Annat F. Ikin

Regulation of beta-amyloid secretion by FE65, an amyloid protein precursor-binding protein.

The principal component of Alzheimer’s amyloid plaques, Aβ, derives from proteolytic processing of the Alzheimer’s amyloid protein precursor (APP). FE65 is a brain-enriched protein that binds to APP. Although several laboratories have characterized the APP-FE65 interaction in vitro, the possible relevance of this interaction to Alzheimer’s disease has remained unclear. We demonstrate here that APP and FE65 co-localize in the endoplasmic reticulum/Golgi and possibly in endosomes. Moreover, FE65 increases translocation of APP to the cell surface, as well as both αAPPs and Aβ secretion. The dramatic (4-fold) FE65-dependent increase in Aβ secretion suggests that agents which inhibit the interaction of FE65 with APP might reduce Aβ secretion in the brain and therefore be useful for preventing or slowing amyloid plaque formation.

The Carboxyl-Terminus of BACE Contains a Sorting Signal That Regulates BACE Trafficking but Not the Formation of Total Aβ

BACE (beta-site APP cleaving enzyme) has been recently proposed as the major aspartyl protease displaying beta secretase activity in neurons. The C-terminal domain of BACE contains a dileucine motif (LL499/500) that can potentially regulate its trafficking and endocytosis, and an adjacent serine, which is a potential phosphorylation site (S498) that could modulate the activity of the LL motif. In this paper we show that S498 is phosphorylated by casein kinase 1 (CKI). Mutating the LL to dialanine (AA) caused an increase in the levels of mature BACE. The LL to AA mutation increased levels of BACE on the cell surface and decreased the internalization of BACE. Mutating the S498 to alanine did not alter levels of cell surface BACE. Mutating either the leucines or the serine did not alter the secretion of A(beta). Our data are consistent with a role for the cytoplasmic domain in regulating BACE trafficking and localization.

Alzheimer Amyloid Protein Precursor Is Localized in Nerve Terminal Preparations to Rab5-containing Vesicular Organelles Distinct from Those Implicated in the Synaptic Vesicle Pathway

In order to localize amyloid protein precursor (APP) in nerve terminals, we have immunoisolated vesicular organelles from nerve terminal preparations using antibodies to Rab5 and synaptophysin. These immunoisolates were then analyzed by electron microscopy and by immunoblotting. The synaptophysin immunoisolates represented a nearly homogeneous population of small synaptic vesicles, with less than 10% contamination by other organelles, and very little APP. In contrast, Rab5 immunoisolates contained, in addition to small synaptic vesicles, substantial numbers of large uni- and bilamellar vesicles and high levels of APP. Thus, it appears that nerve terminal APP is contained predominantly in large vesicular organelles, distinct from synaptic vesicles and from the synaptic vesicle recycling pathway.

A macromolecular complex involving the amyloid precursor protein (APP) and the cytosolic adapter FE65 is a negative regulator of axon branching.

Several studies suggest a role for the amyloid precursor protein (APP) in neurite outgrowth and synaptogenesis, but the downstream interactions that mediate the function of APP during neuron development are unknown. By introducing interaction-deficient FE65 into cultured hippocampal neurons using adenovirus, we show that a complex including APP, FE65 and an additional protein is involved in neurite outgrowth at early stages of neuronal development. Both FE65 that is unable to interact with APP (PID2 mutants) or a WW mutant increased axon branching. Although the FE65 mutants did not affect total neurite output, both mutants decreased axon segment length, consistent with an overall slowing of axonal growth cones. FE65 mutants did not alter the localization of either APP or FE65 in axonal growth cones, suggesting that the effects on neurite outgrowth are achieved by alterations in local complex formation within the axonal growth cone.

Alzheimer's presenilin 1 modulates sorting of APP and its carboxyl-terminal fragments in cerebral neurons in vivo

Studies in continuously cultured cells have established that familial Alzheimer’s disease (FAD) mutant presenilin 1 (PS1) delays exit of the amyloid precursor protein (APP) from the trans‐Golgi network (TGN). Here we report the first description of PS1‐regulated APP trafficking in cerebral neurons in culture and in vivo. Using neurons from transgenic mice or a cell‐free APP transport vesicle biogenesis system derived from the TGN of those neurons, we demonstrated that knocking‐in an FAD‐associated mutant PS1 transgene was associated with delayed kinetics of APP arrival at the cell surface. Apparently, this delay was at least partially attributable to impaired exit of APP from the TGN, which was documented in the cell‐free APP transport vesicle biogenesis assay. To extend the study to APP and carboxyl terminal fragment (CTF) trafficking to cerebral neurons in vivo, we performed subcellular fractionation of brains from APP transgenic mice, some of which carried a second transgene encoding an FAD‐associated mutant form of PS1. The presence of the FAD mutant PS1 was associated with a slight shift in the subcellular localization of both holoAPP and APP CTFs toward iodixanol density gradient fractions that were enriched in a marker for the TGN. In a parallel set of experiments, we used an APP : furin chimeric protein strategy to test the effect of artificially forcing TGN concentration of an APP : furin chimera that could be a substrate for β‐ and γ‐cleavage. This chimeric substrate generated excess Aβ42 when compared with wildtype APP. These data indicate that the presence of an FAD‐associated mutant human PS1 transgene is associated with redistribution of the APP and APP CTFs in brain neurons toward TGN‐enriched fractions. The chimera experiment suggests that TGN‐enrichment of a β‐/γ‐secretase substrate may play an integral role in the action of mutant PS1 to elevate brain levels of Aβ42.

App Localization and Trafficking in the Central Nervous System

One of the hallmarks of Alzheimer’s disease (AD) pathology is amyloid plaque deposition in the brain (reviewed in Sisodia and Price, 1995). The amyloid plaque core consists primarily of a 4 kDa peptide known as β-amyloid or Aβ. Aβ is derived by proteolytic processing of a type I integral membrane protein, the amyloid Aβ protein precursor, or APP. Based on strong genetic and biochemical data (reviewed in Hardy and Duff, 1993), it is widely agreed that at least some forms of AD are caused by excess Aβ deposition in the brain, particularly excess Aβ of 42 or 43 amino acids in length (Aβ 1-42/43). The genetic evidence includes the identification of five distinct mutations in APP, all of which cosegregate with rare forms of AD: four of these mutations have been shown to increase the levels of Aβ 1-42/43. More recently, AD-associated mutations in the presenilin-1 and presenilin-2 genes have also been shown to cause increased levels of Aβl-42/43 (e.g., Scheuner et al., 1996). Finally, it has been suggested that the AD-associated form of apolipoprotein E is involved in the accumulation of Aβ (e.g., Strittmatter et al., 1993; Strittmatter et al., 1993).

Erratum: Alzheimer's presenilin 1 modulates sorting of APP and its carboxyl-terminal fragments in cerebral neurons in vivo (Journal of Neurochemistry (2007) 102, (619-626))

Sam Gandy,* Yun-wu Zhang, Annat Ikin,* Stephen D. Schmidt, Efrat Levy, Roxanne Sheffield,§ Ralph A. Nixon, Francesca-Fang Liao, Paul M. Mathews, Huaxi Xu and Michelle E. Ehrlich* *Farber Institute for Neurosciences and the Department of Neurology, Thomas Jefferson University, Philadelphia, Pennsylvania, USA Burnham Institute for Medical Research, La Jolla, California, USA Center for Dementia Research, Nathan S. Kline Institute, New York University School of Medicine, Orangeburg, New York, USA §Lankenau Institute for Medical Research, Wynnewood, Pennsylvania, USA