OCIAD2 activates γ-secretase to enhance amyloid β production by interacting with nicastrin

The gamma (γ)-secretase holoenzyme is composed of four core proteins and cleaves APP to generate amyloid beta (Aβ), a key molecule that causes major neurotoxicity during the early stage of Alzheimer’s disease (AD). However, despite its important role in Aβ production, little is known about the regulation of γ-secretase. OCIAD2, a novel modulator of γ-secretase that stimulates Aβ production, and which was isolated from a genome-wide functional screen using cell-based assays and a cDNA library comprising 6,178 genes. Ectopic expression of OCIAD2 enhanced Aβ production, while reduction of OCIAD2 expression suppressed it. OCIAD2 expression facilitated the formation of an active γ-secretase complex and enhanced subcellular localization of the enzyme components to lipid rafts. OCIAD2 interacted with nicastrin to stimulate γ-secretase activity. OCIAD2 also increased the interaction of nicastrin with C99 and stimulated APP processing via γ-secretase activation, but did not affect Notch processing. In addition, a cell-permeable Tat-OCIAD2 peptide that interfered with the interaction of OCIAD2 with nicastrin interrupted the γ-secretase-mediated AICD production. Finally, OCIAD2 expression was significantly elevated in the brain of AD patients and PDAPP mice. This study identifies OCIAD2 as a selective activator of γ-secretase to increase Aβ generation.     

Choroid plexus implants rescue Alzheimer’s disease-like pathologies by modulating amyloid-β degradation

The choroid plexuses (CP) release numerous biologically active enzymes and neurotrophic factors, and contain a subpopulation of neural progenitor cells providing the capacity to proliferate and differentiate into other types of cells. These characteristics make CP epithelial cells (CPECs) excellent candidates for cell therapy aiming at restoring brain tissue in neurodegenerative illnesses, including Alzheimer’s disease (AD). In the present study, using in vitro approaches, we demonstrated that CP were able to diminish amyloid-β (Aβ) levels in cell cultures, reducing Aβ-induced neurotoxicity. For in vivo studies, CPECs were transplanted into the brain of the APP/PS1 murine model of AD that exhibits advanced Aβ accumulation and memory impairment. Brain examination after cell implantation revealed a significant reduction in brain Aβ deposits, hyperphosphorylation of tau, and astrocytic reactivity. Remarkably, the transplantation of CPECs was accompanied by a total behavioral recovery in APP/PS1 mice, improving spatial and non-spatial memory. These findings reinforce the neuroprotective potential of CPECs and the use of cell therapies as useful tools in AD.     

APP dimer formation is initiated in the endoplasmic reticulum and differs between APP isoforms

The amyloid precursor protein (APP) is part of a larger gene family, which has been found to form homo- or heterotypic complexes with its homologues, whereby the exact molecular mechanism and origin of dimer formation remains elusive. In order to assess the cellular location of dimerization, we have generated a cell culture model system in CHO-K1 cells, stably expressing human APP, harboring dilysine-based organelle sorting motifs [KKAA-endoplasmic reticulum (ER); KKFF-Golgi], accomplishing retention within early secretory compartments. We show that APP exists as disulfide-bonded dimers upon ER retention after it was isolated from cells, and analyzed by SDS-polyacrylamide gel electrophoresis under non-reducing conditions. In contrast, strong denaturing and reducing conditions, or deletion of the E1 domain, resulted in the disappearance of those dimers. Thus we provide first evidence that a fraction of APP can associate via intermolecular disulfide bonds, likely generated between cysteines located in the extracellular E1 domain. We particularly visualize APP dimerization itself and identified the ER as subcellular compartment of its origin using biochemical or split GFP approaches. Interestingly, we also found that minor amounts of SDS-resistant APP dimers were located to the cell surface, revealing that once generated in the oxidative environment of the ER, dimers remained stably associated during transport. In addition, we show that APP isoforms encompassing the Kunitz-type protease inhibitor (KPI) domain exhibit a strongly reduced ability to form cis-directed dimers in the ER, whereas trans-mediated cell aggregation of Drosophila Schneider S2-cells was isoform independent. Thus, suggesting that steric properties of KPI-APP might be the cause for weaker cis-interaction in the ER, compared to APP695. Finally, we provide evidence that APP/APLP1 heterointeractions are likewise initiated in the ER.     

Neurogenic effects of β-amyloid in the choroid plexus epithelial cells in Alzheimer’s disease

β-amyloid (Aβ) can promote neurogenesis, both in vitro and in vivo, by inducing neural progenitor cells to differentiate into neurons. The choroid plexus in Alzheimer’s disease (AD) is burdened with amyloid deposits and hosts neuronal progenitor cells. However, neurogenesis in this brain tissue is not firmly established. To investigate this issue further, we examined the effect of Aβ on the neuronal differentiation of choroid plexus epithelial cells in several experimental models of AD. Here we show that Aβ regulates neurogenesis in vitro in cultured choroid plexus epithelial cells as well as in vivo in the choroid plexus of APP/Ps1 mice. Treatment with oligomeric Aβ increased proliferation and differentiation of neuronal progenitor cells in cultured choroid plexus epithelial cells, but decreased survival of newly born neurons. These Aβ-induced neurogenic effects were also observed in choroid plexus of APP/PS1 mice, and detected also in autopsy tissue from AD patients. Analysis of signaling pathways revealed that pre-treating the choroid plexus epithelial cells with specific inhibitors of TyrK or MAPK diminished Aβ-induced neuronal proliferation. Taken together, our results support a role of Aβ in proliferation and differentiation in the choroid plexus epithelial cells in Alzheimer’s disease.     

Glycogen synthase kinase-3β regulates fractalkine production by altering its trafficking from Golgi to plasma membrane: implications for Alzheimer’s disease

Glycogen synthase kinase-3β (GSK-3β) is a serine-threonine kinase implicated in multiple processes and signaling pathways. Its dysregulation is associated with different pathological conditions including Alzheimer’s disease (AD). Here we demonstrate how changes in GSK-3β activity and/or levels regulate the production and subsequent secretion of fractalkine, a chemokine involved in the immune response that has been linked to AD and to other different neurological disorders. Treatment of primary cultured neurons with GSK-3β inhibitors such as lithium and AR-A014418 decreased full-length fractalkine in total cell extracts. Opposite effects were observed after neuron transduction with a lentiviral vector overexpressing the kinase. Biotinylation assays showed that those changes mainly affect the plasma membrane-associated form of the protein, an observation that positively correlates with changes in the levels of its soluble form. These effects were confirmed in lithium-treated wild type (wt) mice and in GSK-3β transgenic animals, as well as in brain samples from AD patients, evident as age-dependent (animals) or Braak stage dependent changes (humans) in both the membrane-bound and the soluble forms of the protein. Further immunohistochemical analyses demonstrated how GSK-3β exerts these effects by affecting the trafficking of the chemokine from the Golgi to the plasma membrane, in different and opposite ways when the levels/activity of the kinase are increased or decreased. This work provides for the first time a mechanism linking GSK-3β and fractalkine both in vitro and in vivo, with important implications for neurological disorders and especially for AD, in which levels of this chemokine might be useful as a diagnostic tool.     

Impact of late-onset Alzheimer’s genetic risk factors on beta-amyloid endocytic production

The increased production of the 42 aminoacids long beta-amyloid (Aβ42) peptide has been established as a causal mechanism of the familial early onset Alzheimer’s disease (AD). In contrast, the causal mechanisms of the late-onset AD (LOAD), that affects most AD patients, remain to be established. Indeed, Aβ42 accumulation has been detected more than 30 years before diagnosis. Thus, the mechanisms that control Aβ accumulation in LOAD likely go awry long before pathogenesis becomes detectable. Early on, APOE4 was identified as the biggest genetic risk factor for LOAD. However, since APOE4 is not present in all LOAD patients, genome-wide association studies of thousands of LOAD patients were undertaken to identify other genetic variants that could explain the development of LOAD. PICALM, BIN1, CD2AP, SORL1, and PLD3 are now with APOE4 among the identified genes at highest risk in LOAD that have been implicated in Aβ42 production. Recent evidence indicates that the regulation of the endocytic trafficking of the amyloid precursor protein (APP) and/or its secretases to and from sorting endosomes is determinant for Aβ42 production. Thus, here, we will review the described mechanisms, whereby these genetic risk factors can contribute to the enhanced endocytic production of Aβ42. Dissecting causal LOAD mechanisms of Aβ42 accumulation, underlying the contribution of each genetic risk factor, will be required to identify therapeutic targets for novel personalized preventive strategies.     

Amyloid beta receptors responsible for neurotoxicity and cellular defects in Alzheimer’s disease

Alzheimer’s disease (AD) is the most common neurodegenerative disease. Although a major cause of AD is the accumulation of amyloid-β (Aβ) peptide that induces neuronal loss and cognitive impairments, our understanding of its neurotoxic mechanisms is limited. Recent studies have identified putative Aβ-binding receptors that mediate Aβ neurotoxicity in cells and models of AD. Once Aβ interacts with a receptor, a toxic signal is transduced into neurons, resulting in cellular defects including endoplasmic reticulum stress and mitochondrial dysfunction. In addition, Aβ can also be internalized into neurons through unidentified Aβ receptors and induces malfunction of subcellular organelles, which explains some part of Aβ neurotoxicity. Understanding the neurotoxic signaling initiated by Aβ-receptor binding and cellular defects provide insight into new therapeutic windows for AD. In the present review, we summarize the findings on Aβ-binding receptors and the neurotoxicity of oligomeric Aβ.     

Amyloid precursor protein products concentrate in a subset of exosomes specifically endocytosed by neurons

Amyloid beta peptide (Aβ), the main component of senile plaques of Alzheimer’s disease brains, is produced by sequential cleavage of amyloid precursor protein (APP) and of its C-terminal fragments (CTFs). An unanswered question is how amyloidogenic peptides spread throughout the brain during the course of the disease. Here, we show that small lipid vesicles called exosomes, secreted in the extracellular milieu by cortical neurons, carry endogenous APP and are strikingly enriched in CTF-α and the newly characterized CTF-η. Exosomes from N2a cells expressing human APP with the autosomal dominant Swedish mutation contain Aβ peptides as well as CTF-α and CTF-η, while those from cells expressing the non-mutated form of APP only contain CTF-α and CTF-η. APP and CTFs are sorted into a subset of exosomes which lack the tetraspanin CD63 and specifically bind to dendrites of neurons, unlike exosomes carrying CD63 which bind to both neurons and glial cells. Thus, neuroblastoma cells secrete distinct populations of exosomes carrying different cargoes and targeting specific cell types. APP-carrying exosomes can be endocytosed by receiving cells, allowing the processing of APP acquired by exosomes to give rise to the APP intracellular domain (AICD). Thus, our results show for the first time that neuronal exosomes may indeed act as vehicles for the intercellular transport of APP and its catabolites.     

Clearance of cerebral Aβ in Alzheimer’s disease: reassessing the role of microglia and monocytes

Deficiency in cerebral amyloid β-protein (Aβ) clearance is implicated in the pathogenesis of the common late-onset forms of Alzheimer’s disease (AD). Accumulation of misfolded Aβ in the brain is believed to be a net result of imbalance between its production and removal. This in turn may trigger neuroinflammation, progressive synaptic loss, and ultimately cognitive decline. Clearance of cerebral Aβ is a complex process mediated by various systems and cell types, including vascular transport across the blood–brain barrier, glymphatic drainage, and engulfment and degradation by resident microglia and infiltrating innate immune cells. Recent studies have highlighted a new, unexpected role for peripheral monocytes and macrophages in restricting cerebral Aβ fibrils, and possibly soluble oligomers. In AD transgenic (ADtg) mice, monocyte ablation or inhibition of their migration into the brain exacerbated Aβ pathology, while blood enrichment with monocytes and their increased recruitment to plaque lesion sites greatly diminished Aβ burden. Profound neuroprotective effects in ADtg mice were further achieved through increased cerebral recruitment of myelomonocytes overexpressing Aβ-degrading enzymes. This review summarizes the literature on cellular and molecular mechanisms of cerebral Aβ clearance with an emphasis on the role of peripheral monocytes and macrophages in Aβ removal.     

Transient dynamics of Aβ contribute to toxicity in Alzheimer’s disease

The aggregation and deposition of the amyloid-β peptide (Aβ) in the brain has been linked with neuronal death, which progresses in the diagnostic and pathological signs of Alzheimer’s disease (AD). The transition of an unstructured monomeric peptide into self-assembled and more structured aggregates is the crucial conversion from what appears to be a harmless polypeptide into a malignant form that causes synaptotoxicity and neuronal cell death. Despite efforts to identify the toxic form of Aβ, the development of effective treatments for AD is still limited by the highly transient and dynamic nature of interconverting forms of Aβ. The variability within the in vivo “pool” of different Aβ peptides is another complicating factor. Here we review the dynamical interplay between various components that influence the heterogeneous Aβ system, from intramolecular Aβ flexibility to intermolecular dynamics between various Aβ alloforms and external factors. The complex dynamics of Aβ contributes to the causative role of Aβ in the pathogenesis of AD.     

The molecular link between β- and γ-secretase activity on the amyloid β precursor protein

Alzheimer’s disease (AD) is characterized by an accumulation in the brain of amyloid β peptides (Aβ). The production of Aβ requires two sequential cleavages induced by β- and γ-secretases on the β-amyloid precursor protein (APP). Altered activity of these secretases is involved in the pathogenesis of AD. The expression and activity of β-secretase (BACE1) is augmented in the brain in late-onset sporadic AD. Mutant presenilin 1 (PS1), the major genetic defect of early-onset familial AD (FAD), alters the activity of γ-secretase, leading to increased production of Aβ42. Here we review the role of oxidative stress as a molecular link between the β- and the γ-secretase activities, and provide a mechanistic explanation of the pathogenesis of sporadic late-onset AD. We also discuss evidence for a role of the same mechanism in the pathogenesis of familial AD carrying PS1 mutations.     

Light on the structure of thromboxane A2 receptor heterodimers

The structure-based design of a mutant form of the thromboxane A(2) prostanoid receptor (TP) was instrumental in characterizing the structural determinants of the hetero-dimerization process of this G protein coupled receptor (GPCR). The results suggest that the hetero-dimeric complexes between the TPα and β isoforms are characterized by contacts between hydrophobic residues in helix 1 from both monomers. Functional characterization confirms that TPα-TPβ hetero-dimerization serves to regulate TPα function through agonist-induced internalization, with important implications in cardiovascular homeostasis. The integrated approach employed in this study can be adopted to gain structural and functional insights into the dimerization/oligomerization process of all GPCRs for which the structural model of the monomer can be achieved at reasonable atomic resolution.     

Molecular pathogenesis of apolipoprotein E-mediated amyloidosis in late-onset Alzheimer’s disease

Apolipoprotein E (apoE) ɛ4 allele is a genetic risk factor for late-onset familial and sporadic Alzheimer’s disease (AD). In the central nervous system, apoE is secreted mainly by astrocytes as a constituent of high-density lipoproteins. A recent study using apoE knockout mice provided strong evidence that apoE promotes cerebral deposition of amyloid β protein (Aβ). However, no clear explanation of the pathogenesis of apoE-induced AD has been provided. Here we discuss two possible mechanisms by which apoE might enhance Aβ deposition. One is the intracellular pathway in which apoE is internalized by neurons and induces lysosomal accumulation of Aβ and amyloidogenic APP (amyloid precursor protein) fragments, leading to neuronal death. The other is the extracellular pathway in which apoE-containing lipoproteins are trapped by Aβ1–42 deposits mobilizing soluble Aβ peptides and consequently enlarge amyloid plaques. These two mechanisms may operate at different stages of AD pathogenesis and suggest a chaperone-like function for the apoE molecule. Received 4 February 1999; received after revision 9 April 1999; accepted 23 April 1999     

Analysis of novel endosome-to-Golgi retrieval genes reveals a role for PLD3 in regulating endosomal protein sorting and amyloid precursor protein processing

The processing of amyloid precursor protein (APP) to the neurotoxic pro-aggregatory Aβ peptide is controlled by the mechanisms that govern the trafficking and localisation of APP. We hypothesised that genes involved in endosomal protein sorting could play an important role in regulating APP processing and, therefore, analysed ~ 40 novel endosome-to-Golgi retrieval genes previously identified in a genome-wide siRNA screen. We report that phospholipase D3 (PLD3), a type II membrane protein, functions in endosomal protein sorting and plays an important role in regulating APP processing. PLD3 co-localises with APP in endosomes and loss of PLD3 function results in reduced endosomal tubules, impaired trafficking of several membrane proteins and reduced association of sortilin-like 1 with APP.     

Modes of A�� toxicity in Alzheimer��s disease

Alzheimer's disease (AD) is reaching epidemic proportions, yet a cure is not yet available. While the genetic causes of the rare familial inherited forms of AD are understood, the causes of the sporadic forms of the disease are not. Histopathologically, these two forms of AD are indistinguishable: they are characterized by amyloid-β (Aβ) peptide-containing amyloid plaques and tau-containing neurofibrillary tangles. In this review we compare AD to frontotemporal dementia (FTD), a subset of which is characterized by tau deposition in the absence of overt plaques. A host of transgenic animal AD models have been established through the expression of human proteins with pathogenic mutations previously identified in familial AD and FTD. Determining how these mutant proteins cause disease in vivo should contribute to an understanding of the causes of the more frequent sporadic forms. We discuss the insight transgenic animal models have provided into Aβ and tau toxicity, also with regards to mitochondrial function and the crucial role tau plays in mediating Aβ toxicity. We also discuss the role of miRNAs in mediating the toxic effects of the Aβ peptide.     

Molecular basis of Alzheimer's disease

Despite an exponential production of data, Alzheimer's disease (AD) remains an enigma. Unresolved questions persist in the face of the heterogeneity of this neuropathology. Recent progress in understanding mechanisms for AD results from the study of amyloid precursor protein (APP) metabolism and the involvement of senile plaque-associated proteins. In addition to the amyloid cascade hypothesis, alternative schemes emerge, in which the amyloid peptide is not the primary effector of the disease. Perturbations of vesicular trafficking, the cytoskeletal network, and membrane cholesterol distribution could be central events. Furthermore, since the physiological role of APP, presenilins, and apolipoprotein E in the central nervous system are not completely understood, their involvement in AD etiology remains speculative. New actors have to be found to try to explain sporadic cases and non-elucidated familial cases.     

The potential importance of myeloid-derived suppressor cells (MDSCs) in the pathogenesis of Alzheimer’s disease

The exact cause of Alzheimer’s disease (AD) is still unknown, but the deposition of amyloid-β (Aβ) plaques and chronic inflammation indicates that immune disturbances are involved in AD pathogenesis. Recent genetic studies have revealed that many candidate genes are expressed in both microglia and myeloid cells which infiltrate into the AD brains. Invading myeloid cells controls the functions of resident microglia in pathological conditions, such as AD pathology. AD is a neurologic disease with inflammatory component where the immune system is not able to eliminate the perpetrator, while, concurrently, it should prevent neuronal injuries induced by inflammation. Recent studies have indicated that AD brains are an immunosuppressive microenvironment, e.g., microglial cells are hyporesponsive to Aβ deposits and anti-inflammatory cytokines enhance Aβ deposition. Immunosuppression is a common element in pathological disorders involving chronic inflammation. Studies on cancer-associated inflammation have demonstrated that myeloid-derived suppressor cells (MDSCs) have a crucial role in the immune escape of tumor cells. Immunosuppression is not limited to tumors, since MDSCs can be recruited into chronically inflamed tissues where inflammatory mediators enhance the proliferation and activation of MDSCs. AD brains express a range of chemokines and cytokines which could recruit and expand MDSCs in inflamed AD brains and thus generate an immunosuppressive microenvironment. Several neuroinflammatory disorders, e.g., the early phase of AD pathology, have been associated with an increase in the level of circulating MDSCs. We will elucidate the immunosuppressive armament of MDSCs and present evidences in support of the crucial role of MDSCs in the pathogenesis of AD.     

Redundant regulation of localization and protein stability of DmPar3

Apical–basal polarity is an important characteristic of epithelia and Drosophila neural stem cells. The conserved Par complex, which consists of the atypical protein kinase C and the scaffold proteins Baz and Par6, is a key player in the establishment of apical–basal cell polarity. Membrane recruitment of Baz has been reported to be accomplished by several mechanisms, which might function in redundancy, to ensure the correct localization of the complex. However, none of the described interactions was sufficient to displace the protein from the apical junctions. Here, we dissected the role of the oligomerization domain and the lipid-binding motif of Baz in vivo in the Drosophila embryo. We found that these domains function in redundancy to ensure the apical junctional localization of Baz: inactivation of only one domain is not sufficient to disrupt the function of Baz during apical–basal polarization of epithelial cells and neural stem cells. In contrast, mutation of both domains results in a strongly impaired protein stability and a phenotype characterized by embryonic lethality and an impaired apical–basal polarity in the embryonic epithelium and neural stem cells, resembling a baz-loss of function allele. Strikingly, the binding of Baz to the transmembrane proteins E-Cadherin, Echinoid, and Starry Night was not affected in this mutant protein. Our findings reveal a redundant function of the oligomerization and the lipid-binding domain, which is required for protein stability, correct subcellular localization, and apical–basal cell polarization.     

Amyloid precursor protein and its homologues: a family of proteolysis-dependent receptors

The Alzheimer’s amyloid precursor protein (APP) belongs to a conserved gene family that also includes the mammalian APLP1 and APLP2, the Drosophila APPL, and the C. elegans APL-1. The biological function of APP is still not fully clear. However, it is known that the APP family proteins have redundant and partly overlapping functions, which demonstrates the importance of studying all APP family members to gain a more complete picture. When APP was first cloned, it was speculated that it could function as a receptor. This theory has been further substantiated by studies showing that APP and its homologues bind both extracellular ligands and intracellular adaptor proteins. The APP family proteins undergo regulated intramembrane proteolysis (RIP), generating secreted and cytoplasmic fragments that have been ascribed different functions. In this review, we will discuss the APP family with focus on biological functions, binding partners, and regulated processing.