Subcellular localization of presenilin 2 endoproteolytic C-terminal fragments

Abstract

Mutations in the genes that encode the presenilin 1 and 2 (PS1 and PS2) proteins cause the majority of familial Alzheimer’s disease (FAD). Differential cleavage of the presenilins results in a generation of at least two C-terminal fragments (CTFs). An increase in the smaller of these two CTFs is one of the few changes in presenilin processing associated with FAD mutations in both PS1 and PS2. Interestingly, the phosphorylation of PS2 modulates the production of the smaller, caspase-derived PS2 CTF, which indicates that the generation of this fragment is a regulated, physiologic event. To date, there is no data concerning the subcellular distribution of the caspase-derived PS2 CTF. Because this fragment is normally present at levels that are difficult to detect, we have used cell lines in which the production of wild-type or N141I mutant PS2 is controlled by a tetracycline-regulated promoter in order to assess the subcellular localization of the caspase CTF in relation to the larger, constitutive PS2 CTF and to PS2 holoprotein. We have found that when levels of PS2 are low, the constitutive CTF colocalizes with markers consistent with localization in the early Golgi–ER-Golgi intermediate compartment (ERGIC) while the caspase CTF colocalizes with markers for the endoplasmic reticulum (ER). Following induction of wild-type or mutant PS2, when the levels of PS2 are high, the primary localization of the constitutive CTF appears to shift from the early Golgi–ERGIC in addition to the ER. Interestingly, while the induction of wild-type PS2 resulted in the localization of the caspase CTF primarily in the ER, the induction of mutant PS2 resulted in the localization of the caspase CTF to both the ER and the early Golgi–ERGIC. In summary, these data suggest that the two presenilin 2 CTFs have different patterns of subcellular localization and that the N141I PS2 mutation alters the localization pattern of the PS2 caspase fragment.

Introduction

Although mutations in presenilin-1 (PS1), presenilin-2 (PS2) and the amyloid precursor protein (APP) account for the majority of early-onset familial Alzheimer’s disease (FAD) [31], [24], [38] the pathogenic mechanisms of these mutations remain unclear [44]. The presenilins are multi-transmembrane domain proteins that are situated primarily in the membranes of the endoplasmic reticulum (ER) and Golgi [9], [21], [29], [45]. There have also been reports of presenilin localization to the cell surface [5], and to clathrin-coated vesicles [8], the nucleus [25] and aggresomes [18]. APP is the precursor of the 42 amino acid amyloid β-peptide (Aβ42) that is the major component of the extracellular amyloid plaques that are present in the AD brain. Aβ42 is produced by the coincident actions of β- [14], [26], [43] and γ-secretases on APP (for review see Ref. [36]). The rough ER, the ER-Golgi intermediate compartment (ERGIC), and the trans-Golgi network (TGN) are all believed to serve as major Aβ production sites [3], [16]

PS1 and PS2 undergo endoproteolytic processing which results in the production of fragments [19], [39], [41], [42] that exist as a stable complex [2], [35], [42]. Both proteins can be cleaved at two sites and this differential cleavage results in the production of two different sized C-terminal fragments (CTFs). For PS1 these CTFs migrate at 14 and 18–20 kDa, while for PS2 they migrate at 20 and 25 kDa. Interestingly, increased levels of the smaller PS2 CTF, which is normally present at very low levels, have been associated with the N141I Volga German mutation in PS2 and the M146L mutation in PS1, and the enzyme responsible for the production of the smaller fragment has been demonstrated to be caspase-3 [13], [20], [27]. Notably, the observation that production of the PS2 caspase CTF is regulated by the phosphorylation of PS2 [44] indicates that that the generation of this fragment is a regulated, physiologic event that occurs in the absence of the FAD mutations in the presenilins.

To date, there are no reports concerning either the subcellular distribution of the caspase PS2 CTF in relation to the constitutive CTF or to PS2 holoprotein, or how the localization of these fragments are influenced by the PS2 N141I mutation. Because the caspase fragment is typically present at low levels, we have used inducible PS2 expression and subcellular fractionation, methods that each enrich for the low abundance PS2 CTF, to precisely identify the organelle(s) that this fragment is associated with. Use of this system has also allowed us to assess the effect that overproduction of PS2 has on the localization of the fragments.