**3. Insulin signaling in the brain**

ischemia not only causes increased amyloidogenic cleavage of APP and greater Aβ production,

Decreased Aβ-degrading enzymes in response to hypoxic conditions increase the likelihood of developing pathological levels of Aβ in the brain [113-115]. Aβ serves not only as the end result of a pathological cascade, but Aβ itself has been found to contribute to dysfunction in components of the neurovascular unit. In endothelial cells Aβ was observed to decrease endothelial cell proliferation and accelerate senescence of endothelial cells in vivo and in vitro, inhibit VEGF-induced activation of Akt and eNOS in endothelial cells [116, 117]. Aβ has been found to decrease eNOS (via PKC-dependent pathway) resulting in decreased vascular tonus and decreased substance P-induced vasodilation of the basilar artery[118, 119]. In vascular smooth muscle cells (VSMCs), Aβ affects cellular morphological changes [120] and increases expression of transcription factors, serum response factor and myocardin, resulting in decreased Aβ clearance by downregulating LRP expression [12]. Finally, Aβ has been shown to cause retraction and swelling of astrocyte endfeet in an AD mouse model with CAA [121] as well as increase cholinergic denervation of cortical microvessels which, taken together,

**Type 2 diabetes and vascular dementia** A significant number of population-based studies have indicated an increased risk for the development of dementia attributed to T2DM [23-25]. Due to the importance of insulin in the regulation of several cardiovascular functions, it is unsurprising that insulin resistance plays a role in the cerebrovascular mechanisms of T2DMinduced dementia. The presence of brain infarcts in demented diabetics who did not have AD has been reported 123. Interestingly, the association between T2DM and the development of AD and VaD has been found to be independent of hypertension and hypercholesterolemia [23] indicating that is CVD alone is not sufficient to initiate dementia. Non-cerebrovascular mechanisms such as peripheral hyperinsulinemia and generation of advanced glycation endproducts also play in the etiology of T2DM-related dementia [124]. Studies have shown that the increased risk of developing vascular dementia was greater than developing AD in type 2 diabetics [7, 125, 126], indicating that although symptomatically similar and frequently

**Vascular dementia versus Alzheimer's dementia** The leading cause of dementia is Alzheim‐ er's disease accounting for 70-90% of all cases [127], while vascular dementia (VaD) accounts for the majority of the remaining incidents of dementia [128]. They share common risk factors including hypertension, diabetes mellitus, and hyperlipidemia. [129], highlighting the tight association between these two forms of dementia. In fact, it is now widely believed that AD and VaD are frequently present in the same brain. So-called "mixed dementia" has been observed in elderly people with cardiovascular risk factors in addition to slow progressive

Differing clinical manifestations separate VaD from AD dementia. For example, VaD progres‐ sion appears more varied than AD in relation to symptoms, its rate of progression and the disease outcome [131]. Increased damage to the ganglia-thalamo-cortical circuits specific to VaD results in problems with attention and the planning and speed of mental processing whereas the primary impairments characteristic of AD are memory and language-related

but also impairs Aβ degradation and trafficking [12, 112].

416 Understanding Alzheimer's Disease

results in impaired functional hyperemia [122].

confused [127], their etiologies are distinct.

cognitive decline [130].

**Insulin/IGF-1 pathway activation.** The brain is a major metabolic organ that accounts for ~25% of the body's total glucose use [28, 29]. While glucose uptake in peripheral tissues requires insulin, in the brain this is considered to be an insulin-independent process. Insulin, however, along with Insulin-like Growth Factor-1 (IGF-1), are required for proper brain function as they provide critical neurotrophic support for neurons. IGF-1 and insulin share similar amino acid sequences/ tertiary structures [148] and are known to bind to and activate one anothers' receptors [149]. Both insulin and IGF-1 receptors are tyrosine kinases [150-152] that, when activated, phosphorylate substrate proteins such as IRS. IRS phosphorylation leads to down‐ stream activation of PI3K and Akt, a serine/threonine kinase and key mediator of insulin/ IFG-1's neurotrophic effects. Neuronal processes known to be, at least in part, under the control of insulin/IGF-1 include regulation of apoptotic proteins, transcription of both survival and pro-death genes, neurite outgrowth, and activity of metabolic proteins.

The source of brain insulin remains controversial. While preproinsulin mRNA has been reported in the neurons [153-155], very little insulin is synthesized in the brain [156]. Addi‐ tionally, glial cells have been found not to be involved in insulin production [157], therefore, it is recognized that the majority of insulin in the brain is produced by pancreatic β cells [158-161]. In contrast, IGF-1 is produced locally in the brain and does not depend on growth hormone influence as is the case of liver and other tissues [148].

Neuronal insulin receptors are different than those found in the periphery [162]. Insulin receptors are present in one of two isoforms; the IR-A isoform that lacks exon 11 that the other isoform, IR-B, expresses [163, 164]. A major functional difference between the two isoforms is that IR-A has a higher affinity for the neurotrophic factor Insulin-like Growth Factor – 2 (IGF-II) [165] and a slightly higher affinity for insulin [166] and has also been shown to associate/ dissociate with insulin quicker than IR-B [149]. Brain specific insulin receptors are mainly the IR-A isoform and as result of differential glycosylation have a lower molecular weight than their peripheral counterparts [162].

possess the ability to form heterodimers with one another [208-210], their regulation of apoptosis can be described as a balancing act in which an increase of anti-apoptotic members

Alzheimer's Disease and Diabetes http://dx.doi.org/10.5772/54913 419

Mitochondrial stress incurred by ROS can lead to elevated Ca2+ levels in the mitochondri‐ al matrix [211, 212] resulting in increased mitochondrial membrane permeability and re‐ lease of pro-apoptotic factors such as Cytochrome c, and AIF (apoptosis inducing factor) [213]. Bcl-xL is an anti-apoptotic Bcl-2 family member that prevents Ca2+ induced mito‐ chondrial permeability [214]. In the absence of insulin/IGF-1 stimulation, the survival ef‐ fects of Bcl-xL are blocked as Bcl-xL is complexed with the pro-death Bcl-2 family member Bad [215-217]. Akt liberates Bcl-xL by phosphorylating Bad [195-197, 218] allow‐

Mitochondrial permeability marks a critical event in the cell death cascade. Akt promotes cell survival prior to Cytochrome c release through Bcl-xL activity but has also been found to act post apoptotic factor release. When Cytochrome c is released from the mitochondria, it will associate with Apaf-1, dATP and Caspase-9 forming a structure known as the apoptosome (For review see [219]). Formation of the apoptosome activates the proteolytic activity of caspase-9 which cleaves and activates other caspases critical to the apoptotic process [220,

Bcl-2 is another anti-apoptotic protein under the control of Akt [222]. Bcl-2's role in cell survival is similar to that of Bcl-xL in that in maintains mitochondrial membrane integri‐ ty [223]. Mitochondrial permeability has been linked to an oxidized shift in the mitochon‐ dria [224] while Bcl-2 has been shown to promote a more reduced state [225]. Upregulation of Bcl-2 may lead to higher cell reductive capacity [224] which is supported by the observation that Bcl-2 overexpressing cells show increased amounts of NADPH and

The Bcl-2 promoter contains a cAMP response element site (CRE) that can enhance Bcl-2 expression by binding the transcription factor CREB. Akt is known to phosphorylate CREB which results in increased CREB binding to CBP and increased transcriptional activity [198]. Therefore, the ability of Akt to promote cell survival is mediated, in part, by influence over gene expression such as the up-regulation of Bcl-2 [227-230] and through direct protein

**Akt and transcription factor regulation** Also under CREB transcriptional control is the neurotrophic factor BDNF [231, 232] which is essential for neuronal development, differen‐ tiation, synaptic plasticity, neuroprotection and restoration against a broad range of cellular insults [233]. BDNF has been a focus of AD research for its ability to stimulate non-amyloido‐ genic APP processing pathways [234, 235] in addition to protecting neuronal cultures against the cytotoxic effects of Aβ [236]. This indicates that decreased insulin signaling resulting in reduced BDNF production may be a contributing factor in AD development. In accordance, AD patients have decreased serum BDNF concentrations compared to healthy, elderly subjects [237-241] while reduced BDNF levels were associated with decreased cognitive performance

interactions such as Bad phosphorylation resulting in Bcl-xL liberation [194-197].

221]. Akt blocks apoptosome formation by phosphorylating Caspase 9 [193].

leads to survival while increased pro-death proteins result in apoptosis.

ing for mitochondrial stabilization.

are resistant to ROS generation [226].

in healthy individuals [242].

Structurally, the insulin receptor is a homodimer composed 2α chains and 2β chains held together with disulphide bonds [167-169]. Insulin receptor binding of insulin/IGF-1 results in a conformational change that activates the catalytic tyrosine kinase activity of the β subunits [170]. This activation of the insulin receptor results in autophosphorylation at multiple tyrosine residues [171, 172] including tyrosine 960 in the juxtamembrane region of the β subunit [173, 174]. Phosphorylation at this site is a vital component of the insulin signaling cascade because it provides a binding motif for the phospho-tyrosine binding (PTB) domain of IRS [173, 174]. Once docked to the insulin receptor, IRS is phosphorylated on tyrosine residues [170].

Tyrosine phosphorylation of IRS proteins creates binding sites for Src homology 2 (SH2) domain containing proteins such as PI3K [175]. PI3K catalyzes the production of 3'phosphoi‐ nositide secondary messengers which are critical to the insulin signaling cascade. PI3K is composed of a catalytic p110 subunit and a regulatory p85 subunit that contains SH2 domains that interact with activated IRS [176]. Formation of the IRS/PI3K complex increases the catalytic activity of the p110 subunit [177].

3'phosphoinositides produced by PI3K are important signal conductors that bind to PH (pleckstrin homology) domains on proteins such as IRS [177] and Akt [178]. This interaction is needed to bring IRS and AKT proteins towards the inner layer of the plasma membrane near the juxtamembrane region of the insulin receptor [179] and in close proximity to activating kinases, respectively [180-185]. Furthermore, binding of 3'phosphoinositides is required for Akt to be competent for phosphorylation [184, 186-188].

Akt has two phosphorylation sites, Thr 308 and Ser 473, capable of inducing catalytic activity [189]. PDK1, which also depends on 3'phosphoinosites for its function, phosphorylates Akt at Thr 308 [189, 190].While overexpression of PDK1 has been shown to activate Akt [186], optimal activation of Akt requires additional phosphorylation at Ser 473 by mTORC2 [191] which stabilizes the conformation state of Akt [192].

Akt mediates the neurotrophic effects of insulin/IGF-1, in part, by inhibiting pro-apoptotic machinery [193] and concomitantly activating anti-apoptotic proteins [194-198]. Akt's role in neurotrophic support also involves the regulation of survival transcription factors such as NFκB [199] and CREB [198] as well as those involved in pro-death gene expression such as the FoxO family [200-202]. Moreover, Akt is involved in production of the neurotrophin BDNF [198], activation of proteins involved in neurite outgrowth (for review see: [203]) and regula‐ tion of the metabolic protein GSK-3β [204].

**Akt and Bcl-2 family members** The Bcl-2 family is a structurally related group of proteins that regulate cell death through effects on the mitochondria [205] (for review see [206, 207]). Bcl-2 members include the pro-apoptotic proteins BID, BIM, PUMA, BAD, NOXA, BAX, and BAK [205] along with anti-apoptotic mediators such as Bcl-2 and Bcl-xL [205]. Because Bcl-2 proteins possess the ability to form heterodimers with one another [208-210], their regulation of apoptosis can be described as a balancing act in which an increase of anti-apoptotic members leads to survival while increased pro-death proteins result in apoptosis.

that IR-A has a higher affinity for the neurotrophic factor Insulin-like Growth Factor – 2 (IGF-II) [165] and a slightly higher affinity for insulin [166] and has also been shown to associate/ dissociate with insulin quicker than IR-B [149]. Brain specific insulin receptors are mainly the IR-A isoform and as result of differential glycosylation have a lower molecular weight than

Structurally, the insulin receptor is a homodimer composed 2α chains and 2β chains held together with disulphide bonds [167-169]. Insulin receptor binding of insulin/IGF-1 results in a conformational change that activates the catalytic tyrosine kinase activity of the β subunits [170]. This activation of the insulin receptor results in autophosphorylation at multiple tyrosine residues [171, 172] including tyrosine 960 in the juxtamembrane region of the β subunit [173, 174]. Phosphorylation at this site is a vital component of the insulin signaling cascade because it provides a binding motif for the phospho-tyrosine binding (PTB) domain of IRS [173, 174]. Once docked to the insulin receptor, IRS is phosphorylated on tyrosine residues [170].

Tyrosine phosphorylation of IRS proteins creates binding sites for Src homology 2 (SH2) domain containing proteins such as PI3K [175]. PI3K catalyzes the production of 3'phosphoi‐ nositide secondary messengers which are critical to the insulin signaling cascade. PI3K is composed of a catalytic p110 subunit and a regulatory p85 subunit that contains SH2 domains that interact with activated IRS [176]. Formation of the IRS/PI3K complex increases the catalytic

3'phosphoinositides produced by PI3K are important signal conductors that bind to PH (pleckstrin homology) domains on proteins such as IRS [177] and Akt [178]. This interaction is needed to bring IRS and AKT proteins towards the inner layer of the plasma membrane near the juxtamembrane region of the insulin receptor [179] and in close proximity to activating kinases, respectively [180-185]. Furthermore, binding of 3'phosphoinositides is required for

Akt has two phosphorylation sites, Thr 308 and Ser 473, capable of inducing catalytic activity [189]. PDK1, which also depends on 3'phosphoinosites for its function, phosphorylates Akt at Thr 308 [189, 190].While overexpression of PDK1 has been shown to activate Akt [186], optimal activation of Akt requires additional phosphorylation at Ser 473 by mTORC2 [191] which

Akt mediates the neurotrophic effects of insulin/IGF-1, in part, by inhibiting pro-apoptotic machinery [193] and concomitantly activating anti-apoptotic proteins [194-198]. Akt's role in neurotrophic support also involves the regulation of survival transcription factors such as NFκB [199] and CREB [198] as well as those involved in pro-death gene expression such as the FoxO family [200-202]. Moreover, Akt is involved in production of the neurotrophin BDNF [198], activation of proteins involved in neurite outgrowth (for review see: [203]) and regula‐

**Akt and Bcl-2 family members** The Bcl-2 family is a structurally related group of proteins that regulate cell death through effects on the mitochondria [205] (for review see [206, 207]). Bcl-2 members include the pro-apoptotic proteins BID, BIM, PUMA, BAD, NOXA, BAX, and BAK [205] along with anti-apoptotic mediators such as Bcl-2 and Bcl-xL [205]. Because Bcl-2 proteins

their peripheral counterparts [162].

418 Understanding Alzheimer's Disease

activity of the p110 subunit [177].

Akt to be competent for phosphorylation [184, 186-188].

stabilizes the conformation state of Akt [192].

tion of the metabolic protein GSK-3β [204].

Mitochondrial stress incurred by ROS can lead to elevated Ca2+ levels in the mitochondri‐ al matrix [211, 212] resulting in increased mitochondrial membrane permeability and re‐ lease of pro-apoptotic factors such as Cytochrome c, and AIF (apoptosis inducing factor) [213]. Bcl-xL is an anti-apoptotic Bcl-2 family member that prevents Ca2+ induced mito‐ chondrial permeability [214]. In the absence of insulin/IGF-1 stimulation, the survival ef‐ fects of Bcl-xL are blocked as Bcl-xL is complexed with the pro-death Bcl-2 family member Bad [215-217]. Akt liberates Bcl-xL by phosphorylating Bad [195-197, 218] allow‐ ing for mitochondrial stabilization.

Mitochondrial permeability marks a critical event in the cell death cascade. Akt promotes cell survival prior to Cytochrome c release through Bcl-xL activity but has also been found to act post apoptotic factor release. When Cytochrome c is released from the mitochondria, it will associate with Apaf-1, dATP and Caspase-9 forming a structure known as the apoptosome (For review see [219]). Formation of the apoptosome activates the proteolytic activity of caspase-9 which cleaves and activates other caspases critical to the apoptotic process [220, 221]. Akt blocks apoptosome formation by phosphorylating Caspase 9 [193].

Bcl-2 is another anti-apoptotic protein under the control of Akt [222]. Bcl-2's role in cell survival is similar to that of Bcl-xL in that in maintains mitochondrial membrane integri‐ ty [223]. Mitochondrial permeability has been linked to an oxidized shift in the mitochon‐ dria [224] while Bcl-2 has been shown to promote a more reduced state [225]. Upregulation of Bcl-2 may lead to higher cell reductive capacity [224] which is supported by the observation that Bcl-2 overexpressing cells show increased amounts of NADPH and are resistant to ROS generation [226].

The Bcl-2 promoter contains a cAMP response element site (CRE) that can enhance Bcl-2 expression by binding the transcription factor CREB. Akt is known to phosphorylate CREB which results in increased CREB binding to CBP and increased transcriptional activity [198]. Therefore, the ability of Akt to promote cell survival is mediated, in part, by influence over gene expression such as the up-regulation of Bcl-2 [227-230] and through direct protein interactions such as Bad phosphorylation resulting in Bcl-xL liberation [194-197].

**Akt and transcription factor regulation** Also under CREB transcriptional control is the neurotrophic factor BDNF [231, 232] which is essential for neuronal development, differen‐ tiation, synaptic plasticity, neuroprotection and restoration against a broad range of cellular insults [233]. BDNF has been a focus of AD research for its ability to stimulate non-amyloido‐ genic APP processing pathways [234, 235] in addition to protecting neuronal cultures against the cytotoxic effects of Aβ [236]. This indicates that decreased insulin signaling resulting in reduced BDNF production may be a contributing factor in AD development. In accordance, AD patients have decreased serum BDNF concentrations compared to healthy, elderly subjects [237-241] while reduced BDNF levels were associated with decreased cognitive performance in healthy individuals [242].

The transcription factor NF-κB is also under Akt control [199]. Like CREB, NF-κB plays critical roles in neuron survival [201, 243, 244] and is also involved in neurite outgrowth, myelin formation and axonal regeneration [245]. Genes for antioxidant proteins such as MnSOD [246] and Cu/ZnSOD [247] and anti-apoptotic proteins Bcl-2 and Bcl-xL are targets of NF-κB [248].

dementia in AD [286, 287]. IGF-1 protects neurons from ischemic damage by reducing GSK-3β activity [288] which implies a critical role of Akt in GSK- 3β regulation. Indeed, Akt has been shown to inhibit GSK-3β [204] thus demonstrating a direct role of insulin/IGF-1

Alzheimer's Disease and Diabetes http://dx.doi.org/10.5772/54913 421

**Loss of insulin signaling** While not a cause of death on its own, loss of insulin signaling in the brain leaves neurons vulnerable to a myriad of insults. Insulin signaling is known to protect against oxidative stress, mitochondrial collapse, over-activity of GSK-3β leading to hyperphosphorylation of tau, activation of death promoting transcription factors and forma‐ tion of apoptotic structures. Insulin also results in increased BDNF neurotrophic support as

The mitochondrial permeability transition mediates apoptosis through the release of pro apoptotic factors. Insulin signaling maintains mitochondrial membrane integrity by increas‐ ing levels and activity of anti-apoptotic Bcl-2 family members [194-197, 227-230]. In the ab‐ sence of insulin signaling, the balance of Bcl-2 proteins tips in favor of pro-apoptotic members resulting in cell death. Post mitochondrial collapse, normal insulin signaling can still prevent apoptosis by blocking formation of apoptotic complexes [193, 229] while a state

Even under normal circumstances, ROS are produced in respiratory chain reactions in the mitochondria [289]. However, if not properly managed, ROS can cause oxidative damage to proteins, lipids, and nucleic acids. Insulin supplies cells with antioxidant proteins capable of diffusing the oxidative effects of ROS by activating protective transcription factors such as NF-κB [246, 247, 263]. Insulin resistance not only results in reduced antioxidants but also leaves cells susceptible to ROS mediated mitochondrial collapse because of the before men‐

The FoxO family of transcription factors is known to play a role in the cell's response to oxi‐ dative stress, however, their prolonged activation results in apoptosis [290]. Insulin signal‐ ing inactivates FoxO transcription factors through phosphorylation by Akt. Absence of insulin signaling allows FoxO members to remain in the nucleus and sustain transcription of

Insulin resistance is linked to structural changes in AD by overactive GSK-3β. Neurofibril‐ lary tangles are a pathological hallmark of AD [283] and produced by hyperphosphorylation of tau by GSK-3β. Under normal insulin signaling, GSK-3β is inactivated by Akt. Neurofi‐ brillary tangles are one of two significant pathological characteristics of AD the other being accumulation of Aβ [291]. Aβ toxicity and aggregation into plaques has devastating conse‐ quences in the brain such as synaptic disruption [292] and inhibition of LTP [293], interfer‐ ence of detoxifying enzymes [294], increased ROS and oxidative stress [295], increased vulnerability to calcium overload [296] and the before mentioned effects on brain vascula‐ ture. Aβ also depresses insulin signaling [297] which results in further loss of neurotrophic support. Insulin signaling, on the other, hand is involved in Aβ clearance [298] introducing a

of insulin resistance allows this process to continue unimpeded.

signaling in the prevention of AD pathology.

well as increased neurite outgrowth.

tioned lack of anti-apoptotic Bcl-2 members.

convoluted relationship between insulin and Aβ.

pro-death genes [201, 255-257].

In its inactive form, NF-κB is bound to IκB proteins that sequester it to the cytosol (for review see [249, 250]). NF-κB is activated when IκB proteins are phosphorylated by IκB Kinase (IKK) complexes and targeted for degradation which allows NF-κB to translocate to the nucleus where it binds to regulatory DNA sequences [251]. The IKK complex consists of catalytic IKKα and IKKβ subunits and a regulatory IKKγ subunit [251]. Akt facilitates NF-κB activation by phosphorylating IKKα at a critical regulatory site that promotes IKK activation [252] and subsequent IκB degradation.

Akt influence is not limited to only survival transcription factors but extends to pro-death modulators as well [253, 254].The forkhead box class O (FoxO) family of transcription factors contribute to apoptosis through the induction of pro-death genes such as Fas L [201, 255, 256] and the Bcl-2 member BIM-1 [257]. Fas L facilitates apoptosis by activation of caspases [258] while BIM-1 activates the pro-apoptotic Bcl-2 family memeber BAX [259]. In the absence of Akt, FoxO transcription factors are transcriptionally active in the nucleus [200-202]. Akt phosphorylates FoxO family members at a conserved c-terminal sequence [253] which leads to nuclear exclusion and inhibition of transcriptional activity.

p53,another pro-death transcription factor known to be inactivated by Akt, [260] induces the expression of the pro-apoptotic Bcl-2 family member BAX. BAX proteins form oligomers that insert into the outer mitochondrial membrane which provide a passageway for Cytochrome c and other pro-apoptotic proteins to escape through [261]. Increased p53 activity leading to BAX expression has been linked to neuronal deprivation of neurotrophic factors [262].

**Akt and neurite outgrowth** Akt effects extend beyond apoptosis regulation as Akt also contributes to neurite outgrowth (for review see [203]). In hippocampal neurons Akt enhances characteristics such as dendritic length/complexity, caliber, and branching [263-267] with similar effects, excluding dendritic length, observed in dorsal root ganglia neurons [268-271]. Akt substrates implicated in neurite outgrowth include GSK-3β [272, 273], CREB [198], mTOR [274], peripherin [275], and β-catenin [276]. Akt may also work in conjunction with other pathways involved in neurite outgrowth. For example, Akt has been found to be complexed with Hsp-27 (heat shock protein) in spinal motor neurons following nerve injury [277] as well as in areas of regeneration following sciatic nerve axotomy [278].

**Akt and GSK-3β** Activity of the metabolic protein GSK-3β is also influenced by Akt. GSK-3β was originally identified for decreasing glycogen production through inhibition of glycogen synthase [272, 279-281]. However, GSK-3β is also involved in protein synthesis, cell proliferation/differentiation, microtubule dynamics, cell motility and apoptosis. Of particular interest, GSK-3β has also been shown to phosphorylate cytoskeletal associated tau proteins [282] which, in a diseased state, result in protein aggregates known as neurofibrillary tangles [283]. Neurofibrillary tangles have been linked to increased oxidative stress, mitochondrial dysfunction and apoptosis [284, 285] and are the most significant structural correlates of dementia in AD [286, 287]. IGF-1 protects neurons from ischemic damage by reducing GSK-3β activity [288] which implies a critical role of Akt in GSK- 3β regulation. Indeed, Akt has been shown to inhibit GSK-3β [204] thus demonstrating a direct role of insulin/IGF-1 signaling in the prevention of AD pathology.

The transcription factor NF-κB is also under Akt control [199]. Like CREB, NF-κB plays critical roles in neuron survival [201, 243, 244] and is also involved in neurite outgrowth, myelin formation and axonal regeneration [245]. Genes for antioxidant proteins such as MnSOD [246] and Cu/ZnSOD [247] and anti-apoptotic proteins Bcl-2 and Bcl-xL are targets of NF-κB [248].

In its inactive form, NF-κB is bound to IκB proteins that sequester it to the cytosol (for review see [249, 250]). NF-κB is activated when IκB proteins are phosphorylated by IκB Kinase (IKK) complexes and targeted for degradation which allows NF-κB to translocate to the nucleus where it binds to regulatory DNA sequences [251]. The IKK complex consists of catalytic IKKα and IKKβ subunits and a regulatory IKKγ subunit [251]. Akt facilitates NF-κB activation by phosphorylating IKKα at a critical regulatory site that promotes IKK activation [252] and

Akt influence is not limited to only survival transcription factors but extends to pro-death modulators as well [253, 254].The forkhead box class O (FoxO) family of transcription factors contribute to apoptosis through the induction of pro-death genes such as Fas L [201, 255, 256] and the Bcl-2 member BIM-1 [257]. Fas L facilitates apoptosis by activation of caspases [258] while BIM-1 activates the pro-apoptotic Bcl-2 family memeber BAX [259]. In the absence of Akt, FoxO transcription factors are transcriptionally active in the nucleus [200-202]. Akt phosphorylates FoxO family members at a conserved c-terminal sequence [253] which leads

p53,another pro-death transcription factor known to be inactivated by Akt, [260] induces the expression of the pro-apoptotic Bcl-2 family member BAX. BAX proteins form oligomers that insert into the outer mitochondrial membrane which provide a passageway for Cytochrome c and other pro-apoptotic proteins to escape through [261]. Increased p53 activity leading to BAX expression has been linked to neuronal deprivation of neurotrophic factors [262].

**Akt and neurite outgrowth** Akt effects extend beyond apoptosis regulation as Akt also contributes to neurite outgrowth (for review see [203]). In hippocampal neurons Akt enhances characteristics such as dendritic length/complexity, caliber, and branching [263-267] with similar effects, excluding dendritic length, observed in dorsal root ganglia neurons [268-271]. Akt substrates implicated in neurite outgrowth include GSK-3β [272, 273], CREB [198], mTOR [274], peripherin [275], and β-catenin [276]. Akt may also work in conjunction with other pathways involved in neurite outgrowth. For example, Akt has been found to be complexed with Hsp-27 (heat shock protein) in spinal motor neurons following nerve injury [277] as well

**Akt and GSK-3β** Activity of the metabolic protein GSK-3β is also influenced by Akt. GSK-3β was originally identified for decreasing glycogen production through inhibition of glycogen synthase [272, 279-281]. However, GSK-3β is also involved in protein synthesis, cell proliferation/differentiation, microtubule dynamics, cell motility and apoptosis. Of particular interest, GSK-3β has also been shown to phosphorylate cytoskeletal associated tau proteins [282] which, in a diseased state, result in protein aggregates known as neurofibrillary tangles [283]. Neurofibrillary tangles have been linked to increased oxidative stress, mitochondrial dysfunction and apoptosis [284, 285] and are the most significant structural correlates of

to nuclear exclusion and inhibition of transcriptional activity.

as in areas of regeneration following sciatic nerve axotomy [278].

subsequent IκB degradation.

420 Understanding Alzheimer's Disease

**Loss of insulin signaling** While not a cause of death on its own, loss of insulin signaling in the brain leaves neurons vulnerable to a myriad of insults. Insulin signaling is known to protect against oxidative stress, mitochondrial collapse, over-activity of GSK-3β leading to hyperphosphorylation of tau, activation of death promoting transcription factors and forma‐ tion of apoptotic structures. Insulin also results in increased BDNF neurotrophic support as well as increased neurite outgrowth.

The mitochondrial permeability transition mediates apoptosis through the release of pro apoptotic factors. Insulin signaling maintains mitochondrial membrane integrity by increas‐ ing levels and activity of anti-apoptotic Bcl-2 family members [194-197, 227-230]. In the ab‐ sence of insulin signaling, the balance of Bcl-2 proteins tips in favor of pro-apoptotic members resulting in cell death. Post mitochondrial collapse, normal insulin signaling can still prevent apoptosis by blocking formation of apoptotic complexes [193, 229] while a state of insulin resistance allows this process to continue unimpeded.

Even under normal circumstances, ROS are produced in respiratory chain reactions in the mitochondria [289]. However, if not properly managed, ROS can cause oxidative damage to proteins, lipids, and nucleic acids. Insulin supplies cells with antioxidant proteins capable of diffusing the oxidative effects of ROS by activating protective transcription factors such as NF-κB [246, 247, 263]. Insulin resistance not only results in reduced antioxidants but also leaves cells susceptible to ROS mediated mitochondrial collapse because of the before men‐ tioned lack of anti-apoptotic Bcl-2 members.

The FoxO family of transcription factors is known to play a role in the cell's response to oxi‐ dative stress, however, their prolonged activation results in apoptosis [290]. Insulin signal‐ ing inactivates FoxO transcription factors through phosphorylation by Akt. Absence of insulin signaling allows FoxO members to remain in the nucleus and sustain transcription of pro-death genes [201, 255-257].

Insulin resistance is linked to structural changes in AD by overactive GSK-3β. Neurofibril‐ lary tangles are a pathological hallmark of AD [283] and produced by hyperphosphorylation of tau by GSK-3β. Under normal insulin signaling, GSK-3β is inactivated by Akt. Neurofi‐ brillary tangles are one of two significant pathological characteristics of AD the other being accumulation of Aβ [291]. Aβ toxicity and aggregation into plaques has devastating conse‐ quences in the brain such as synaptic disruption [292] and inhibition of LTP [293], interfer‐ ence of detoxifying enzymes [294], increased ROS and oxidative stress [295], increased vulnerability to calcium overload [296] and the before mentioned effects on brain vascula‐ ture. Aβ also depresses insulin signaling [297] which results in further loss of neurotrophic support. Insulin signaling, on the other, hand is involved in Aβ clearance [298] introducing a convoluted relationship between insulin and Aβ.

**4. Generation of Aβ**

[324].

**Background** Aβ is a small peptide 38-43 amino acids in size long believed to have a major role in neurodegeneration and pathology of AD (for review see [299]). In sporadic AD (sAD), which accounts for over 90% of AD cases, Aβ's role in pathogensis is still under heavy investigation. The cause of familial AD (fAD), however, has been linked to 3 mutations involved in Aβ processing; presinilins 1 and 2 (PS1/PS2), which are part of Aβ producing complexes, and amyloid precursor protein (APP) from which Aβ is derived [300]. Successive cleavages of APP by β- and γ-secretases produce toxic Aβ peptides (for review see [301]) while cleavage by α-

Alzheimer's Disease and Diabetes http://dx.doi.org/10.5772/54913 423

While the physiological role of APP remains unknown, it has been suggested that APP plays a part in neurite outgrowth, synaptogenesis, neuronal trafficking along the axon, transmem‐ brane signal transduction, cell adhesion and calcium metabolism, all of which still require in vivo evidence (for review see [303]). APP concentrations are elevated in the brain during the prenatal period in mice which implies a role of APP in brain development [304]. In the adult brain, APP is expressed in regions of synaptic modification [304] and has been shown to

APP belongs to a family of transmembrane proteins that includes APP-like protein 1 and 2 (APPLP1/APPLP2). All APP family members are processed in a similar fashion by α, β, and γ secretases [306-308], however the Aβ domain is unique to APP. Three isoforms of APP have been identified consisting of 695, 751, or 770 amino acids which arise from alternative splicing of the same gene located on chromosome 21 [309]. APP 751 and APP 770 are expressed in most tissues and contain a 56 amino acid Kunitz Protease inhibitor (KPI) domain not found in the neuron specific 695 isoform [310, 311]. mRNA levels of the 2 KPI containing isoforms are

Synthesis of APP occurs in the endoplasmic reticulum where it is then transported through the golgi apparatus to the trans golgi network where the highest concentrations of APP are found in neurons [313-315]. From there, APP can be transported in secretory vesicles to the cell surface where α-secretases are located, however, Aβ production occurs within the trans

**APP cleavage** Aβ generation requires cleavage of APP by β-secretase which has been inden‐ tified to be BACE1 [319-322]. Several studies have found that regions of the brain affected by AD have elevated BACE1 activity and levels [319, 320]. Once identified, BACE1 became a popular therapeutic target for AD treatment. However, BACE1 knockout mice have shown reduced survivability after birth and were smaller than wild-type littermates [323]. BACE1 knockouts also present with hyperactive behavior [323] and other abnormalities such as hypomyelination of peripheral nerves, reduced grip strength and elevated pain sensitivity

APP cleavage by BACE1 results in two fragments: sAPPβ and Beta Carboxyl Terminal Fragment (βCTF) [301, 325]. sAPPβ has been identified as a ligand for Death Receptor 6 which mediates axonal pruning and neuronal death [326]. The remaining βCTF can be cleaved by

secretase produces the neuroprotective product Secreted APP alpha (sAPPα) [302].

increase hippocampal neuronal response to glutamate [305].

elevated in AD brains and are associated with Aβ deposition [312].

golgi network where γ-secretase complexes are thought to reside [315-318].

**Figure 2.** Insulin receptor binding of insulin triggers a complex signaling cascade (in blue) leading to activation of the serine/threonine kinase Akt. Upon binding of insulin, insulin receptors are autophophorylated and subsequently bind IRS proteins. IRS proteins are then phsophorylated by activated insulin receptors and complex with PI3K resulting in PI3K activation. Activated PI3K produces phospholipid secondary messengers by catalyzing the conversion of phos‐ phatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-trisphosphate (PIP3). PIP3 messengers activate PDK1 which phosphorylates Akt at Threonine 308. Akt is further activated by phosphorylation at Ser 473 by mammali‐ an target of rapamyicin 2 (mTORC2). Targets of activated Akt include pro-apoptotic mediators (in red) as well as prosurvival machinery (in green). Loss of insulin signaling (at sites labeled with numbers 1-6 in purple) allows FoxO and p53 transcription factors to remain active and (1) transcribe genes for pro-apoptotic proteins such as BIM, BAX and FasL. Akt inhibits the activity of GSK-3β that, when active, (2) causes increased amyloidogenic processing and hyper‐ phosphorylation of tau. Other pro-apoptotic proteins inhibited by Akt include (3) caspase-9, which forms an apoptotic structure known as the apoptosome, and (6) Bad, which blocks activity of the ant-apoptotic protein Bcl-xL. Pro-survival modulators regulated by Akt include CREB and NF-κB. Reduction of CREB transcriptional activity as a result of a loss of insulin signaling leads to (4) decreased BDNF and Bcl-2 expression while inhibition of NF-κB leads to (5) reduced ex‐ pression of anti-oxidants such as MnSOD and CuSOD as well as anti-apoptotic Bcl-2 family members.
