**3. Physiological function of amyloid precursor protein**

Although APP is a part of the pathophysiological processes involved in AD, it is clear that the protein also carries out several natural physiological functions, particularly within the regulation of the synaptic transmission. It has been proved that transgenic mice with knock-out gene for APP exhibited an inability to transmit signals to the neuromuscular junction. Despite this fact, mice with upregulated expression of APP show better cognitive functions and spatial orientation. This is often rationalized by overproduction of AICD given by γ-secretase. The activity of APP is also put in a close connection with the metabolism of cholesterol. The neuroprotective character of APP was also demonstrated by suppression of the cyclin-dependent kinase 5 (CDK-5) activity in the process of tau hyperhosphorylation [9].

## **4. Pathological features of amyloid precursor protein**

The pathological role of APP is generally associated with the amyloidogenic way of its splitting. In general, many mutations of APP cause the autosomal dominant form of AD with early onset. Interestingly, genetic mutations in the adjacent part of the β-site of the APP gene induce neuroprotective effects, because Aβ is then produced only in a small extent. On the other hand, an excessive expression of the mutated APP forms associated with FAD (a redox cofactor in a number of biochemical reactions) leads to a loss of sense of smell, without dissemination of amyloid plaques, though. This observation is in a line with the loss of sense of smell, which occurs in some patients in the early stages of AD [9].

#### **5. β-secretase**

**Figure 1.** Scheme of the amyloidogenic processing of APP.

the developed AD [3].

**2. Amyloid precursor protein**

2 Alzheimer's Disease - The 21st Century Challenge

beta-amyloid (Aβ) [2]. Among all these theories, the amyloid metabolic cascade or the amyloid hypothesis and posttranslational modification of tau protein are considered as the main pathophysiological theories elucidating the outbreak of AD, although none of them is able to sufficiently explain the diversity of the biochemical and pathological abnormalities associated with

According to the amyloid hypothesis, slow accumulation of extracellular senile plaques, composed of Aβ deposits, occurs in the beginning and further progresses into AD. On the other hand, a direct link between the toxic influence of Aβ, the impaired neuronal functions and the decline in memory functions still has not been fully clarified, but it is broadly accepted that

Amyloid precursor protein (APP) is an integral membrane glycoprotein that is expressed in the brain and the central nervous system (CNS). APP can be cleaved by specific proteases in two different pathways: α-path and β-path [5]. In most cases, APP is cleaved in the α-path with the participation of enzymes α- and γ-secretases. The cleavage of APP by α-secretase proceeds in the way, which can be described as non-amyloidogenic one, while the cleavage in the β-way leads to formation of the toxic fragments of Aβ. In the case the non-amyloidogenic path, APP

Aβ undoubtedly plays a key role in the neuropathology of AD [4].

β-secretase (BACE-1; also referred to as Asp2 or memapsin 2) is an enzyme that breaks down APP in the site called β into the *C*-terminal fragment, from which monomers of Aβ are subsequently formed in the neurons. BACE-1 and the homologous BACE-2 are regulated differently and also control different processes. A disrupted intracellular calcium homeostasis may stimulate the genetic expression of BACE-1 *via* triggering the nuclear factor of activated T-cells of type 1 (NFAT1), which leads to over-production of Aβ. Expression of the BACE-1 can also be controlled by the level of Aβ1–42 (but not by the Aβ1–40) through some transcription factors. In addition, some plaques containing Aβ1–42 even increase the levels of BACE-1 in the adjacent neurons just before their death [10]. The homologous enzyme BACE-2 shares 64% of the sequence identity with BACE-1. The action of BACE-2 is in many aspects similar to the activity of α-secretase. BACE-2 triggers a cascade of cleavage of APP by the non-amyloidogenic way. Its physiological function is associated with the organ pigmentation [11].

BACE-2 knockout (−/−) mice showed the same phenotype. Double-knockout mice, that is, mice with deactivated genes for BACE-1 (−/−) as well as for BACE-2 (−/−), are not phenotypically very different from mice without the gene for the BACE-1, with the exception of an increased number of dying mice freshly after birth. The results of this study therefore assume that nonselective inhibitors of both subtypes of the enzyme BACE may be well tolerated at least from the perspective of the inhibition of BACE-2. The latest research has shown that BACE-2 is expressed in the pancreatic β cells and BACE-2 knockout mice exhibit an improved glycemic regulation due to the increased production of insulin. These findings imply the possible use of BACE-2 inhibitors

Amyloid Beta Hypothesis: Attention to β- and γ-Secretase Modulators

http://dx.doi.org/10.5772/intechopen.75629

5

Currently, BACE-1 inhibitors have an exclusive position regarding the therapeutic options for introduction into clinical practice to treat AD [16]. Their mechanism of action is based on reducing the levels of Aβ in the brain. Although several of these inhibitors had already reached clinical testing, there are still important questions to answer, for instance, about their safety, the optimum degree of inhibition of BACE-1 needed to achieve the desired therapeutic effect without the presence of side effects, and the stage of the disease when these compounds

Aβ is produced by neurons in the brain, partly also by astrocytes and other glial cells, which are involved in the formation of this protein in particular during the stress conditions accompanying the AD development. For the production of Aβ, the activity of both enzymes, BACE-1 and γ-secretase, is necessary [10]. The biochemical processes involving the activity of these enzymes are often referred in the literature as the amyloid pathway. Importantly, modulation or inhibition of these enzymes can reduce the formation of Aβ in the brain of patients with AD. On the other hand, activation of the non-amyloidogenic pathway by supporting the α-secretase activity may also reduce the formation of Aβ and currently it is alternatively considered as a promising approach for therapy of AD. An important role for the accumulation of Aβ is also played by the genetic aspects of AD. Nowadays, more than 200 autosomal-dominant mutations in APP and presenilin (PS) have been identified which contribute to the occurrence of familial forms of AD [18]. Without any exception, all these mutations increase the production of all Aβ isoforms, in particular the toxic Aβ containing 42 amino acids (Aβ1–42). An example might be seen in Swedish mutation of APP in the amino acids Lys670 and Met671, that is, the places where BACE-1 enzyme cleaves APP. This mutation results in higher proteolytic efficacy of BACE-1, which promotes an increased rate of the C99 fragment formation and thereby the total production of Aβ [19]. The *APOE-ε4* allele represents a major genetic risk factor for the development of AD with the late onset and it is also associated with an increased production and accumulation of Aβ. Similarly, mutation of ADAM10 metaloprotein, which is endowed with physiologically similar activity to that of α-secretase in neurons, causes the late onset of the AD by suppressing this enzyme activity, while the amyloidogenic cleavage of APP by BACE-1 prevails [20]. Recently, at least five different genes whose mutation contributes significantly to the increased formation of Aβ have been identified. Based on all

for the treatment of diabetes mellitus of type 2 [15].

**6. BACE-1 inhibitors in the treatment of Alzheimer's disease**

are to be indicated in order to achieve the greatest assets [17].

In order to clearly demonstrate the involvement of BACE-1 in the pathogenesis of AD, many prominent scientific groups worldwide dealt with developing a mouse model that had deactivated the gene for the production of BACE-1 (i.e., BACE-1 knockout (−/−) mice). At first, these strains of mice were viable, capable of reproduction, with the normal morphology of the body, without any obvious signs of damage of the tissues and normal blood picture [12]. This finding supported the idea that inhibition of BACE-1 can bring about the desired therapeutic effect without adverse effects. The results of this study also point to the fact that the related BACE-2 fails to offset the activity of BACE-1 in the formation of Aβ. It is interesting that hybridization of these BACE-1 knockout (−/−) mice with transgenic mice having the APP gene, which increasingly produce amyloid plaques, provided a generation, the newly born individuals of which did not exhibit the formation of Aβ, Aβ deposits or signs of memory impairment caused by production/accumulation of Aβ. As already mentioned, BACE-1 is located mostly in the presynaptic endings of neurons, where its physiological effects is assumed to occur. Over time, however, it was found that BACE-1 knockout (−/−) mice had impaired axonal conduction, experiencing hypomyelinization (i.e., disrupted formation of myelin, the substance that surrounds the axons and nerve fibers), memory disorders, disturbed neurochemical balance, pathological neurogenesis, astrogenesis, degeneration of neurons with increasing age, pathological changes in the retina and schizophrenic symptoms. All these discoveries observed in BACE-1 knockout (−/−) mice can serve as a model that reflects the potential adverse effects associated with the administration of BACE-1 inhibitors for normal animals or people [13].

The substrates subject to proteolysis by BACE-1 are in particular the membrane-bound proteins like APP. Many of these BACE-1 substrates undergo a process called ectodomain shedding (ectodomain is a part of a membrane protein which protrudes to the extracellular space), while at the same time, these substrates can be cleaved by proteases, called also disintegrins, and ADAM-related metaloproteases. The extent of cleavage of the substrate by ADAM related proteases or BACE-1 depends on the nature of the particular substrate. All the possible side effects caused by inhibition of BACE-1 thus may not be always exhibited, assuming that some substrates are hydrolyzed by another protease [14].

The homology between BACE-1 and BACE-2 gave rise to arguments that BACE-1 inhibitors may simultaneously inhibit non-selectively also BACE-2. For this reason, transgenic BACE-2 knockout (−/−) mice were developed to clarify the physiological role of BACE-2 and to explore the benefits offered by inhibition of this enzyme. Similar to the BACE-1 knockout (−/−) mice, the BACE-2 knockout (−/−) mice showed the same phenotype. Double-knockout mice, that is, mice with deactivated genes for BACE-1 (−/−) as well as for BACE-2 (−/−), are not phenotypically very different from mice without the gene for the BACE-1, with the exception of an increased number of dying mice freshly after birth. The results of this study therefore assume that nonselective inhibitors of both subtypes of the enzyme BACE may be well tolerated at least from the perspective of the inhibition of BACE-2. The latest research has shown that BACE-2 is expressed in the pancreatic β cells and BACE-2 knockout mice exhibit an improved glycemic regulation due to the increased production of insulin. These findings imply the possible use of BACE-2 inhibitors for the treatment of diabetes mellitus of type 2 [15].
