Amyloid Precursor Protein In Brain Research Articles

Modeling Alzheimer's disease in transgenic mice: effect of age and of presenilin1 on amyloid biochemistry and pathology in APP/London mice.

In transgenic mice that overexpress mutant Amyloid Precursor Protein [V717I], or APP/London (APP/Lo) (1999a. Early phenotypic changes in transgenic mice that overexpress different mutants of Amyloid Precursor Protein in brain. J. Biol. Chem. 274, 6483-6492; 1999b. Premature death in transgenic mice that overexpress mutant Amyloid precursor protein is preceded by severe neurodegeneration and apoptosis. Neuroscience 91, 819-830) the AD related phenotype of plaque and vascular amyloid pathology is late (12-15 months). This typical and diagnostic pathology is thereby dissociated in time from early symptoms (3-9 months) that include disturbed behavior, neophobia, aggression, glutamate excitotoxicity, defective cognition and decreased LTP. The APP/Lo transgenic mice are therefore a very interesting model to study early as well as late pathology, including the effect of age. In ageing APP*Lo mice, brain soluble and especially "insoluble" amyloid peptides dramatically increased, while normalized levels of secreted APPsalpha and APPsbeta, as well as cell-bound beta-C-stubs, remained remarkably constant, indicating normal alpha- and beta-secretase processing of APP. In double transgenic mice, i.e. APP/LoxPS1, clinical mutant PS1[A246E] but not wild-type human PS1 increased Abeta, and plaques and vascular amyloid developed at age 6-9 months. The PS1 mutant caused increasing Abeta42 production, while ageing did not. Amyloid deposits are thus formed, not by overproduction of Abeta, but by lack of clearance and/or degradation in the brain of ageing APP/Lo transgenic mice. The clearance pathways of the cerebral amyloid peptides are therefore valuable targets for fundamental research and for therapeutic potential. Although hyper-phosphorylated protein tau was evident in swollen neurites around the amyloid plaques, neurofibrillary pathology is not observed and the "tangle" aspect of AD pathology is therefore still missing from all current transgenic "amyloid" models. Also the "ApoE4" risk for late onset AD remains a problem for modeling in transgenic mice. We have generated transgenic mice that overexpress human ApoE4 (2000. Expression of Human Apolipoprotein E4 in neurons causes hyperphosphorylation of Protein tau in the brains of transgenic mice. Am. J. Pathol. 156 (3) 951-964) or human protein tau (1999. Prominent axonopathy in the brain and spinal cord of transgenic mice overexpressing four-repeat human tau protein. Am. J. Pathol. 155, 2153-2165) in their neurons. Both develop a similar although not identical axonopathy, with progressive degeneration of nerves and with muscle wasting resulting in motoric problems. Remarkably, ApoE4 transgenic mice are, like the tau transgenic mice, characterized by progressive hyper-phosphorylation of protein tau also in motor neurons which explains the motoric defects. Further crossing with the APP/Lo transgenic mice is ongoing to yield "multiple" transgenic mouse strains to study new aspects of amyloid and tau pathology.

Abnormal Glutamate Transport Function in Mutant Amyloid Precursor Protein Transgenic Mice

Recent studies have shown that amyloid precursor protein (APP), which plays a central role in Alzheimer's disease (AD), protects against excitotoxic neuronal injuries by regulating the function of the glial glutamate transporters. The mechanisms underlying these effects and their relationship to the neurodegenerative process in AD are under intense scrutiny. In this context, the main objective of the present study was to determine if overexpression of mutant human APP in transgenic mouse brains results in altered functioning of the excitatory amino acid transporters (EAATs). Transgenic mice expressing the 695 amino acid form of the human APP from the Thy-1 promoter showed a significant decrease in Bmax and KD for aspartate uptake when compared to nontransgenic controls. This decrease in glutamate transporter activity was associated with decreased protein expression of glial specific glutamate transporters, EAAT1 and 2, but did not affect mRNA levels. These results suggest that expression of mutant forms of APP disturbs astroglial transport of excitatory amino acids at the posttranscriptional level leading, in turn, to increased susceptibility to glutamate toxicity.

Distribution of β-amyloid and amyloid precursor protein in the brain of spawning (senescent) salmon: a natural, brain-aging model

Brain amyloid precursor protein (APP), a normal constituent of neurons, glial cells and cerebrospinal fluid, has several proposed functions (e.g., in neuronal growth and survival). It appears, however, that altered processing of APP is an initial or downstream step in the neuropathology of brain aging, Alzheimer's disease (AD), and Down's syndrome (DS). Some studies suggest that proteolytic cleavage of APP, producing β-amyloid (Aβ 1–42), could have neurotoxic or neuroprotective effects. In this study, we utilized antibodies to human APP 695 and Aβ 1–42, and Congo red staining, to search for amyloid deposition in the brain of semelparous spawning kokanee salmon ( Oncorhynchus nerka kennerlyi). Intracellular APP 695 immunoreactivity (APP-ir) was observed in brain regions involved in gustation (glomerulosus complex), olfaction (putative hippocampus, olfactory bulb), vision (optic tectum), the stress response (nucleus preopticus and nucleus lateralis tuberis), reproductive behavior (nucleus preopticus magnocellularis, nucleus preopticus periventricularis, ventral telencephalon), and coordination (cerebellum). Intra- and extra-neuronal Aβ 1–42 immunoreactivity (Aβ-ir) were present in all APP-ir regions except the nucleus lateralis tuberis and Purkinje cells of the cerebellum (coordination). Thus, the relationship between APP and Aβ deposition during brain aging could shed light on the processing of APP into Aβ, neurodegeneration, and possible protection of neurons that are functioning in spawning but senescent salmon. Pacific salmon, with their predictable and synchronized life history, could provide research options not available with the existing models for studies of brain aging and amyloidosis.

Upregulation of amyloid precursor protein and its mRNA in an experimental model of paediatric head injury

Amyloid precursor protein (APP), a membrane spanning glycoprotein which plays an important role in neuronal growth and synaptic plasticity, is increased after traumatic brain injury (TBI) and has been used as a sensitive marker of neuronal damage in an adult sheep head impact model. We hypothesised that APP expression would similarly be increased in lambs, suggesting that in the immature injured brain APP is also upregulated as an acute phase response to trauma.Ten anaesthetised and ventilated 4–5 week old Merino lambs sustained a left temporal head impact from a humane stunner. At 2 h after impact, there was widespread and intense neuronal cell body APP immunoreactivity which was more widely distributed than axonal APP. APP messenger RNA (mRNA) expression was also markedly increased with a distribution similar to that of APP antigen. These results demonstrate that APP antigen and mRNA are sensitive early indicators of TBI in paediatric cases.

Behavioral Disturbances without Amyloid Deposits in Mice Overexpressing Human Amyloid Precursor Protein with Flemish (A692G) or Dutch (E693Q) Mutation

The contribution of mutations in the amyloid precursor protein (APP) gene known as Flemish (APP/A692G) and Dutch (APP/E693Q) to the pathogenesis of Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis of the Dutch type, respectively, was studied in transgenic mice that overexpress the mutant APP in brain. These transgenic mice showed the same early behavioral disturbances and defects and increased premature death as the APP/London (APP V717I), APP/Swedish (K670N, M671L), and other APP transgenic mice described previously. Pathological changes included intense glial reaction, extensive microspongiosis in the white matter, and apoptotic neurons in select areas of the brain, while amyloid deposits were absent, even in mice over 18 months of age. This contrasts with extensive amyloid deposition in APP/London transgenic mice and less pronounced amyloid deposition in APP/Swedish transgenic mice generated identically. It demonstrated, however, that the behavioral deficiencies and the pathological changes in brain resulting from an impaired neuronal function are caused directly by APP or its proteolytic derivative(s). These accelerate or impinge on the normal process of aging and amyloid deposits per se are not essential for this phenotype.

Early Phenotypic Changes in Transgenic Mice That Overexpress Different Mutants of Amyloid Precursor Protein in Brain

Transgenic mice overexpressing different forms of amyloid precursor protein (APP), i.e. wild type or clinical mutants, displayed an essentially comparable early phenotype in terms of behavior, differential glutamatergic responses, deficits in maintenance of long term potentiation, and premature death. The cognitive impairment, demonstrated in F1 hybrids of the different APP transgenic lines, was significantly different from nontransgenic littermates as early as 3 months of age. Biochemical analysis of secreted and membrane-bound APP, C-terminal "stubs," and Abeta(40) and Abeta(42) peptides in brain indicated that no single intermediate can be responsible for the complex of phenotypic dysfunctions. As expected, the Abeta(42) levels were most prominent in APP/London transgenic mice and correlated directly with the formation of amyloid plaques in older mice of this line. Plaques were associated with immunoreactivity for hyperphosphorylated tau, eventually signaling some form of tau pathology. In conclusion, the different APP transgenic mouse lines studied display cognitive deficits and phenotypic traits early in life that dissociated in time from the formation of amyloid plaques and will be good models for both early and late neuropathological and clinical aspects of Alzheimer's disease.

Modulation of Secreted β-Amyloid Precursor Protein and Amyloid β-Peptide in Brain by Cholesterol

The effects of dietary cholesterol on brain amyloid precursor protein (APP) processing were examined using an APP gene-targeted mouse, genetically humanized in the amyloid beta-peptide (Abeta) domain and expressing the Swedish familial Alzheimer's disease mutations. These mice express endogenous levels of APP holoprotein and abundant human Abeta. Increased dietary cholesterol led to significant reductions in brain levels of secreted APP derivatives, including sAPPalpha, sAPPbeta, Abeta1-40, and Abeta1-42, while having little to no effect on cell-associated species, including full-length APP and the COOH-terminal APP processing derivatives. The changes in levels of sAPP and Abeta in brain all were negatively correlated with serum cholesterol levels and levels of serum and brain apoE. These results demonstrate that secreted APP processing derivatives and Abeta can be modulated in the brain of an animal by diet and provide evidence that cholesterol plays a role in the modulation of APP processing in vivo. APP gene-targeted mice lacking apoE, also have high serum cholesterol levels but do not show alterations in APP processing, suggesting that effects of cholesterol on APP processing require the presence of apoE.

Increased secretion of the amino-terminal fragment of amyloid precursor protein in brains of rats with a constitutive up-regulation of protein kinase C.

Protein kinase C (PKC) activation stimulates release of secreted amyloid precursor protein (APPs) in several cell lines. To ascertain the role of PKC in regulating APP metabolism in vivo, we used an animal model (methylazoxymethanol-treated rats; MAM rats) in which PKC is permanently hyperactivated in selected brain areas, i.e., cortex and hippocampus. A significant decrease in membrane-bound APP concentration was found in synaptosomes derived from cortex and hippocampus of MAM rats, where PKC is up-regulated, with a concomitant increase in APPs production in soluble fractions of the same brain areas. In contrast, in a brain area not affected by MAM treatment (i.e., cerebellum), APP secretion is similar in control and MAM rats, indicating that altered metabolism of APP is restricted to only those areas in which the PKC system is up-regulated. In addition, phorbol esters or H-7 modulate APPs release in hippocampal slices from both control and MAM rats, further supporting an in vivo role for this enzyme in regulating metabolism of mature APP.

The Cytoplasmic Domain of Alzheimer’s Amyloid Precursor Protein Is Phosphorylated at Thr654, Ser655, and Thr668 in Adult Rat Brain and Cultured Cells

The cytoplasmic domain of the Alzheimer's disease amyloid precursor protein (APP) is phosphorylated in vitro at Thr654 and Ser655, and both in vitro and in intact cells at Thr668 (numbering for APP695 isoform). We have developed phosphorylation state-specific antibodies to each of the sites, and we have used these to analyze the phosphorylation of APP in adult rat brain and in cultured cell lines. We demonstrate that all three sites in APP are phosphorylated in adult rat brain. Phosphorylation at Thr654, Ser655, and Thr668 was also observed in several cultured cell lines. In PC12 cells, phosphorylation at Ser655 was increased more than 10-fold by treatment with okadaic acid, a specific inhibitor of protein phosphatases 1 and 2A, but was not affected by activators of protein kinase C. In HeLa cells, phosphorylation at Thr668 was regulated in a cell cycle-dependent manner with near-stoichiometric phosphorylation being observed at the G2/M phase of the cell cycle. In general, phosphorylation at Ser655 was found to be highest in mature APP isoforms, whereas phosphorylation of Thr668 was highest in immature APP isoforms in cultured cells. The results demonstrate that phosphorylation of the cytoplasmic domain of APP occurs at Thr654, Ser655, and Thr668 under physiological conditions. The further characterization of APP phosphorylation using phosphorylation-specific antibodies may help in the elucidation of the biological function of APP.

Expression of the Amyloid Precursor Protein of Alzheimer's Disease on the Surface of Transfected HeLa Cells

The principal component of the amyloid which accumulates in Alzheimer's Disease brain is a 4-kDa βA4 fragment of the amyloid precursor protein (APP). Although APP has the structural features of an integral transmembrane receptor, there has been limited evidence for expression of APP at the plasma membrane. The function of APP and related molecules is unknown. Using rabbit antisera to purified human brain APP, surface labeling of APP is demonstrable in HeLa cells transfected with the APP695 isoform. Indirect immunofluorescence indicates the presence of APP at the surface of unfixed or aldehyde-fixed cells; preembedding immunoelectron microscopy using 5- or 1-nm gold particles and silver enhancement confirms plasma membrane labeling as well as labeling within intracellular membrane vesicles. Immunolabeling of unfixed cells at 4°C followed by incubation at 37°C shows APP within endocytic vesicles. Transfected HeLa cells with prominent surface APP were larger with more extensive microvilli than nonimmunoreactive HeLa cells. This is consistent with the postulated role of APP as a mediator of cell surface adhesion and membranematrix stabilization.

Calcium-activated neutral proteinase (calpain) system in aging and Alzheimer's disease.

Calpains (CANPs) are a family of calcium-dependent cysteine proteases under complex cellular regulation. By making selective limited proteolytic cleavages, they activate or alter the regulation of certain enzymes, including key protein kinases and phosphatases, and induce specific cytoskeletal rearrangements, accounting for their suspected involvement in intracellular signaling, vesicular trafficking, and structural stabilization. Calpain activity has been implicated in various aging phenomena, including cataract formation and erythrocyte senescence. Abnormal activation of the large stores of latent calpain in neurons induces cell injury and is believed to underlie neurodegeneration in excitotoxicity, Wallerian degeneration, and certain other neuropathologic states involving abnormal calcium influx. In Alzheimer's disease, we found the ratio of activated calpain I to its latent precursor isoform in neocortex to be threefold higher than that in normal individuals and those with Huntington's or Parkinson's disease. Immunoreactivity toward calpastatin, the endogenous inhibitor of calpain, was also markedly reduced in layers II-V of the neocortex in Alzheimer's disease. The excessive calpain system activation suggested by these findings represents a potential molecular basis for synaptic loss and neuronal cell death in the brain in Alzheimer's disease given the known destructive actions of calpain I and its preferential neuronal and synaptic localization. In surviving cells, persistent calpain activation may also contribute to neurofibrillary pathology and abnormal amyloid precursor protein trafficking/processing through its known actions on protein kinases and the membrane skeleton. The degree of abnormal calpain activation in the brain in Alzheimer's disease strongly correlated with the extent of decline in levels of secreted amyloid precursor protein in brain. Cytoskeletal proteins that are normally good calpain substrates become relatively calpain resistant when they are hyperphosphorylated, which may contribute to their accumulation in neurofibrillary tangles. As a major effector of calcium signals, calpain activity may mirror disturbances in calcium homeostasis and mediate important pathologic consequences of such disturbances.

Secretion of amyloid precursor protein and laminin by cultured astrocytes is influenced by culture conditions

Although normally quiescent, astrocytes in the adult brain respond to various types of brain injury by rapidly dividing, swelling, extending cellular processes, and expressing increased amounts of glial fibrillary acidic protein (GFAP). These phenomena are collectively referred to as "astrogliosis." Similarly, astroglia in primary culture stop dividing when they attain confluency, yet, as seen in situ, they retain their proliferative capacity for extended periods and resume rapid division when subcultured. To examine the impact of glial division on secretion of neurite-promoting factors, conditioned medium (CM) was removed from subconfluent, newly confluent, and long-term confluent ("aged") neonatal rat astrocyte cultures, and from aged confluent cultures that had been repassaged, "lesioned" (scraping with a rubber policeman), or triturated 3 days before harvest. Secretion of neurite-promoting factor(s) by glial cells into these CM was then assayed by treating neuroblastoma cultures with these various CM and quantitating neurite elaboration. Extensive neurite sprouting was elicited by CM from cultures just reaching confluency and from repassaged, lesioned, or triturated cultures. CM from aged confluent cultures did not induce sprouting. These results indicate that secretion of neurite-promoting factor(s) is regulated by glial division, and suggest that gliosis in situ may contribute to neurite sprouting by similar mechanisms. Immunoblot analysis demonstrated the presence in CM of varying amounts of laminin and amyloid precursor protein (APP), including isoforms containing the Kunitz-type protease inhibitor domain. CM from subconfluent cultures contained trace amounts of these proteins, but CM from cultures just reaching confluency contained significant amounts. Although CM from aged cultures contained barely detectable levels of either protein, trituration or repassage of aged cultures dramatically increased secretion of these proteins. APP- and laminin-enriched CM fractions promoted neuritogenesis to a similar level as respective unfractionated CM; anti-APP and anti-laminin antisera blocked this effect. Purified human brain APP promoted neuritogenesis when added to non-conditioned medium and aged CM. Increased secretion of APP and laminin therefore mediates at least a portion of CM-induced neuronal sprouting; these proteins may perform analogous functions during astrogliosis in situ.

Accumulation of amyloid precursor protein in damaged neuronal processes and microglia following intracerebral administration of aluminum salts

Alzheimer's disease is characterized by neurofibrillary tangles and amyloid deposits, with the latter probably occuring because of abnormal accumulation and/or processing of amyloid precursor protein (APP). Aluminumsalts are known to be neurotoxic and to be capable of inducing neurofibrillary tangles. We explored the effects of intraventricular or intrastriatal injections of AlCl 3 on the immunodistribution of APP in rat brain. There was a striking and long-lasting accumulation of APP in affected neurites, as well as in activated microglia/macrophages. Abnormal neurites also showed argentophilic changes, neurofilament accumulation, andAlz 50 immunoreactivity. However, no extracellular amyloid fibrils were seen. The results, taken together with previous studies on colchicine, are consistent with the hypothesis that interruption of axoplasmic flow can lead to both APP accumulation and cytoskeletal changes.