Subcellular localization of amyloid precursor protein in senile plaques of Alzheimer''s disease.

The authors have previously shown that amyloid precursor protein (APP) accumulates in neurites present in senile plaques of Alzheimer's disease (AD). In this ultrastructural immunocytochemical study, we describe the subcellular site of APP accumulation. Vibratome sections of glutaraldehyde-paraformaldehyde fixed hippocampi from five cases of AD were pretreated with methanol and immunostained with an antibody raised against recombinant APP 770 by using either indirect immunogold or peroxidase methods. Immunolabeling was localized in cell processes filled with amorphous, irregular-shaped materials, which were identified as dense bodies deformed by postmortem autolysis and methanol treatment, as well as multilamellar membranous bodies. Identification of these bodies was obtained with comparative ultrastructural examination of biopsy and autopsy tissue fixed with and without methanol treatment. These electron-dense organellae were positive for the lysosomal marker, acid phosphatase. At light microscopy, acid phosphatase and APP colocalized to the same cell processes in senile plaques. Many of those cell processes contained abnormal straight or paired helical filaments supporting their neuritic nature. These results suggest that APP accumulates in the lysosomal system of the dystrophic neurites present in senile plaques and are consistent with a neuronal origin of the APP forming the amyloid in senile plaques.     

Caspase-cleaved amyloid precursor protein in Alzheimer's disease

Caspase-3 mediated cleavage of the amyloid precursor protein (APP) has been proposed as a putative mechanism underlying amyloidosis and neuronal cell death in Alzheimer's disease (AD). We utilized an antibody that selectively recognizes the neo epitope generated by caspase-3 mediated cleavage of APP (alphadeltaC(csp)-APP) to determine if this proteolytic event occurs in senile plaques in the inferior frontal gyrus and superior temporal gyrus of autopsied AD and age-matched control brains. Consistent with a role for caspase-3 activation in AD pathology, alphadeltaC(csp)-APP immunoreactivity colocalized with a subset of TUNEL-positive pyramidal neurons in AD brains. AlphadeltaC(csp)-APP immunoreactivity was found in neurons and glial cells, as well as in small- and medium-size particulate elements, resembling dystrophic terminals and condensed nuclei, respectively, in AD and age-matched control brains. There were a larger number of alphadeltaC(csp)-APP immunoreactive elements in the inferior frontal gyrus and superior temporal gyrus of subjects with AD pathology than age-matched controls. AlphadeltaC(csp)-APP immunoreactivity in small and medium size particulate elements were the main component colocalized with 30% of senile plaques in the inferior frontal gyrus and superior temporal gyrus of AD brains. In some control brains, alphadeltaC(csp)-APP immunoreactivity appeared to be associated with a clinical history of metabolic encephalopathy. Our results suggest that apoptosis contributes to cell death resulting from amyloidosis and plaque deposition in AD.     

Alzheimer''s disease. Beta-amyloid precursor protein expression in the nucleus basalis of Meynert.

The nucleus basalis of Meynert (nbM) was examined using immunocytochemistry for beta-amyloid precursor protein (beta APP) expression in Alzheimer's disease (AD). In mild AD cases, light labeling of the cell body and proximal processes was observed, and small intracellular structures were labeled rarely. In the more severe cases, intense cytoplasmic beta APP labeling was seen, often along with small beta APP-positive structures. Double-labeling experiments demonstrated that in the more severe cases these small structures were also decorated by a neurofibrillary tangle (NFT) antiserum. Other neurons in the severe cases showed incorporation of beta APP into large inclusions, which were also labeled with the NFT antiserum. However, some large inclusions in the severe cases were labeled by the NFT antiserum but contained no beta APP. Extraneuronal NFTs did not show beta APP labeling and did not react with an antibody to the beta-amyloid peptide. These results suggest that increased expression of beta APP coincides with intracellular NFT formation in the nbM, but that the formation of extraneuronal NFTs results in a loss of beta APP immunoreactivity.     

NMDA receptor/amyloid precursor protein interactions: a comparison between wild-type and amyloid precursor protein mutations associated with familial Alzheimer's disease

Two recent reports showed that amyloid precursor protein (APP) may contribute to postsynaptic mechanisms via the regulation of the surface trafficking of excitatory N-methyl-D-aspartate (NMDA) receptors. Here we have investigated the interactions and surface trafficking of NR1-1a/NR2A and NR1-1a/NR2B NMDA receptor subtypes with three APP mutations linked to familial Alzheimer's disease, APP695(Indiana), APP695(London) and APP695(Swedish). Flag-tagged mutated APP695s were generated and shown to be expressed at equivalent levels to wild-type APP695 in mammalian cells. Each APP mutant co-precipitated with NR1-1a/NR2A and NR1-1a/NR2B receptors following co-expression in mammalian cells. Further, as found for wild-type APP695, each enhanced NMDA receptor surface expression with no concomitant increase in total NR1-1a, NR2A or NR2B subunit expression. Thus these three familial APP mutations behave as wild-type APP695 with respect to their association with assembled NMDA receptors and their APP695-enhanced receptor cell surface trafficking.     

Phospholipase D1 is associated with amyloid precursor protein in Alzheimer's disease

Amyloid precursor protein (APP) is a widely expressed transmembrane protein of unknown function that is involved in the pathogenesis of Alzheimer's disease (AD). We investigated the involvement of phospholipase D (PLD) in the pathophysiology of AD. We showed dramatic upregulation of PLD1 immunoreactivity in reactive astroglial cells in brain tissue sections from authentic AD patients. Expression and activity of PLD1 were up-regulated in brain tissues from AD patients, especially caveolae membrane fraction, compared with those of control brains. Interestingly, PLD1 physically interacts and colocalizes with APP and caveolin-3. We found that APP was associated with the pleckstrin homology domain of PLD1, and the amyloid region of APP interacted with PLD. Elevated expression of APP stimulated PLD activity in human astroglioma cells. These results suggest that up-regulation of PLD might have a role in the neuronal pathology associated with AD.     

The amyloid precursor protein of Alzheimer disease in human brain and blood.

Studies of the metabolism and function of the amyloid precursor protein (APP) and its proteolytic fragment A beta in cultured cells, transgenic mice, and post-mortem brain tissue have advanced our understanding of Alzheimer disease (AD). However, the molecular pathogenesis of the disease is still not clear, and we are a long way from finding a cure for the disease. Studies carried out on human platelets and leukocytes have also helped shed light on APP and A beta metabolism and function. Platelet and leukocyte APP isoforms are processed using mechanisms similar to those in neuronal cells to generate A beta and soluble forms of APP. The activation of platelets and leukocytes leads to the secretion of APP and A beta, resulting in higher levels of these proteins in serum. APP and A beta in the circulation may be involved in the regulation of platelet function and in the modulation of immune responses. Because human platelets and lymphocytes produce all forms of APP and secrete amyloidogenic A beta peptides, these tissues may be useful in monitoring responses to therapeutic interventions directed at APP metabolism. Although not of neuronal origin, further studies on the more accessible ex vivo tissues, including platelets and leukocytes and other blood components, may reveal potential peripheral markers for AD and will further our understanding of the molecular pathogenesis of AD.     

Amyloid precursor protein and ubiquitin immunoreactivity in dystrophic axons is not unique to Alzheimer''s disease.

A distinctive feature of Alzheimer's disease (AD) is the presence of dystrophic neurites that immunoreact with antibodies to amyloid precursor protein (APP) and ubiquitin (Ub). The authors examined dystrophic axons (DA) present in other chronic conditions such as familial infantile neuroaxonal dystrophy (INAD), aging, cystic fibrosis, and biliary obstruction as well as in conditions of shorter duration such as human immunodeficiency virus (HIV) leucoencephalopathy, infarction and radiation therapy to determine whether APP and Ub immunoreactivity was unique to the DA of AD. A large number of DA immunoreacted with antibodies to the A4, C- and N-terminal regions of APP as well as to Ub. Ub and APP immunoreactivities often, but not always, colocalized. "Acute" DA generally reacted more intensely and in larger number with antibodies to APP than to Ub, whereas the reverse was true for "chronic" DA. Structureless DA immunostained diffusely. In DA with cores or granules, the Ub immunoreaction was occasionally limited to these structures, whereas reaction with antibodies to APP was more diffuse. In view of the contention that impairment of proteolysis is the common pathogenetic step in the formation of DA, Ub immunoreactivity in all DA may indicate a vicarious attempt to degrade accumulated components through an activation of the Ub system. The role of APP in the formation of DA remains to be determined.     

Phosphorylation of Alzheimer amyloid precursor protein by protein kinase C.

The beta/A4 amyloid precursor protein is a membrane protein with one transmembrane domain. The accumulation and deposition of beta/A4 amyloid protein in Alzheimer's disease is thought to be brought about by altered processing of beta/A4 amyloid precursor protein. Activation of protein kinase C and/or inhibition of protein phosphatases 1 and 2A results in an increase in the proteolytic processing and secretion of beta/A4 amyloid precursor protein. These effects might result either from phosphorylation of beta/A4 amyloid precursor protein by protein kinase C or from phosphorylation of components of the beta/A4 amyloid precursor protein processing apparatus. We have previously reported phosphorylation by protein kinase C of a synthetic peptide corresponding to part of the cytoplasmic domain of beta/A4 amyloid precursor protein. However, it was not known whether beta/A4 amyloid precursor protein holoprotein was phosphorylated in its native conformation in the cell membrane. Using a PC12 (rat pheochromocytoma) semi-intact cell system, we now report that mature isoforms of beta/A4 amyloid precursor protein are phosphorylated by protein kinase C at Ser655. Five COOH-terminal fragments which are generated by processing of mature beta/A4 amyloid precursor protein were also phosphorylated by protein kinase C at Ser655. The results support the idea that the beta/A4 amyloid precursor protein haloprotein is a physiological substrate for protein kinase C. These observations should facilitate our understanding of the relationship between altered protein phosphorylation and beta/A4 amyloid production.     

Immunohistochemical identification of thrombospondin in normal human brain and in Alzheimer''s disease.

Thrombospondin is part of a family of adhesive glycoproteins and is involved in a number of physiologic processes such as angiogenesis and neurite outgrowth. Immunohistochemical localization of thrombospondin in normal human brains was investigated in the hippocampus and inferior temporal cortex. Two antibodies (one polyclonal and one monoclonal) against thrombospondin-labeled microvessels, glial cells, and a subpopulation of pyramidal neurons. The distribution of thrombospondin staining in patients with Alzheimer's disease was found to be comparable to control subjects. However, in patients with Alzheimer's disease a subset of pyramidal neurons that may be vulnerable in Alzheimer's disease exhibited decreased staining. This decrease in the intensity of labeling might constitute a marker for a neuronal population prone to early degeneration. In addition, thrombospondin staining was demonstrated in senile plaques in Alzheimer's disease. These results suggest that thrombospondin may be involved in the process of neuronal degeneration and senile plaque formation.     

Brain amyloid in normal aging and cerebral amyloid angiopathy is antigenically related to Alzheimer''s disease beta-protein.

Amyloid deposition is a prominent feature of a number of brain disorders, in which amyloid fibrils are found within blood vessel walls, the neuropil (neuritic plaques), neurons (neurofibrillary tangles). These include Alzheimer's disease (AD), AD changes associated with Down's syndrome, neurologically asymptomatic amyloidosis, Parkinson dementia of Guam, hereditary cerebral hemorrhage with amyloidosis of Icelandic origin (HCHWA-I), hereditary cerebral hemorrhage with amyloidosis of Dutch origin (HCHWA-D), and sporadic cerebral amyloid angiopathy (SCAA). Recently it was shown that the amyloid deposits in AD, Parkinson dementia of Guam, and HCHWA-D are formed by a similar 4-kd polypeptide called beta-protein. Because the nature of the amyloid deposits in other types of cerebral amyloidosis is not known, we have conducted immunocytochemical studies on brains from autopsy cases of AD, HCHWA-D, SCAA and neurologically asymptomatic elderly individuals. Brains from two subjects without neurologic involvement were used as controls. Sections from these specimens were incubated with rabbit polyclonal antibodies against 1) a synthetic peptide of 28 residues (anti-SP28), homologous to the NH2-terminal sequence of the beta-protein, 2) the main amyloid component of the HCHWA-I, a variant of cystatin C, and 3) purified fraction of neurofibrillary tangles. In all cases, anti-SP28 antibody specifically stained amyloid deposits in leptomeningeal and cortical vessels and neuritic plaques. These findings demonstrate that the amyloid deposits of SCAA and aged brains are composed of a protein antigenically similar to AD, HCHWA-D, and Parkinson dementia of Guam beta-protein, suggesting that all of these clinically and etiologically different morbid conditions are pathogenetically related. On this basis, they can be tentatively grouped as beta-protein deposition diseases. In addition, we found that HCHWA-D and SCAA vessels were mainly affected, while in AD parenchymal involvement predominates. These differences in the localization and extent of beta-protein deposits may account from the predominance of vascular complications in HCHWA-D and SCAA and of dementia in AD.     

Beta-protein amyloid is widely distributed in the central nervous system of patients with Alzheimer''s disease.

To clarify the distribution, morphology, and density of amyloid deposits in patients with Alzheimer's disease (AD), tissue sections from various areas of the central nervous system of 14 patients with AD and from 20 nondemented aged controls were investigated immunohistochemically using anti-beta protein antiserum. beta-protein amyloid deposits were present not only in the cores of the senile plaques and in the vascular wall (amyloid angiopathy), but also in various sized plaque-shaped fibrillary, perivascular, subpial, and subependymal deposits. Amyloid deposits were found mainly in the cerebral cortex in nondemented controls, while in AD they were distributed widely in the regions that were not affected in nondemented controls. The positivity of amyloid deposits in AD was 100% in the cerebral cortex, hippocampus, amygdala, thalamus, caudate nucleus, claustrum, hypothalamus, nucleus basalis of Meynert, and cerebellar cortex. Putamen and brain-stem nuclei were affected frequently, and the spinal cord, dentate nucleus, and globus pallidus were sometimes (less than 50%) affected. This result provides an evidence that Alzheimer's disease is a beta-protein amyloidosis of the central nervous system. An assessment of the distribution of amyloid deposits should prove to be useful for the histopathologic diagnosis of AD.     

Coexistence of Alzheimer's amyloid precursor protein and amyloid protein in cerebral vessel walls

Polyclonal antibodies to synthetic peptides homologous to amino acid residues 45-62, 597-624, and 676-695 of the predicted sequence of Alzheimer's amyloid precursor protein (APP) were used to investigate the site of origin of APP, and the relationship between APP and amyloid protein in Alzheimer's disease (AD) and hereditary cerebral hemorrhage with amyloidosis, Dutch type (HCHWA-D). Cortical sections as well as homogenates of isolated leptomeningeal and cortical microvessels from three patients with AD, two patients with HCHWA-D, and two nondemented controls were probed. In vessel extracts of both groups of patients and the controls, APP was detected as a set of proteins with electrophoretic mobility of 105 to 135 kilodaltons. In cortical sections of all subjects, APP immunoreactivity was found in leptomeningeal and cortical vessel walls. In patients with AD and HCHWA-D, APP and amyloid fibrils coexisted in the same vessels. Moreover, APP immunoreactivity was found in association with 50% of senile plaques in AD brains, but was not evidenced in parenchymal amyloid deposits in patients with HCHWA-D. These data suggest that the vascular system is a source of APP and that the processing of APP into insoluble fibrils in AD and HCHWA-D may take place in situ.     

Calcium regulates the interaction of amyloid precursor protein with Homer3 protein

Ca²⁺ dysregulation is an important factor implicated in Alzheimer's disease pathogenesis. The mechanisms mediating the reciprocal regulation of Ca²⁺ homeostasis and amyloid precursor protein (APP) metabolism, function, and protein interactions are not well known. We have previously shown that APP interacts with Homer proteins, which inhibit APP processing toward amyloid-β. In this study, we investigated the effect of Ca²⁺ homeostasis alterations on APP/Homer3 interaction. Influx of extracellular Ca²⁺ upon treatment of HEK293 cells with the ionophore A23187 or addition of extracellular Ca²⁺ in cells starved of calcium specifically reduced APP/Homer3 but not APP/X11a interaction. Endoplasmic reticulum Ca²⁺ store depletion by thapsigargin followed by store-operated calcium entry also decreased the interaction. Interestingly, application of a phospholipase C stimulator, which causes inositol 1,4,5-trisphosphate-induced endoplasmic reticulum Ca²⁺ release, caused dissociation of APP/Homer3 complex. In human neuroblastoma cells, membrane depolarization also disrupted the interaction. This is the first study showing that changes in Ca²⁺ homeostasis affect APP protein interactions. Our results suggest that Ca²⁺ and Homers play a significant role in the development of Alzheimer's disease pathology.     

Aggregation of the amyloid precursor protein within degenerating neurons and dystrophic neurites in Alzheimer's disease.

Using a monoclonal antibody raised against purified, native, human protease nexin-2/amyloid precursor protein, which recognizes an amino terminal epitope on the amyloid precursor protein and detects all major isoforms of amyloid precursor protein, we examined the localization of the amyloid precursor protein within Alzheimer's and aged control brains. Very light cytoplasmic neuronal amyloid precursor protein staining but no neuritic staining was visible in control brains. In the Alzheimer's brain, we detected numerous amyloid precursor protein-immunopositive neurons with moderate to strong staining in select regions. Many neurons also contained varying levels of discrete granular, intracellular accumulations of amyloid precursor protein, and a few pyramidal neurons in particular appeared completely filled with amyloid precursor protein granules. "Ghost"-like deposits of amyloid precursor protein granules arranged in pyramidal, plaque-like shapes were identified. We detected long, amyloid precursor protein-immunopositive neurites surrounding and entering plaques. Many contained swollen varicosities along their length or ended in bulbous tips. Amyloid precursor protein immunoreactivity in the Alzheimer's brain was primarily present as granular deposits (plaques). The amyloid precursor protein granules do not appear to co-localize within either astrocytes or microglia, as evidenced by double-labeling immunohistochemistry with anti-glial fibrillary acidic protein and anti-leukocyte common antigen antibodies or Rinucus cummunicus agglutin lectin. Amyloid precursor protein could occasionally be detected in blood vessels in Alzheimer's brains. The predominantly neuronal and neuritic localization of amyloid precursor protein immunoreactivity indicates a neuronal source for much of the amyloid precursor protein observed in Alzheimer's disease pathology, and suggests a time-course of plaque development beginning with neuronal amyloid precursor protein accumulation, then deposition into the extracellular space, subsequent processing by astrocytes or microglia, and resulting in beta-amyloid peptide accumulation in plaques.     

Neurite outgrowth and the amyloid protein precursor

Neurite outgrowth in Alzheimer's disease may well be a multifactorial phenomenon. Recent evidence suggests possible roles for the protease inhibitor domain of the 751 amino acid amyloid precursor protein (APP), as well as for the 695 amino acid APP.     

Neurofibrillary tangles in some cases of dementia pugilistica share antigens with amyloid beta-protein of Alzheimer''s disease.

Formalin-fixed, paraffin-embedded temporal lobe sections from eight former boxers' brains were examined using an immunohistochemical method with antibodies to amyloid beta protein. In accord with recent observations in Alzheimer's disease, significant numbers of beta-protein immunoreactive neurofibrillary tangles (NFT) were observed in three cases. Most of these immunoreactive NFTs appeared to be tombstone tangles, although not all such tangles were stained. This immunoreaction was completely abolished by preincubation of antibodies with synthetic beta-protein peptides, and the identity of the immunostained NFTs was confirmed by polarization microscopy of sections counterstained with Congo red. However, it is not yet clear if the beta-protein antigens are, in fact, an integral part of paired helical filaments. These observations, together with our recent finding of beta-immunoreactive plaque-like lesions in dementia pugilistica, also emphasize the many similarities in pathology between this condition and Alzheimer's disease.     

Amyloid beta interacts with the amyloid precursor protein: a potential toxic mechanism in Alzheimer's disease

Amyloid beta protein (Abeta) deposition in the brain is a hallmark of Alzheimer's disease (AD). The fibrillar form of Abeta is neurotoxic, although the mechanism of its toxicity is unknown. We showed that conversion of Abeta to the fibrillar form markedly increased binding to specific neuronal membrane proteins, including amyloid precursor protein (APP). Nanomolar concentrations of fibrillar Abeta bound cell-surface holo-APP in cortical neurons. Reduced vulnerability of cultured APP-null neurons to Abeta neurotoxicity suggested that Abeta neurotoxicity involves APP. Thus Abeta toxicity may be mediated by the interaction of fibrillar Abeta with neuronal membrane proteins, notably APP. An Abeta-APP interaction reminiscent of the pathogenic mechanism of prions may thus contribute to neuronal degeneration in AD.     

Immunohistochemical study of cerebral amyloid angiopathy: use of an antiserum to a synthetic 28-amino-acid peptide fragment of the Alzheimer's disease amyloid precursor

A polyclonal antibody to a synthetic peptide representing a 28-amino-acid sequence of the previously isolated and described Alzheimer's disease amyloid precursor was raised in rabbits. The antibody was used in conjunction with an avidin-biotin-peroxidase technique to stain cerebral microvessels involved by amyloid angiopathy and senile "neuritic" plaque amyloid cores. The staining method has significant advantages over standard histologic techniques used to demonstrate brain amyloid and might have important practical applications in the study of microvascular lesions associated with cerebral amyloid angiopathy, as well as in studies on the pathogenesis of Alzheimer's disease or senile dementia of Alzheimer type.