Troubleshooting methods for APP processing in vitro

Abstract

Introduction

The amyloid hypothesis states that Aβ is the main trigger for Alzheimer's disease. This report is focused in the study of the processing of the Amyloid Precursor Protein (APP) as a procedure to investigate the molecular mechanisms that may result in changes in the levels of Aβ.

Methods

Here we analyse different methodologies for Aβ determination, soluble APP, APP-Carboxy terminus fragments (CTFs) and enzymes for synthesis (secretases) and degradation of Aβ. In addition the advantages and disadvantages of different methodologies are discussed.

Discussion

The potential value of these procedures is described in the context of the function of APP and the different fragments derived from its cleavage.

Introduction

Alzheimer's disease (AD) is a neurodegenerative disorder whose main pathological hallmarks are the presence of neuritic plaques, of β-amyloid peptide (Aβ), and neurofibrillary tangles, of hyperphosphorylated Tau. Although there is still no clear connection between both of these cerebral lesions, a great deal of evidence suggests that Aβ deposition could be the primary event. This hypothesis is known as the “amyloid hypothesis” (Walsh and Selkoe, 2004). Therefore, many of the studies related to AD have focused their attention on the regulation of Aβ generation.

Aβ is generated from the cleavage of a larger protein called Amyloid Precursor Protein (APP), which is alternatively cut by two enzymes called β-secretase also called BACE (from beta-APP cleaving enzyme) and γ-secretase. Alternatively, Aβ production can be eluded if APP is cleaved by an α-secretase (see reviews, Jacobsen & Iverfeldt, 2009, Sastre et al., 2008). Several proteins from the ADAM family have been implicated in this α-secretase cleavage, such as ADAM10 and ADAM17 (see review Sastre et al., 2008). The secretases are located in different cellular compartments. α-secretase cleavage and some γ-secretase cleavage of APP take place at the plasma membrane, while β-secretase activity is mainly localized in late endosomes. Therefore, a way to regulate Aβ generation is by modifying APP subcellular distribution. APP is processed through the secretory pathway, during which it is glycosylated and phosphorylated. APP can be also ubiquitinated and degraded by the proteasome (see review Jacobsen and Iverfeldt, 2009).

There are several forms of Aβ but the most common are Aβ1-40 and Aβ1-42, the latter being the most toxic and fibrillogenic. Aβ is physiologically secreted and can be found in physiological fluids such as blood and CSF as well as in tissue culture media from various cell types. In addition, there have been a large number of studies on post-mortem AD, Down syndrome and transgenic mouse brains which have revealed the presence of intracellular Aβ within neurons (see review LaFerla, Green, & Oddo, 2007), most of which has been found to be of the 1–42 subtype, and not 1–40 (Gouras et al., 2000). Furthermore, immunogold electron microscopy has been carried out to demonstrate that Aβ42 can be found in multivesicular bodies (MVBs) of neurons in the human brain, where it is associated with synaptic pathology (Takahashi et al., 2002). Recently longer intracellular Aβ forms have been detected, which seem to be the precursors of Aβ40 and 42, such as Aβ43, Aβ45, Aβ46, and Aβ48 (Qi-Takahara et al., 2005).

As seen in Fig. 1A, proteolysis of APP by α-secretase or β-secretase leads to the secretion of α-APPs (which has neurotrophic properties) or β-APPs respectively. Both secretases generate C-terminal fragments of 10 kDa and 12 kDa respectively, which are localized within the membrane (see review Walter, Kaether, Steiner, & Haass, 2001) (Fig. 1A). These fragments can be cut by the γ-secretase to release the peptides P3 and Aβ as well as a cytoplasmic fragment identified as the APP intracellular domain (AICD). This site is similar to the S3 cleavage site for Notch and was named ε-site (Weidemann et al., 2002). Recently, a new cleavage site was described for γ-secretase (Sastre et al., 2001). The ξ-cleavage occurs between the γ- and ε-cleavage sites and generates longer Aβ forms within cells and in the brain, including Aβ43, Aβ45, Aβ46, and Aβ48 (Qi-Takahara et al., 2005, Zhao et al., 2004).

Aβ can be degraded by different systems, which include enzymatic degradation or receptor-mediated clearance (Tanzi, Moir, & Wagner, 2004). Several peptidases have been identified, which are able to degrade soluble Aβ, such as the Insulin degrading enzyme (IDE) and neprylisin. In addition, Aβ can be cleared via phagocytosis by activated microglia and by transport through the blood–brain barrier. This process can be enhanced by binding to chaperons such as ApoE and α2-macroglobulin.

Therefore, studying APP processing provides information about the mechanisms by which Aβ levels are modified by a factor or treatment. In this paper, we give some tips how to analyse the processing of APP, using different methodology.