Research reportSubcellular localization of presenilin 2 endoproteolytic C-terminal fragments
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.
Section snippets
Mammalian cells
H4 human neuroglioma cells transfected with wild-type or N141I PS2 mutant pUHD constructs that allowed for tetracycline repressible promoter regulation of protein production were generated as described and tested for inducible expression of PS2 and constitutive expression by Western blot analyses.
Subcellular fractionation
H4 neuroglioma cells at 80% confluency were induced to produce PS2 for 24 h, then fractionated and separated on 10–28% Iodixinol (Nycomed Optiprep™, Gibco BRL) density gradients optimized for ER and
Results
The fact that the generation of a PS2 caspase derived 20 kDa CTF is modulated by phosphorylation, suggests that this process is a regulated physiologic event. Cultured cells do not contain readily detectable levels of this fragment unless they are transfected with PS2 or unless pharmacologic agents geared towards apoptotic induction are employed. Therefore, inducible PS2 expression and subcellular fractionation, methods that enrich low abundance molecules, were used to precisely localize the
Discussion
Cells that express FAD mutant forms of PS2 produce increased levels of a 20-kDa caspase CTF relative to the levels of a 25 kDa constitutive CTF [20]. This difference in PS2 CTF levels is one of the few documented effects of FAD mutations on the presenilins themselves. Because the enzyme responsible for the cleavage that produces the 20 kDa CTF is caspase-3, it is clear that this cleavage is associated with apoptotic pathways. However, the fact that the phosphorylation of PS2 modulates the
Uncited references
[1]; [11]; [12]; [22]; [47]
Acknowledgements
We thank Nikhat Zaidi, Eun-Kyoung Choi, Rob Moir, Tae-Wan Kim, Suzanne Guenette and Rudolph Tanzi, for constructive discussions and Dennis Selkoe for cell lines. This work was supported by grants from the NIA (TLT; F32 AG05817-02), the NINDS (WW; NS35978) and the Alzheimer’s Association (WW). WW is a Pew Biomedical Foundation Scholar and TLT is a John Douglas French Alzheimer’s Foundation fellowship recipient.
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