected by AICD. These results also indicated that the observed differences in expression were not due to techni‐

retinol transporter from the liver to extrahepatic tissues

**Table 2.** Expression levels of 7 upregulated and 7 downregulated genes, as well as 3 housekeeping genes

essential for motility of cilia and flagellae 133 41 43

putative glucose transporter -232 -325 -306

**Relative Expression Levels (fold) Undifferentiated Aggregated Differentiated**


1 -1.1 1.2

906 204 116

16 296 222

5.8 244 54


69 80 43

90 54 40




**Figure 5.** RT-PCR analysis of representative 7 upregulated genes and 7 downregulated genes, as well as 3 housekeep‐ ing genes, in P19 cells overexpressing AICD. The RNA samples same as applied to DNA microarray analysis was used in this RT-PCR analysis.

We performed Gene Ontology (GO) analysis and classified these upregulated and downre‐ gulated genes according to the GO terms [30]; however, we did not find genes that were sig‐ nificantly related to cell death among those with altered expression. Furthermore, we evaluated AICD-induced changes in the expression of genes thought to be involved in cell death in AD [30]; however, we found no significant changes in expression of these genes. Thus, it is likely that AICD does not directly induce the expression of genes involved in cell death, but the extreme dynamic changes in gene expression disrupt the homeostasis of cer‐ tain neurons and thus give rise to neuron-specific cell death. Taken together, these results strongly suggest the existence of APP signaling.

ment have been proposed, these results lead to the idea that both soluble and insoluble

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Based on these observations, it has been suggested that AD may be caused by an APP-de‐ rived fragment, just not necessarily Aβ [105]. As both extracellular fragments and AICD are generated at the same time as Aβ, acceleration of proteolytic processing leads to overpro‐ duction of not only Aβ but also of both the extracellular fragments and AICD. Therefore, it is likely that the extracellular fragments and/or AICD are responsible for the onset and pro‐ gression of AD. Indeed, AICD has been shown to induce neuron-specific apoptosis, which

In addition, it has also been proposed that APP is a ligand of Death receptor 6 (DR6) [106], which mediates cell death and is expressed at high levels in the human brain regions most affected by AD. APP is cleaved by β-secretase, releasing the extracellular domain (sAPPβ), which is further cleaved by an as yet unknown mechanism to release a 35 kDa N-terminal fragment (N-APP). This N-APP fragment binds DR6 to trigger neurodegeneration through caspase 6 in axons and caspase 3 in cell bodies [106]. These results suggest that N-APP may

Through this chapter, we discussed the possibility of the existence of APP signaling. It is likely that disorders of this signaling mechanism are involved in the onset and progression of AD. As AICD is generated at the same time as Aβ, acceleration of proteolytic processing leads to overproduction of not only Aβ but also AICD in AD brain as discussed above. Fur‐ thermore, we showed that AICD alters the expression of certain genes and induces neuron-

If the APP signaling hypothesis is correct, certain molecules involved in APP signaling may be attractive candidates for the targets of drug discovery for treating AD. Fig.6 is a schemat‐ ic model of APP signaling. As mentioned above, after cleavage within the JM domain by αor β-secretase, AICD is released from the membrane by γ-secretase. Inhibiters for these

As mentioned in section 4.3, non-phosphorylated AICD can bind to the nuclear adaptor pro‐ tein Fe65 [92, 93], which is essential for translocation of AICD to the nucleus. However, phosphorylated AICD cannot bind to Fe65. These results suggest the possibility that a cer‐ tain stimulus controls APP signaling through phosphorylation and dephosphorylation of AICD. It has also been shown that the majority of cell membrane-associated APP is phos‐ phorylated specifically at Thr668 in neurons [107]. Therefore, phosphorylated AICD, which is released from the cell membrane to the cytoplasm by γ-secretase, cannot bind to Fe65 and thus cannot translocate to the nucleus. Phosphorylated AICD left in the cytosol is rapidly degraded, probably by the proteasome and/or IDE [88]. However, if AICD is dephosphory‐ lated by certain phosphatase, AICD can binds to Fe65. Thus, AICD/Fe65 complexes may im‐

forms of Aβ may not be involved in the onset and progression of AD.

leads to AD pathology, as mentioned above.

also be involved in the onset and progression of AD.

**7. The model of APP signaling**

proteases are being studied extensively.

specific apoptosis [29, 30].
