The flotillins/reggie proteins are associated with noncaveolar membrane microdomains and have been implicated in the regulation of a clathrin-and caveolin-independent endocytosis pathway. Endocytosis is required for the amyloidogenic processing of the amyloid precursor protein (APP) and thus to initiate the release of the neurotoxic -amyloid peptide (A), the major component of extracellular plaques found in the brains of Alzheimer's disease patients. Here, we report that small interference RNA-mediated downregulation of flotillin-2 impairs the endocytosis of APP, in both neuroblastoma cells and primary cultures of hippocampal neurons, and reduces the production of A. Similar to tetanus neurotoxin endocytosis, but unlike the internalization of transferrin, clathrin-dependent endocytosis of APP requires cholesterol and adaptor protein-2 but is independent of epsin1 function. Moreover, on a nanoscale resolution using stimulated emission depletion microscopy and by Förster resonance energy transfer with fluorescence lifetime imaging microscopy, we provide evidence that flotillin-2 promotes the clustering of APP at the cell surface. We show that the interaction of flotillin-2 with APP is dependent on cholesterol and that clustering of APP enhances its endocytosis rate. Together, our data suggest that cholesterol/flotillindependent clustering of APP may stimulate the internalization into a specialized clathrin-dependent endocytosis pathway to promote amyloidogenic processing.
Alzheimer's disease (AD) is a devastating neurodegenerative disorder and a major medical problem. Here, we have investigated the impact of amyloid- (A) oligomers, AD-related neurotoxins, in the brains of rats and adult nonhuman primates (cynomolgus macaques). Soluble A oligomers are known to accumulate in the brains of AD patients and correlate with disease-associated cognitive dysfunction. When injected into the lateral ventricle of rats and macaques, A oligomers diffused into the brain and accumulated in several regions associated with memory and cognitive functions. Cardinal features of AD pathology, including synapse loss, tau hyperphosphorylation, astrocyte and microglial activation, were observed in regions of the macaque brain where A oligomers were abundantly detected. Most importantly, oligomer injections induced AD-type neurofibrillary tangle formation in the macaque brain. These outcomes were specifically associated with A oligomers, as fibrillar amyloid deposits were not detected in oligomer-injected brains. Human and macaque brains share significant similarities in terms of overall architecture and functional networks. Thus, generation of a macaque model of AD that links A oligomers to tau and synaptic pathology has the potential to greatly advance our understanding of mechanisms centrally implicated in AD pathogenesis. Furthermore, development of disease-modifying therapeutics for AD has been hampered by the difficulty in translating therapies that work in rodents to humans. This new approach may be a highly relevant nonhuman primate model for testing therapeutic interventions for AD.
Alzheimer's disease (AD) is a devastating neurological disorder that still lacks an effective treatment, and this has stimulated an intense pursuit of disease‐modifying therapeutics. Given the increasingly recognized link between AD and defective brain insulin signaling, we investigated the actions of liraglutide, a glucagon‐like peptide‐1 (GLP‐1) analog marketed for treatment of type 2 diabetes, in experimental models of AD. Insulin receptor pathology is an important feature of AD brains that impairs the neuroprotective actions of central insulin signaling. Here, we show that liraglutide prevented the loss of brain insulin receptors and synapses, and reversed memory impairment induced by AD‐linked amyloid‐β oligomers (AβOs) in mice. Using hippocampal neuronal cultures, we determined that the mechanism of neuroprotection by liraglutide involves activation of the PKA signaling pathway. Infusion of AβOs into the lateral cerebral ventricle of non‐human primates (NHPs) led to marked loss of insulin receptors and synapses in brain regions related to memory. Systemic treatment of NHPs with liraglutide provided partial protection, decreasing AD‐related insulin receptor, synaptic, and tau pathology in specific brain regions. Synapse damage and elimination are amongst the earliest known pathological changes and the best correlates of memory impairment in AD. The results illuminate mechanisms of neuroprotection by liraglutide, and indicate that GLP‐1 receptor activation may be harnessed to protect brain insulin receptors and synapses in AD. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
The amyloid precursor protein (APP) is implied both in cell growth and differentiation and in neurodegenerative processes in Alzheimer disease. Regulated proteolysis of APP generates biologically active fragments such as the neuroprotective secreted ectodomain sAPP␣ and the neurotoxic -amyloid peptide. Furthermore, it has been suggested that the intact transmembrane APP plays a signaling role, which might be important for both normal synaptic plasticity and neuronal dysfunction in dementia. To understand APP signaling, we tracked single molecules of APP using quantum dots and quantitated APP homodimerization using fluorescence lifetime imaging microscopy for the detection of För-ster resonance energy transfer in living neuroblastoma cells. Using selective labeling with synthetic fluorophores, we show that the dimerization of APP is considerably higher at the plasma membrane than in intracellular membranes. Heparan sulfate significantly contributes to the almost complete dimerization of APP at the plasma membrane. Importantly, this technique for the first time structurally defines the initiation of APP signaling by binding of a relevant physiological extracellular ligand; our results indicate APP as receptor for neuroprotective sAPP␣, as sAPP␣ binding disrupts APP dimers, and this disruption of APP dimers by sAPP␣ is necessary for the protection of neuroblastoma cells against starvation-induced cell death. Only cells expressing reversibly dimerized wild-type, but not covalently dimerized mutant APP are protected by sAPP␣. These findings suggest a potentially beneficial effect of increasing sAPP␣ production or disrupting APP dimers for neuronal survival. The amyloid precursor protein (APP)4 is known both for its important role in the development and plasticity of the nervous system (1-6) and for its involvement in Alzheimer disease (AD) (7,8). Despite intensive research efforts, the initial events that lead to the prevalent sporadic, i.e. non-familial, forms of AD are still unclear. Furthermore, although a higher gene dose of APP (9) or the presence of pathological APP mutations is sufficient to induce familial AD (for review, see Ref. 10), the exact pathological mechanism that is triggered by APP is still under debate.Some fragments of APP, such as the -amyloid peptide (A), are thought to contribute to synaptic dysfunction and neurotoxicity (11,12). On the other hand, the ␣-secretasederived extracellular fragment of APP (sAPP␣), which is present at lower levels in AD patients than in controls (13), has been shown to be beneficial for memory function, to possess neuroprotective properties, and to counteract the effects of A (14 -18).Signaling by transmembrane APP may directly contribute to neurodegeneration in AD (19 -24); however, the signal transduction pathway for transmembrane APP remains unknown, although several potential regulatory proteins, glycosaminoglycans, and metal ions are known to bind with high affinity to APP and sAPP␣ (25,26). The most common form of signal transduction for single-pass transm...
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