Alzheimer's disease-associated ␤-amyloid peptides (A␤) are generated by the sequential proteolytic processing of amyloid precursor protein (APP) by ␤-and ␥-secretases. There is growing evidence that cholesterol-and sphingolipid-rich membrane microdomains are involved in regulating trafficking and processing of APP. BACE1, the major ␤-secretase in neurons is a palmitoylated transmembrane protein that resides in lipid rafts. A subset of APP is subject to amyloidogenic processing by BACE1 in lipid rafts, and this process depends on the integrity of lipid rafts. Here we describe the association of all four components of the ␥-secretase complex, namely presenilin 1 (PS1)-derived fragments, mature nicastrin, APH-1, and PEN-2, with cholesterol-rich detergent insoluble membrane (DIM) domains of non-neuronal cells and neurons that fulfill the criteria of lipid rafts. In PS1 ؊/؊ /PS2 ؊/؊ and NCT ؊/؊ fibroblasts, ␥-secretase components that still remain fail to become detergent-resistant, suggesting that raft association requires ␥-secretase complex assembly. Biochemical evidence shows that subunits of the ␥-secretase complex and three TGN/endosomeresident SNAREs cofractionate in sucrose density gradients, and show similar solubility or insolubility characteristics in distinct non-ionic and zwitterionic detergents, indicative of their co-residence in membrane microdomains with similar protein-lipid composition. This notion is confirmed using magnetic immunoisolation of PS1-or syntaxin 6-positive membrane patches from a mixture of membranes with similar buoyant densities following Lubrol WX extraction or sonication, and gradient centrifugation. These findings are consistent with the localization of ␥-secretase in lipid raft microdomains of postGolgi and endosomes, organelles previously implicated in amyloidogenic processing of APP.Alzheimer's disease, a neurodegenerative dementing disorder, is pathologically characterized by the cerebral deposition of 39 -42 amino acid peptides termed ␤-amyloid (A␤) 1 peptides.

␥-Secretase facilitates the regulated intramembrane proteolysis of select type I membrane proteins that play diverse physiological roles in multiple cell types and tissue. In this study, we used biochemical approaches to examine the distribution of amyloid precursor protein (APP) and several additional ␥-secretase substrates in membrane microdomains. We report that APP C-terminal fragments (CTFs) and ␥-secretase reside in Lubrol WX detergent-insoluble membranes (DIM) of cultured cells and adult mouse brain. APP CTFs that accumulate in cells lacking ␥-secretase activity preferentially associate with DIM. Cholesterol depletion and magnetic immunoisolation studies indicate recruitment of APP CTFs into cholesterol-and sphingolipid-rich lipid rafts, and co-residence of APP CTFs, PS1, and syntaxin 6 in DIM patches derived from the trans-Golgi network. Photoaffinity cross-linking studies provided evidence for the preponderance of active ␥-secretase in lipid rafts of cultured cells and adult brain. Remarkably, unlike the case of APP, CTFs derived from Notch1, Jagged2, deleted in colorectal cancer (DCC), and N-cadherin remain largely detergent-soluble, indicative of their spatial segregation in non-raft domains. In embryonic brain, the majority of PS1 and nicastrin is present in Lubrol WXsoluble membranes, wherein the CTFs derived from APP, Notch1, DCC, and N-cadherin also reside. We suggest that ␥-secretase residence in non-raft membranes facilitates proteolysis of diverse substrates during embryonic development but that the translocation of ␥-secretase to lipid rafts in adults ensures processing of certain substrates, including APP CTFs, while limiting processing of other potential substrates. Sequential processing of amyloid precursor protein (APP)1 by ␤-and ␥-secretases releases the 39 -42-amino acid-long ␤-amyloid (A␤) peptides, which accumulate in the brains of aged individuals and patients with Alzheimer disease (AD) (1). The major ␤-secretase in neurons is an aspartyl protease termed BACE-1, which cleaves APP within the luminal domain, generating the N terminus of A␤ (2). The C terminus of A␤ is generated by intramembranous cleavage of APP C-terminal fragments (CTFs) by ␥-secretase, a multiprotein complex made of four essential components, presenilin (PS) 1 (or PS2), nicastrin, PEN-2, and APH-1 (3). Mutations in PSEN1 and PSEN2, encoding multipass membrane proteins PS1 and PS2, respectively, co-segregate with the majority of cases of autosomal dominant familial early-onset AD (4). Familial AD-linked PS1 and PS2 variants elevate the production of highly fibrillogenic A␤42 peptides (1). A role for PS1 in Notch function was first discovered in Caenorhabditis elegans screens and involves intramembranous cleavage of the Notch receptor, analogous to APP processing (5, 6). Nicastrin is a type I membrane protein independently identified as a novel component of the GLP-1/ Notch signaling pathway in C. elegans early embryos and in the biochemical characterization of proteins that interacted with PS1 (7,8). Multitransmembra...

FUS-proteinopathies, a group of heterogeneous disorders including ALS-FUS and FTLD-FUS, are characterized by the formation of inclusion bodies containing the nuclear protein FUS in the affected patients. However, the underlying molecular and cellular defects remain unclear. Here we provide evidence for mitochondrial localization of FUS and its induction of mitochondrial damage. Remarkably, FTLD-FUS brain samples show increased FUS expression and mitochondrial defects. Biochemical and genetic data demonstrate that FUS interacts with a mitochondrial chaperonin, HSP60, and that FUS translocation to mitochondria is, at least in part, mediated by HSP60. Down-regulating HSP60 reduces mitochondrially localized FUS and partially rescues mitochondrial defects and neurodegenerative phenotypes caused by FUS expression in transgenic flies. This is the first report of direct mitochondrial targeting by a nuclear protein associated with neurodegeneration, suggesting that mitochondrial impairment may represent a critical event in different forms of FUS-proteinopathies and a common pathological feature for both ALS-FUS and FTLD-FUS. Our study offers a potential explanation for the highly heterogeneous nature and complex genetic presentation of different forms of FUS-proteinopathies. Our data also suggest that mitochondrial damage may be a target in future development of diagnostic and therapeutic tools for FUS-proteinopathies, a group of devastating neurodegenerative diseases.

FUS (fused in sarcoma) proteinopathy is a group of neurodegenerative diseases characterized by the formation of inclusion bodies containing the FUS protein, including frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Previous studies show that mitochondrial damage is an important aspect of FUS proteinopathy. However, the molecular mechanisms by which FUS induces mitochondrial damage remain to be elucidated. Our biochemical and genetic experiments demonstrate that FUS interacts with the catalytic subunit of mitochondrial ATP synthase (ATP5B), disrupts the formation of ATP synthase complexes, and inhibits mitochondrial ATP synthesis. FUS expression activates the mitochondrial unfolded protein response (UPRmt). Importantly, down-regulating expression of ATP5B or UPRmt genes in FUS transgenic flies ameliorates neurodegenerative phenotypes. Our data show that mitochondrial impairment is a critical early event in FUS proteinopathy, and provide insights into the pathogenic mechanism of FUS-induced neurodegeneration.

Alzheimer's disease (AD) is the most common cause of age-related dementia. Pathologically, AD is characterized by the deposition in the brain of amyloid-beta peptides derived from proteolysis of amyloid precursor protein (APP) by beta-site APP cleaving enzyme 1 (BACE1) and gamma-secretase. A growing body of evidence implicates cholesterol and cholesterol-rich membrane microdomains in amyloidogenic processing of APP. Here, we review recent findings regarding the association of BACE1, gamma-secretase and APP in lipid rafts, and discuss potential therapeutic strategies for AD that are based on knowledge gleaned from the membrane environment that fosters APP processing.