Retromer binds the FANSHY sorting motif in SorLA to regulate amyloid precursor protein sorting and processing

sorLA is a sorting receptor for amyloid precursor protein (APP) genetically linked to Alzheimer's disease (AD). Retromer, an adaptor complex in the endosome-to-Golgi retrieval pathway, has been implicated in APP transport because retromer deficiency leads to aberrant APP sorting and processing and levels of retromer proteins are altered in AD. Here we report that sorLA and retromer functionally interact in neurons to control trafficking and amyloidogenic processing of APP. We have identified a sequence (FANSHY) in the cytoplasmic domain of sorLA that is recognized by the VPS26 subunit of the retromer complex. Accordingly, we characterized the interaction between the retromer complex and sorLA and determined the role of retromer on sorLA-dependent sorting and processing of APP. Mutations in the VPS26 binding site resulted in receptor redistribution to the endosomal network, similar to the situation seen in cells with VPS26 knockdown. The sorLA mutant retained APP-binding activity but, as opposed to the wild-type receptor, misdirected APP into a distinct non-Golgi compartment, resulting in increased amyloid processing. In conclusion, our data provide a molecular link between reduced retromer expression and increased amyloidogenesis as seen in patients with sporadic AD.     

Role of the VPS35 D620N mutation in Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disorder involving the loss of dopaminergic neurons in the brain. Following the discovery of the PD-causing D620N mutation in the VPS35 (Vacuolar sorting protein 35) gene, dysfunction in the subcellular retromer complex has been strongly implicated in pathogenesis of PD. Although the function and dysfunction of the retromer has been a focus of study for some time, the role of this complex in the development of PD is not fully understood. Investigating cellular alterations that occur when the retromer is rendered dysfunctional, such as when the D620N disease-causing mutation is introduced into various model systems, shows that endosomal processing defects are major contributors to the disease phenotype. Altered trafficking of retromer cargo molecules, reduced cellular survival and altered processing of alpha-synuclein have all been observed in the presence of the D620N mutation. In addition, interactions between the retromer and the protein products of other familial Parkinsonism-related genes, has made the retromer a prime target of research in PD. This review gives an overview of the changes in retromer function, identified thus far, that may contribute to the neurodegeneration observed in PD.     

Relevance of abeta1-42 intrahippocampal injection as an animal model of inflamed Alzheimer's disease brain

Injection of amyloid-beta peptide (Abeta1-42) into hippocampal and cortical regions of brain may have utility as an animal model of Alzheimer's disease (AD) emphasizing the inflammatory component of disease pathology. This review summarizes recent evidence supporting the relevance of the peptide injection model to describe inflammatory conditions in AD brain. A wide spectrum of responses are considered from effects of Abeta1-42 on animal behavior and cognitive performance to peptide actions at the cellular and molecular levels. In the latter case a particular focus is placed on inflammatory responses mediated by activated microglia. Specific pharmacological modulations of microglial signaling pathways and factors and how they shape patterns of inflammatory reactivity in peptide-injected brain are included. Overall, the considerations for the validity and limitations of Abeta1-42 injection as an animal model for AD pathology are also discussed.     

Phosphorylation of amyloid-beta at the serine 26 residue by human cdc2 kinase.

The amyloid-beta (Abeta) peptide has been implicated in the pathology of Alzheimer's disease (AD). Using an antisense peptide approach a novel interaction between Abeta and the human cdc2 kinase was identified. The Abeta 1-42, 1-40 and 25-35 peptides were shown to be substrates for the cdc2 kinase and phosphorylated on the Serine 26 residue. Phosphorylated Abeta (pSAbeta) was found in extracts from NT-2 neurons and AD brain. In NT-2 neurons the levels of pSAbeta were increased in the presence of exogenous Abeta and this increase was prevented by a cdc2 protein kinase inhibitor, olomoucine, that also prevented Abeta cytotoxicity. The results from this study suggest that Abeta phosphorylation by cdc2 could play a role in the brain pathology of AD.     

The location and trafficking routes of the neuronal retromer and its role in amyloid precursor protein transport

The retromer complex plays an important role in intracellular transport, is highly expressed in the hippocampus, and has been implicated in the trafficking of the amyloid precursor protein (APP). Nevertheless, the trafficking routes of the neuronal retromer and the role it plays in APP transport in neuronal processes remain unknown. Here we use hippocampal neuronal cultures to address these issues. Using fluorescence microscopy, we find that Vps35, the core element of the retromer complex, is in dendrites and axons, is enriched in endosomes and trans-Golgi network, and is found in APP-positive vesicles. Next, to identify the role the neuronal retromer plays in cargo transport, we infected hippocampal neurons with a lentivirus expressing shRNA to silence Vps35. By live fluorescence imaging, Vps35 deficiency was found to reduce the frequency, but not the kinetics, of long-range APP transport within neuronal processes. Supporting the interpretation that retromer promotes long-range transport, Vps35 deficiency led to increased APP in the early endosomes, in processes but not the soma. Finally, Vps35 deficiency was associated with increased levels of Aβ, a cleaved product of APP, increased colocalization of APP with its cleaving enzyme BACE1 in processes, and caused an enlargement of early endosomes. Taken together, our studies clarify the function of the neuronal retromer, and suggest specific mechanisms for how retromer dysfunction observed in Alzheimer's disease affects APP transport and processing.     

Increased expression of the oligopeptidase THOP1 is a neuroprotective response to Abeta toxicity

In a comprehensive proteomics study aiming at the identification of proteins associated with amyloid-beta (Abeta)-mediated toxicity in cultured cortical neurons, we have identified Thimet oligopeptidase (THOP1). Functional modulation of THOP1 levels in primary cortical neurons demonstrated that its overexpression was neuroprotective against Abeta toxicity, while RNAi knockdown made neurons more vulnerable to amyloid peptide. In the TgCRND8 transgenic mouse model of amyloid plaque deposition, an age-dependent increase of THOP1 expression was found in brain tissue, where it co-localized with Abeta plaques. In accordance with these findings, THOP1 expression was significantly increased in human AD brain tissue as compared to non-demented controls. These results provide compelling evidence for a neuroprotective role of THOP1 against toxic effects of Abeta in the early stages of AD pathology, and suggest that the observed increase in THOP1 expression might be part of a compensatory defense mechanism of the brain against an increased Abeta load.     

Deposits of fibrillar Aβ do not cause neuronal loss or ferritin expression in adult rat brain

In Alzheimer's disease (AD), senile plaques containing amyloid-beta (Abeta) are associated with neurodegeneration, yet little quantitative data are available concerning the spatiotemporal patterns of neuronal death that result from exposure to Abeta deposits. Furthermore, plaques are accompanied by ferritin-rich cells but no data exist regarding the spatiotemporal expression of ferritin in response to Abeta. The present study has obtained such data after injecting aged Abeta peptide into the parietal cortex of adult rats. Injected deposits of fibrillar Abeta (1 microliter of 1 mM in saline) were cleared within 7 days but did not cause a significant increase in ferritin expression. Counts of dying neurones showed that human Abeta1-40 killed as many neurones as control injections of saline, while human Abeta1-42 and rat Abeta1-40 killed significantly less. We conclude that the fibrillar Abeta in plaques is not likely to be directly responsible for the neurodegeneration and ferritin expression that occurs in AD.     

Nogo receptor interacts with brain APP and Abeta to reduce pathologic changes in Alzheimer's transgenic mice

Pathophysiologic hypotheses for Alzheimer's disease (AD) are centered on the role of the amyloid plaque Abeta peptide and the mechanism of its derivation from the amyloid precursor protein (APP). As part of the disease process, an aberrant axonal sprouting response is known to occur near Abeta deposits. A Nogo to Nogo-66 receptor (NgR) pathway contributes to determining the ability of adult CNS axons to extend after traumatic injuries. Here, we consider the potential role of NgR mechanisms in AD. Both Nogo and NgR are mislocalized in AD brain samples. APP physically associates with the NgR. Overexpression of NgR decreases Abeta production in neuroblastoma culture, and targeted disruption of NgR expression increases transgenic mouse brain Abeta levels, plaque deposition, and dystrophic neurites. Infusion of a soluble NgR fragment reduces Abeta levels, amyloid plaque deposits, and dystrophic neurites in a mouse transgenic AD model. Changes in NgR level produce parallel changes in secreted APP and AB, implicating NgR as a blocker of secretase processing of APP. The NgR provides a novel site for modifying the course of AD and highlights the role of axonal dysfunction in the disease.     

Inhibitory effect of green tea extract on beta-amyloid-induced PC12 cell death by inhibition of the activation of NF-kappaB and ERK/p38 MAP kinase pathway through antioxidant mechanisms

Beta-amyloid peptide (Abeta) is considered responsible for the pathogenesis of Alzheimer's disease (AD). Several lines of evidence support that Abeta-induced cytotoxicity is mediated through the generation of reactive oxygen species (ROS). Thus, agents that scavenge ROS level may usefully impede the development or progress of AD. Green tea extract has been known to have such antioxidant properties. Our previous studies demonstrate that green tea extract protected ischemia/reperfusion-induced brain cell death by scavenging oxidative damages of macromolecules. In this study, we investigated the effects of green tea extract on Abeta-induced oxidative cell death in cultured rat pheochromocytoma (PC12) cells. PC12 cells treated with Abeta25-35 (10-50 microM) showed intracellular ROS elevation, the formation of 8-oxodG (an oxidized form of DNA), and underwent apoptotic cell death in a dose-dependent manner. Abeta(25-35) treatment upregulated pro-apoptotic p53 at the gene level, and Bax and caspase-3 at the protein level, but downregulated anti-apoptotic Bcl-2 protein. Interestingly, co-treated green tea extract (10-50 microg/ml) dose-dependently attenuated Abeta(25-35) (50 microM)-induced cell death, intracellular ROS levels, and 8-oxodG formation, in addition to p53, Bax, and caspase-3 expression, but upregulated Bcl-2. Furthermore, green tea extract prevented the Abeta(25-35)-induced activations of the NF-kappaB and ERK and p38 MAP kinase pathways. Our study suggests that green tea extract may usefully prevent or retard the development and progression of AD.     

Increased expression of the urokinase plasminogen-activator receptor in amyloid beta peptide-treated human brain microglia and in AD brains

The urokinase plasminogen-activator receptor (uPAR) is involved in many processes in inflammation including the migration of inflammatory-associated cells to sites of tissue damage. This receptor, also designated as CD87, is induced in response to a range of stimuli and is a marker of macrophage activation. Its role in inflammatory responses of microglia in Alzheimer's disease (AD) has not been previously investigated. In this study we demonstrate that uPAR mRNA and protein expression is induced following incubation of human post-mortem brain-derived microglia with fibrillar amyloid beta (Abeta) peptide. This response was stronger with Abeta peptide than with other tested pro-inflammatory agents. Induction of uPAR surface expression by microglia was inhibited by the antioxidant N-acetyl-cysteine, indicating that this gene may be induced as a result of oxidative stress-related mechanisms. The significance of these findings to AD was investigated. UPAR protein levels were significantly increased in human brain tissues from the hippocampus, superior frontal gyrus and inferior temporal gyrus of AD cases compared with similar tissues from non-demented cases. Increased uPAR expression was not demonstrated in AD cerebellum. Finally, increased uPAR immunoreactivity was demonstrated in activated microglia in AD brain samples using two different antibodies to uPAR. These results provide a connection between the induction of oxidative stress in AD and microglial activation, and establish a possible involvement of uPAR in AD pathogenesis.     

Increased amyloid beta protein levels in children and adolescents with Down syndrome

BACKGROUND: Persons with Down syndrome (DS) (40 years and older) have neuropathological changes characteristic of Alzheimer disease (AD). Soluble forms of amyloid beta (Abeta) peptide generated from amyloid precursor protein (APP) end at C-terminal residues 40 and 42. The presence of the apolipoprotein E (ApoE) epsilon4 allele is a significant risk factor for the development of sporadic AD. Although preliminary studies have shown an association of plasma Abeta42 and ApoE epsilon4 allele in older persons with DS who have dementia, the relationship between plasma Abeta40 and Abeta42 levels and ApoE phenotypes in children with DS has not been examined. Inflammation might play a role in the growth of DS brains. Neopterin is an immune activation marker for the cell-mediated immune response. OBJECTIVE: To examine the levels of plasma Abeta40, Abeta42, and neopterin in children or adolescents with DS or controls. MATERIALS AND METHODS: Blood was collected from DS (N=35; 7+/-3.8 years old) and their siblings (N=34; 10+/-4.5). Plasma Abeta40 and Abeta42, and neopterin levels were quantitated by sandwich ELISA. RESULTS: Abeta40 and Abeta42 levels were higher in DS than controls. The ratio of Abeta42/Abeta40 was lower in DS than in controls. There were significant negative correlations between age and Abeta40 in DS and controls, and between age and Abeta42 levels in DS but not in controls. There was no association of Abeta40 or Abeta42 levels with Apo E in either group. Neopterin levels were higher in DS than controls, and the levels were not correlated with Abeta40 and Abeta42 levels in DS or controls. CONCLUSIONS: The over expression of APP gene in DS leads to increases in plasma Abeta40 and Abeta42 levels before plaque formation in DS brain. Higher neopterin concentrations in DS reflect inflammatory cell activation. Further studies are needed to determine whether DS children with lower plasma Abeta42/Abeta40 ratios are at increased risk of developing AD during aging than those with higher ratios.     

Pathogenic role of mitochondrial [correction of mitochondral] amyloid-beta peptide

Metabolic dysfunction is one of the early features in Alzheimer's disease (AD) affected brain. Amyloid-beta peptide (Abeta), a major peptide deposited in neuritic plaques, has been considered as an important initiating molecule in the pathogenesis of AD. However, the pathogenic role of Abeta remains to be determined. Here, we review current studies showing that progressive accumulation of Abeta occurs within the mitochondria of both transgenic mice overexpressing mutant Abeta peptide precursor protein and autopsied brains from AD patients. Interaction of Abeta with Abeta-binding alcohol dehydrogenase (ABAD), a short-chain alcohol dehydrogenase in the mitochondrial matrix, leads to mitochondrial dysfunction evidenced by increased reactive oxygen species generation, mitochondrial membrane permeability formation and caspase-3-like activity induction, and decreased activities of the Krebs cycle. These effects can be blocked by intracellular transduction of the ABAD decoy peptide. We hypothesize that Abeta-induced and mitochondria-dependent cytotoxic pathways might play an important role in AD pathogenesis and could be a potential therapeutic target.     

Transforming growth factor-β signaling pathway as a therapeutic target in neurodegeneration

Neurodegenerative diseases are becoming an increasing social and economical burden as our population ages; but current knowledge of the processes leading to these diseases is still limited, and no effective treatments are available. Neurodegeneration in Alzheimer's disease (AD) is the most common cause of dementia and afflicts an estimated 4 million people in this country alone. Because accumulation of beta-amyloid (Abeta) peptide appears central to AD pathogenesis, large efforts have been directed at understanding and interfering with Abeta production or aggregation. These efforts have largely identified the processes resulting in Abeta production from the larger amyloid precursor protein (APP) and have revealed that Abeta peptide is also produced at low levels in the healthy brain. Interestingly, Abeta production is rapidly increased after neuronal injury, and traumatic brain injury is a known risk factor for AD and Parkinson's disease. In contrast, brain injury in young individuals does not seem to result in AD, and brain injury in animal models can promote Abeta clearance. This suggests that certain factors associated with injury might be able to reduce the accumulation of Abeta. Accumulation of Abeta peptide might be reduced either directly by stimulating phagocytes or other Abeta-degrading processes, or indirectly, by reducing neuronal injury and thus lowering the production of Abeta peptide. Directing the brain's natural mechanisms for clearing Abeta or increasing neuroprotection might therefore be reasonable approaches in interfering with AD pathogenesis.     

Lack of neprilysin suffices to generate murine amyloid-like deposits in the brain and behavioral deficit in vivo

Accumulation of the beta-amyloid peptide (Abeta) in the brain is a major pathological hallmark of Alzheimer's disease (AD), leading to synaptic dysfunction, neuronal death, and memory impairment. The levels of neprilysin, a major Abeta-degrading enzyme, are decreased in AD brains and during aging. Because neprilysin cleaves Abeta in vivo, its down-regulation may contribute to the pathophysiology of AD. The aim of this study was to assess the consequences of neprilysin deficiency on accumulation of murine Abeta in brains and associated pathologies in vivo by investigating neprilysin-deficient mice on biochemical, morphological, and behavioral levels. Aged neprilysin-deficient mice expressed physiological amyloid precursor protein (APP) levels and exhibited elevated brain Abeta concentrations and amyloid-like deposits in addition to signs of neuronal degeneration in their brains. Behaviorally, neprilysin-deficient mice acquired a significantly weaker conditioned taste aversion that extinguished faster than the aversion of age-matched controls. Our data establish that, under physiological APP expression levels, neprilysin deficiency is associated with increased Abeta accumulation in the brain and leads to deposition of amyloid-like structures in vivo as well as with signs of AD-like pathology and with behavioral deficits.     

The degradation of Abeta(25-35) by the serine protease plasmin is inhibited by aluminium

The catabolism of amyloid beta peptides (Abeta) may be important in their accumulation in the brain in both early and late-onset Alzheimer's disease (AD). The serine protease plasmin is one of a suite of proteases implicated in AD. It is a promoter of alpha-cleavage of the amyloid beta precursor protein (AbetaPP) and will degrade Abeta in vitro. Herein we have demonstrated cleavage of the amyloidogenic Abeta(25-35) by plasmin to produce the non-amyloidogenic fragment Abeta(29-35). The activity of plasmin was halved by pre-mixing it with aluminium (Al) prior to its addition to the peptide. An interaction between Al and proteases involved in the catabolism of Abeta might define the putative link between Al and AD.     

Therapeutic treatment of Alzheimer's disease using metal complexing agents

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by deposition of extracellular amyloid plaques, formation of intracellular neurofibrillary tangles and neuronal dysfunction in the brain. A growing body of evidence indicates a central role for biometals such as copper in many critical aspects of AD. The amyloid beta (Abeta) peptide and its parental molecule, the amyloid precursor protein (APP) both modulate Cu and Zn metabolism in the brain. Therefore, aberrant changes to APP or Abeta metabolism could potentially alter biometal homoestasis in AD, leading to increased free radical production and neuronal oxidative stress. Modulation of metal bioavailability in the brain has been proposed as a potential therapeutic strategy for treatment of AD patients. The lipid permeable metal complexing agent, clioquinol (CQ), has shown promising results in animal models of AD and in small clinical trials involving AD patients. Moreover, a new generation of metal-ligand based therapeutics is currently under development. Patents now cover the generation of novel metal ligand structures designed to modulate metal binding to Abeta and quench metal-mediated free radical generation. However, the mechanism by which CQ and other metal complexing agents slows cognitive decline in AD animal models and patients is unknown. Increasing evidence suggests that ligand-mediated redistribution of metals at a cellular level in the brain may be important. Further research will be necessary to fully understand the complex pathways associated with efficacious metal-based pharmaceuticals for treatment of AD.     

Lipoprotein effects on Abeta accumulation and degradation by microglia in vitro.

An inflammatory response involving activated microglia in neuritic beta-amyloid plaques is found in Alzheimer's disease (AD) brain. Because HDL lipoproteins have been shown to carry the beta-amyloid peptide (Abeta) in plasma and CSF, we have investigated the influence of plasma high-density lipoprotein (HDL) and lipidated ApoE and ApoJ particles on the interaction of cultured rat microglia with Abeta1-42. Microglia degraded Abeta via a pathway sensitive to cytochalasin D and the scavenger receptor inhibitor, fucoidan. HDL increased the degradation of Abeta and the ratio of multimeric/monomeric Abeta in a dose-dependent manner. In contrast, lipidated ApoJ and ApoE decreased the degradation of Abeta, and the effects were ApoE isoform-dependent. Immuno-electron microscopy revealed internalized Abeta in endosomes and lysosomes as well as cell-associated Abeta in deep invaginations, which may be related to caveolae and surface-connected compartments. These data suggest that lipoprotein-dependent Abeta trafficking to microglia could be relevant to plaque pathogenesis in AD.     

In vivo effects of apoE and clusterin on amyloid-β metabolism and neuropathology

The epsilon4 allele of apolipoprotein E APOE is a risk factor for Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA), and the epsilon2 allele is associated with a decreased risk for AD. There is strong evidence to suggest that a major, if not the main, mechanism underlying the link between apoE and both AD and CAA is related to the ability of apoE to interact with the amyloid-beta (Abeta) peptide and influence its clearance, aggregation, and conformation. In addition to a number of in vitro studies supporting this concept, in vivo studies with amyloid precursor protein (APP) transgenic mice indicate that apoE and a related molecule, clusterin (also called apolipoprotein J), have profound effects on the onset of Abeta deposition, as well as the local toxicity associated with Abeta deposits both in the brain parenchyma and in cerebral blood vessels. Taken together, these studies suggest that altering the expression of apoE and clusterin in the brain or the interactions between these molecules and Abeta would alter AD pathogenesis and provide new therapeutic avenues for prevention or treatment of CAA and AD.