The recycling of the amyloid precursor protein (APP) from the cell surface via the endocytic pathways plays a key role in the generation of amyloid β-peptide (Aβ) in Alzheimer's Disease (AD). We report here that inherited variants in the SORL1 neuronal sorting receptor are associated with late-onset AD. These variants, which occur in at least two different clusters of intronic sequences may regulate tissue-specific expression of SORL1. We also show that SORL1 directs trafficking of APP into recycling pathways, and that when SORL1 is under-expressed, APP is sorted into Aβ-generating compartments. These data suggest that inherited or acquired changes in SORL1 expression or function are mechanistically involved in causing AD.

The presenilin proteins (PS1 and PS2) and their interacting partners nicastrin, aph-1 (refs 4, 5) and pen-2 (ref. 5) form a series of high-molecular-mass, membrane-bound protein complexes that are necessary for gamma-secretase and epsilon-secretase cleavage of selected type 1 transmembrane proteins, including the amyloid precursor protein, Notch and cadherins. Modest cleavage activity can be generated by reconstituting these four proteins in yeast and Spodoptera frugiperda (sf9) cells. However, a critical but unanswered question about the biology of the presenilin complexes is how their activity is modulated in terms of substrate specificity and/or relative activities at the gamma and epsilon sites. A corollary to this question is whether additional proteins in the presenilin complexes might subsume these putative regulatory functions. The hypothesis that additional proteins might exist in the presenilin complexes is supported by the fact that enzymatically active complexes have a mass that is much greater than predicted for a 1:1:1:1 stoichiometric complex (at least 650 kDa observed, compared with about 220 kDa predicted). To address these questions we undertook a search for presenilin-interacting proteins that differentially affected gamma- and epsilon-site cleavage events. Here we report that TMP21, a member of the p24 cargo protein family, is a component of presenilin complexes and differentially regulates gamma-secretase cleavage without affecting epsilon-secretase activity.

The intramembrane cleavage of ␤-amyloid precursor protein by ␥-secretase is the final step in the generation of amyloid ␤-protein. A 59-or 57-residue C-terminal fragment called CTF␥ is produced concomitantly. Putative CTF␥ generated in rat brain membrane preparations was purified and sequenced. Instead of CTF␥, shorter 50-and 49-residue fragments were identified. In addition, we found similar C-terminal fragments of ␤-amyloid precursor-like proteins 1 and 2; these were also cleaved at corresponding sites. This newly identified cleavage occurs at a site two to five residues inside the cytoplasmic membrane boundary, which is very similar to ␥-secretase-like cleavage of Notch 1. Cerebral deposition of amyloid ␤-protein (A␤)1 is an early, invariant, and essential event in the pathogenesis of Alzheimer's disease (AD) (1). A␤ is generated from ␤-amyloid precursor protein (APP) when ␤-secretase cleavage at the extracellular domain produces a 99-residue C-terminal fragment called CTF99 or CTF␤. Subsequent cleavage of CTF␤ in the middle of the transmembrane domain by ␥-secretase primarily produces either a 40-residue protein (A␤40) or a 42-residue protein (A␤42). A␤42 has the higher aggregation potential and is the first species to accumulate in aged and AD brains (2-4). Intramembrane ␥-secretase cleavage of CTF␤ should yield a 59-or 57-residue cytoplasmic C-terminal fragment (CTF41-99 or CTF43-99; APP712-770 or APP714 -770 according to the numbering of the APP770 isoform) called CTF␥. However, CTF␥has not yet been identified in either brain homogenates or cell lysates, presumably because of its instability in vivo. To learn more about the properties of ␥-cleavage, we decided to characterize CTF␥. EXPERIMENTAL PROCEDURES Subcellular Fractionation of Newborn Rat Brains and Chinese Hamster Ovary (CHO) CellsStably Expressing APP751-Fresh newborn (day 1) rat brains were homogenized on ice with 20 strokes in a glassTeflon homogenizer in eight volumes of Buffer H (20 mM Hepes, 150 mM NaCl, 10% glycerol, 5 mM EDTA, pH 7.4). All subsequent steps were carried out at 4°C unless indicated otherwise. The nuclear fractions were collected by centrifugation of the homogenates at 2,500 ϫ g (brains) or at 800 ϫ g (CHO cells) for 10 min, followed by a brief washing in Buffer H with or without 0.4% Triton X-100. The postnuclear supernatant was separated into cytosolic and membranous fractions by centrifugation at 100,000 ϫ g for 1 h. Each fraction was adjusted to an equal volume for Western blotting.Preparation of Active Membrane Fractions-The postnuclear supernatant from newborn rat brains, as prepared above, was spun at 10,000 ϫ g for 15 min to collect membrane fraction F2P, with additional subsequent spin at 100,000 ϫ g for 1 h to collect F3P. F2P was collected from the CHO cells by centrifugation at 100,000 ϫ g for 1 h. The membrane fractions, after being washed once with the buffer, were incubated at 37°C in Buffer H containing a mixture of protease inhibitors (5 mM EDTA, 5 mM EGTA, 5 mM 1,10-phenanthroline, 10 M bestatin, 10 M amasta...

Mutations in the PTEN-induced kinase 1 (PINK1) gene have recently been implicated in autosomal recessive early onset Parkinson Disease (1, 2). To investigate the role of PINK1 in neurodegeneration, we designed human and murine neuronal cell lines expressing either wild-type PINK1 or PINK1 bearing a mutation associated with Parkinson Disease. We show that under basal and staurosporine-induced conditions, the number of terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)-positive cells was lower in wild-type PINK1 expressing SH-SY5Y cells than in mock-transfected cells. This phenotype was due to a PINK1-mediated reduction in cytochrome c release from mitochondria, which prevents subsequent caspase-3 activation. We show that overexpression of wild-type PINK1 strongly reduced both basal and staurosporine-induced caspase 3 activity. Overexpression of wild-type PINK1 also reduced the levels of cleaved caspase-9, caspase-3, caspase-7, and activated poly(ADP-ribose) polymerase under both basal and staurosporine-induced conditions. In contrast, Parkinson disease-related mutations and a kinase-inactive mutation in PINK1 abrogated the protective effect of PINK1. Together, these results suggest that PINK1 reduces the basal neuronal pro-apoptotic activity and protects neurons from staurosporine-induced apoptosis. Loss of this protective function may therefore underlie the degeneration of nigral dopaminergic neurons in patients with PINK1 mutations. Parkinson disease (PD)2 is the most common neurodegenerative movement disorder, affecting ϳ1% of the population by age 65 years (3, 4). It is characterized by the predominant degeneration of midbrain dopaminergic neurons. Although most patients with PD are sporadic, familial cases represent ϳ10% of all diagnoses. To date, six genes responsible for inherited forms of PD have been identified. Mutations in the ␣-synuclein (5), LRRK2 (leucine-rich repeat kinase 2) and UCH-L1 (ubiquitin C-terminal esterase L1) genes cause dominant forms of familial PD. In contrast, mutations in parkin (6), DJ-1 (7,8), and the newly identified PTEN (phosphatase and tensin homologue on chromosome 10)-induced kinase 1 (PINK1) (1, 2) are responsible for recessive forms of familial PD.PINK1 encodes a highly conserved, 581-amino acid, putative serinethreonine protein kinase and is a member of a small family of novel kinases including CLIK1 (CLP-36 interacting kinase)/PDLIM1 kinases. Bioinformatic analysis suggests that residues Gly-193 to Leu-507 comprise the catalytic domain, residues Gly-193 to Lys-219 form the ATPbinding cassette (with Tyr-166 as an autophosphorylated regulatory residue), and residues Asp-384 to Glu-417 form an activation loop (Fig. 1). PINK1 is transcriptionally transactivated by the PTEN gene (9) and is expressed at variable levels in different cancer cell types. Valente et al. showed that overexpressed, epitope-tagged PINK1 localized to mitochondria and may have a protective function against cell death (1).To further investigate the role of PINK1 in neuronal ap...