Biology

Vita

Carsten Rupp - Diplom-Biologe

FB Biologie, Humanbiologie und Humangenetik; AG Kins, TU Kaiserslautern
Erwin-Schrödinger-Straße
67663 Kaiserslautern

Phone: +49 (0)631 205 4724
rupp@nar.uni-heidelberg.de

Fellows: Prof. Dr. Stefan Kins

The pathophysiological function of APP in Alzheimer’s Disease

A broad range of neurodegenerative disorders is characterized by neuronal damage that may be caused by toxic, aggregate-prone proteins (Taylor et al., 2002). Neurodegenerative diseases typically involving such a disease pattern include Parkinson’s disease (PD), motor neuron diseases like amyotrophic lateral sclerosis (ALS), polyglutamine diseases which includes Huntington’s disease (HD), prion diseases like Creutzfeldt-Jacob’s disease (CJD), frontotemporal dementia and Alzheimer’s disease (AD), which is the most common form of dementia in the elderly.

In all of these diseases, characteristic deposits of protein aggregates (for review see (Ross and Poirier, 2005)) are found in the brain, which can be cytoplasmic, nuclear or extracellular. The common characteristics of these neurodegenerative disorders suggests parallel approaches for treatment, based on an understanding of the cellular mechanisms for disposing of unwanted and potentially noxious proteins or protein aggregates. The most relevant finding for molecular biology research in AD was the identification of the main constituent of the frequent, roughly spherical, extracellular depositions in AD brains, called Amyloid plaques. These are primarily composed of amyloid fibrils, which are highly insoluble aggregations of a protein fragment with 40- 42 amino acids termed Amyloid-β (Aβ40-42) originating from the Amyloid Precursor Protein, APP (Glenner and Wong, 1984; Masters et al., 1985).

Moreover, numerous studies found different physical forms of soluble Aβ, including Aβ42, Aβ40 and also longer or shorter versions to be toxic to neurons and to induce apoptosis (for review see (Cleary et al., 2005; Walsh et al., 2005)). Although some researchers now believe that oligomeric Aβ42 is the crucial agent causing the disease (Selkoe, 2002b), it is still unclear which physical forms of Aβ are pathological relevant to the Alzheimer neurodegenerative process. Thus the precise molecular mechanism leading to AD is still unknown.

APP is a ubiquitously expressed type I transmembrane protein with a large ectodomain and a short cytoplasmic tail (Kang et al., 1987), thus resembling a ubiquitously expressed cell surface receptor. The encoding gene is part of a large multi-gene family. In mammals, two paralogues of APP have been identified called Amyloid Precursorlike Proteins, APLP1 and APLP2 (Paliga et al., 1997; Wasco et al., 1993; Wasco et al., 1992). All APP homologues are type I transmembrane proteins and share high sequence homology and similar protein domain organization with APP (Bayer et al., 1999; Coulson et al., 2000).

The major proteases involved in APP cleavage are α-, β- and γ-secretase,and cleavage by all three proteases occurs under physiological as well as pathological conditions (Haass et al., 1992; Seubert et al., 1993). In the amyloidogenic pathway, APP is processed by β-secretase BACE1 (β-site APP cleaving enzyme 1) (Hussain et al., 1999; Sinha et al., 1999; Vassar et al., 1999; Yan et al., 1999), resulting in the release of the soluble β-cleaved ectodomain (sAPPβ). Subsequently, γ-secretase cleaves the remaining C-terminal fragment of APP (β-CTF, C99) within the transmembrane region (Weidemann et al., 2002), thus setting free the Aβ-peptides ending at Val40 or Ala42 (numbering of C99) and the APP intracellular domain (AICD). Whether APP is first cleaved by α- or β-secretase, strongly depends on its subcellular localization. Besides the Golgi apparatus, the plasma membrane is one of the two major sites where α-secretase cleavage takes place (Lammich et al., 1999; Skovronsky et al., 2000). In contrast, BACE1 is most active at mildly acidic pH (pH 4.5) (Vassar et al., 1999), making endosomes the most likely location for BACE1 cleavage of APP. Moreover, BACE1 has been reported to be cycling between the cell surface and endosomes (Huse et al., 2000). While the occurrence of APP processing in the secretory pathway is discussed controversially, internalization of APP and trafficking through the endocytic pathway is well accepted to be an important step in APP proteolysis (Koo and Squazzo, 1994; LeBlanc and Gambetti, 1994; Perez et al., 1999) The most notable feature of the short intracellular domain of all three APP family members is a common, highly conserved YENPTY sequence. This sequence is in particular important since it has been demonstrated to be essential for efficient clathrin dependent endocytosis of APP (Koo and Squazzo, 1994; Perez et al., 1999) (Lai et al., 1995), and it is required for binding a number of intracellular key proteins In addition to the YENPTY sequence, a basolateral sorting sequence (BaSS) has been identified in the juxtamembrane region of AICD (Haass et al., 1995). In epithelial cells the tyrosine based BaSS was reported to be essential for normal sorting of APP-holoprotein to the basolateral surface (Haass et al., 1995; Lai et al., 1998; Lai et al., 1995; Tienari et al., 1996). Further the BaSS has also been reported to be involved in APP endocytosis (Lai et al., 1995).

The aim of this PhD work is to investigate the functionality of the postulated sorting signals in relation to APP sorting and endocytosis. For this purpose APP, APLP1 andAPLP2 mutants lacking different regions of interest have to be generated and their subcellular localization will be investigated in cell culture. To elucidate a role of the putative sorting signals in APP endocytosis an antibody uptake assay will be used. We want to get a better understanding of the sorting and endocytosis of APP to discover potential sites of interest that could be helpful for the pharmacological treatment of Alzheimer’s disease.

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