APP Isoforms Research Articles

Xylosylation of alternatively spliced isoforms of Alzheimer APP by xylosyltransferase.

Acceptor affinities of UDP-D-xylose:proteoglycan core protein beta-D-xylosyltransferase (XT) recognition signals in synthetic L-APP and L-APLP2 homologous peptides were determined. The Michaelis-Menten constants (KM) of the L-APP peptide TENEGSGLTNIK and the L-APLP2 peptide SENEGSGMAEQK were 20.1 and 18.9 microM, respectively. Therefore, the peptides proved to be as good acceptors for XT as the bikunin aminoterminus homologous peptide (KM = 22 microM). Due to the occurrence of L-APP and L-APLP2 transcripts in human brain tissue, XT activity was measured in human liquor cerebrospinalis. Mean values were calculated as 0.22 mU/L in males and 0.47 mU/L in females without disturbance of blood brain barrier. In addition, in homogenized rat brain tissue a mean XT activity of 0.75 mU/L was determined. Furthermore, XT activity was investigated in 21 different human cell lines. In 7 cell lines an enzyme activity was not detected in either extracellular space or cytoplasma. Our findings indicate that XT is not ubiquitously expressed in human cell types.

Post-ischemic changes in the expression of Alzheimerʼs APP isoforms in rat cerebral cortex

A significant porportion (25%) of patients with Alzheimer's disease (AD) also shows vascular pathology. Recent ultrastructural studies demonstrated characteristic and extensive angio-architectural distortions of cerebral capillaries in AD brains. We examined the expression of APP mRNA isoforms of cerebral cortex after transient ischemia by middle cerebral artery occlusion, using RT-PCR. Neuronal damage and glial fibrillary acidic protein immunohistochemistry were also examined histologically. After transient ischemia, the Kunitz protease inhibitor-bearing isoforms (KPI-APP) were increased whereas APP 695, which lacks KPI domain, was decreased. Neuronal damage and GFAP-immunoreactive astrocytes were also observed. These results show that focal, transient ischemia alters KPI-APP/APP 695 ratio in cerebral cortex and this shift in APP isoforms could be related to neurodegeneration and/or activation of astrocytes during the ischemic process.

The effects of excitotoxicity on the expression of the amyloid precursor protein gene in the brain and its modulation by neuroprotective agents

This work has explored the relationship between excitotoxicity and the amyloid precursor protein gene (APP) which may be relevant to future therapeutic developments in Alzheimer's disease. The excitotoxic effects of kainic acid (KA) and pentylenetetrazole (PTZ) have been compared and contrasted on the two major mRNA isoforms of APP using in situ hybridization and quantitative analysis of gene expression in rat brain. The Kunitz Protease Inhibitor containing isoform APP 770 KPI+, the major glial cell isoform, has been shown to be stimulated after KA and was related to neuronal loss and astrocyte activation as gauged by GFAP mRNA. This was associated with reduced expression of APP695 KPI- isoform, the major neuronal isoform. These changes were not observed after PTZ where there was no neuronal loss and no glial reaction. The KA induced changes in APP were prevented by pretreatment with the non-competitive NMDA receptor antagonist dizocilpine and the barbiturate pentobarbitone, but not with the kappa-opioid receptor agonist enadoline. These findings were related to the suppression of seizures and the survival of neurons. In conclusion, excitotoxic stimulation leading to neuronal death was associated with increased expression of APP KPI+ mRNA and decreased APP KPI- mRNA, a finding which may relate to the plasticity of the central nervous system.

3-12-30 APP isoforms in platelets: A peripheral marker of Alzheimer disease and a measure of the disease severity

3-12-30 APP isoforms in platelets: A peripheral marker of Alzheimer disease and a measure of the disease severity

Cell-specific expression of β-amyloid precursor protein isoform mRNAs and proteins in neurons and astrocytes

The abnormal accumulation of β-amyloid (A β) in senile plaques appears to be a central pathological process in Alzheimer's disease. A β is formed by proteolysis of β-amyloid precursor protein (APP) with several isoforms generated by alternative splicing of exons 7, 8 and 15. A semi-quantitative reverse transcription (RT)-polymerase chain reaction (PCR) analysis showed that APP695 mRNA lacking exon 7 and 8 was most abundant in primary cultures of rat neurons, while APP770 and APP751 representing, respectively, the full length and exon 8 lacking isoforms predominated in cultured astroglial cells. Antisera AP-2 and AP-4 were produced by immunizing rabbits with keyhole limpet haemocyanin coupled with synthetic peptides representing KPI region APP301-316 and A β region APP670-686 of APP770, respectively. These polyclonal antisera were purified against the corresponding peptide using affinity chromatography. Western blot analysis of homogenates of relatively enriched neuronal and astroglial cultures showed that these antibodies discretely stained bands of proteins in a cell-specific manner. Dot-blot analysis using AP-2, AP-4 and 22C11 antibodies indicated that, in comparison with neurons, cultured astrocytes contained 3-fold greater KPI-containing APP isoform proteins. The amount of total APP proteins, which include both KPI-containing and KPI-lacking APP isoforms, was ≈90% higher in astrocytes than in neurons. Consistent with these in vitro findings in cultured astrocytes, in fimbria-fornix lesioned rat hippocampus, labelling with AP-2 antibody, which specifically reacts with KPI-containing APP proteins, was mainly observed in glial fibrillary acidic protein-positive reactive astrocytes in vivo. The results showed that APP isoforms are expressed in a cell type-specific manner in the brain and, since deposition of A β is closely associated with the expression of KPI-containing APP isoforms, provide further evidence for the involvement of astrocytes in plaque biogenesis.

Proteolytical processing of mutated human amyloid precursor protein in transgenic mice

The evidence that βA4 is central to the pathology of Alzheimer's disease (AD) came from the identification of several missense mutations in the amyloid precursor protein (APP) gene co-segregating with familial AD (FAD). In an attempt to study the proteolytical processing of mutated human APP in vivo, we have created transgenic mice expressing the human APP695 isoform with four FAD-linked mutations. Expression of the transgene was controlled by the promoter of the HMG-CR gene. Human APP is expressed in the brain of transgenic mice as shown by Western blot and immunohistology. The proteolytic processing of human APP in the transgenic mice leads to the generation of C-terminal APP fragments as well as to the release of βA4. Despite substantial amounts of βA4 detected in the brain of the transgenic mice, neither signs of Alzheimer's disease-related pathology nor related behavioural deficits could be demonstrated.

Altered Amyloid Protein Processing in Platelets of Patients With Alzheimer Disease

beta-Amyloid peptide, the core component of neuritic plaques in brain areas in patients with Alzheimer disease (AD), is 1 cleavage product of the beta-amyloid precursor protein (APP) in neurons and platelets. Alternate cleavage products of intact 140- to 150-kd APPs in platelets include nonamyloidogenic 120- to 130-kd and 110-kd isoforms. The possible differential significance of these 2 isoforms, structurally similar to protease nexin II, is unknown. To determine whether the ratio of the 120- to 130-kd APP isoform to the 110-kd APP isoform as processed in platelets correlates with the presence of AD and/or the apolipoprotein E4 (ApoE4) allele, which is a major risk factor for AD. The Alzheimer Disease Center at The University of Texas Southwestern Medical Center at Dallas. The APP isoforms were quantitated with the use of 2 different Western blot detection methods in platelets from 15 patients with AD and 19 control subjects in whom genotyping of apolipoprotein E was performed. The mean ratio of the 120- to 130-kd APP isoform to the 110-kd APP isoform in the patients with AD was significantly lower than that of the control subjects (5.98 vs 7.64; P = .03 [method 1] and 5.98 vs 7.92; P = .01 [method 2]) after adjusting for age and the increased incidence of ApoE4 in patients with AD. The lower APP ratios were also associated with increased age and with the presence of an ApoE4 allele. The APP processing in platelets of patients with AD is different from that of control subjects. This difference, largely caused by factors other than the ApoE4 genotype, may reflect chronic platelet activation in patients with AD. The use of these data to estimate "AD risk," by using the APP isoform ratio, indicates an odds ratio of 1.75, suggesting possible utility as an adjunct in the diagnosis of AD. Moreover, these findings may relate to analogous alterations in APP processing that may occur in brain areas affected by AD.

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.

Amyloid precursor proteins protect neurons of transgenic mice against acute and chronic excitotoxic injuries in vivo

The β-amyloid protein precursor (APP) is well conserved across different species and may fulfill important physiological functions within the CNS. While high-level neuronal expression of amyloidogenic forms of human APP results in β-amyloid production and neurodegeneration, lower levels of neuronal human APP expression in neurons of transgenic mice may primarily accentuate physiological functions of this molecule. To assess the neuroprotective potential of human APP in vivo, mice from seven distinct transgenic lines expressing different human APP isoforms from the neuron-specific enolase promoter were challenged with systemic kainate injections ( n=30) or transgene-mediated glial expression of gp120 ( n=32), an HIV-1 protein capable of inducing excitotoxic neuronal damage. To quantitate human APP-mediated neuroprotection, the area of neuropil occupied by presynaptic terminals and neuronal dendrites in the neocortex and hippocampus of each mouse was determined using laser scanning confocal microscopy of double-immunolabelled brain sections and computer-aided image analysis. Compared with gp120 singly transgenic controls, mice from three of three human APP751/gp120 bigenic lines expressing the 751 amino acid form of human APP at low levels showed significant protection against degeneration of presynaptic terminals; two of these lines also showed significantly less damage to neuronal dendrites. Two of three human APP695/gp120 bigenic lines expressing human APP695 at low levels were protected against presynaptic and dendritic damage, whereas one low expressor line and a human APP695/gp120 bigenic line expressing human APP695 at higher levels showed no significant protection. In the corresponding human APP singly transgenic lines, overexpressing only specific human APP isoforms, significant protection against kainate-induced degeneration of presynaptic terminals and neuronal dendrites was found in two of three human APP751 lines and not in any of the four human APP695 lines tested. These results indicate that human APP can protect neurons against chronic and acute excitotoxic insults in vivo and that human APP isoforms differ in their neuroprotective potential, at least with respect to specific forms of neural injury. It is therefore possible that impairments of neuroprotective human APP functions or aberrant shifts in human APP isoform ratios could contribute to neurodegeneration.

Expression of beta-amyloid precursor protein mRNAs following transient focal ischaemia.

beta-Amyloid precursor protein (beta APP) can be alternatively processed to result in release of either neurotoxic amyloid beta-peptide, or secreted forms of beta APP, which might have a role in neuronal plasticity and survival. Four different forms of beta APP mRNAs have been described in rodents: APP695, APP714, APP751 and APP770. The two larger forms contain a Kunitz-type serine protease inhibitor domain (KPI). Since previous studies have shown increased APP immunoreactivity following brain ischaemia, we used in situ hybridization histochemistry to determine whether induction of any APP mRNA transcripts take place following focal brain ischaemia. While the hybridization signal of all APP isoforms in the infarct was lost 4-24 h following the insult, APP770 mRNA was slightly upregulated in the ipsilateral cortex and striatum 3 days after 90 min ischaemia. At 7 days post-ischaemia APP770 and APP751 mRNAs were induced in the infarct core and in a thin perifocal zone. KPI-containing APP forms are differentially induced following focal brain ischaemia, possibly as a neuroprotective response to neuronal injury.

Serum deprivation alters the expression and the splicing at exons 7, 8 and 15 of the beta-amyloid precursor protein in the C6 glioma cell line.

Amyloid deposition characterizes the pathological lesions of Alzheimer's disease. We investigated the effect of serum deprivation on the regulation of beta-amyloid precursor protein (APP) mRNA expression in C6 glioma cells. Serum deprivation increased APP mRNA levels approximately 4-fold over controls. This increase was accompanied by changes in the pattern of alternative splicing, including the novel alternatively spliced site at exon 15. The proportion of isoforms containing exons 7 and 8 significantly increased from 61% to 68%, while isoforms lacking these exons decreased from 14% to 8%. The proportion of leukocyte-derived APP, which is a novel alternatively spliced isoform lacking exon 15, significantly increased from 19% to 40%. Among the six major isoforms produced by the two independent splicing sites, L-APP752 which contains exons 7 and 8, but lacks exon 15, increased the most (approximately 10-fold). Our findings provide evidence linking APP expression to alterations in alternative splicing at exon 15. These results demonstrate that in glial cells, APP mRNA regulation involves the alteration in alternative splicing at exons 7, 8 and 15, suggesting that not only increased expression but also an imbalance in the relative abundance of the six APP isoforms in stressed condition might affect the amyloidogenesis in Alzheimer's disease.

Beta-amyloid precursor protein (APP) and APP-RNA are rapidly affected by glutamate in cultured neurons: selective increase of mRNAs encoding a Kunitz protease inhibitor domain.

Alternative splicing of beta-amyloid precursor protein (APP) RNA generates APP isoforms with or without a Kunitz protease inhibitor (KPI) domain. Previously, we showed that KPI (+) APP RNA, but not KPI (-) APP RNA, is upregulated in response to experimental lesions in which neurotoxicity is dependent on NMDA receptor activation and in Alzheimer's disease hippocampus. Recent studies by Mucke et al. (1995) showed that neuronal expression of human KPI (+) APP, but not KPI (-) APP, in transgenic mice is neuroprotective against experimental lesions. In this study we examined the direct effects of the excitotoxic amino acid Glu on alternatively, spliced APP RNAs and the corresponding protein isoforms in cultured rat cortical neurons. Glu treatment rapidly induced (4.5 h) KPI (+) APP RNA but not KPI (-) APP RNA. Induction of KPI (+) RNA preceded Glu-induced neuronal cell death and was partially blocked by an NMDA-receptor antagonist. In contrast to the RNA, cellular levels of KPI (+) APP were not changed by 4.5 h of Glu treatment. Instead, the cellular full-length form of the protein KPI (-) APP was reduced by approximately 50% after 2 h of Glu treatment and remained depleted after 24 h of treatment. Cellular levels of KPI (+) forms of amyloid precursor-like protein 2 (APLP2) were not changed by Glu treatment. Our data are consistent with the hypothesis that sustained NMDA-receptor activation can regulate alternative splicing of the APP pre-mRNA in neurons.

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.

Cell-surface beta-amyloid precursor protein stimulates neurite outgrowth of hippocampal neurons in an isoform-dependent manner

beta-Amyloid precursor protein (beta APP) is an integral membrane polypeptide expressed in many neural and non-neural cells. beta APP occurs in part at the cell surface and undergoes proteolytic processing to release the large soluble ectodomain (APPs) and the amyloid beta-peptide (A beta), both of which have apparent trophic activity in vitro. Despite intense interest in beta APP expression and metabolism, there is limited knowledge about the function mediated by beta APP inserted at the cell-surface. We established a coculture system in which beta APP-transfected CHO cells serve as a substrate for the growth of primary rat hippocampal neurons. Compared to nontransfected CHO cells, the increased surface beta APP of the transfectants stimulated short-term neuronal adhesion and longer-term neurite outgrowth, whereas the increased amount of secreted APPs and A beta in conditioned medium produced no such effects when neurons were grown either on untransfected CHO cells or on a polylysine substrate. Moreover, a peptide which has been shown to block the trophic effects of secreted APPs (Ninomiya et al., 1993) failed to interrupt the neurite promoting activity mediated by the surface-expressed beta APP. Surface-expressed beta APP751 or beta APP770 isoforms mediated more neurite outgrowth than did the beta APP695 isoform. Antibody blocking and regional deletion experiments indicated that the mid-region of the beta APP ectodomain (residues 361-648) is involved in promoting neurite outgrowth. We conclude that surface-expressed cellular beta APP has a neurite-promoting function which is distinct from the trophic function of the secreted beta APP derivatives and may have special significance during brain development.

The Vacuolar H+-ATPase Inhibitor Bafilomycin A1 Differentially Affects Proteolytic Processing of Mutant and Wild-type β-Amyloid Precursor Protein

We analyzed the effect of the vacuolar H(+)-ATPase inhibitor bafilomycin A1 (bafA1) on the processing of beta-amyloid precursor protein (beta APP). In kidney 293 cells stably transfected with the wild-type beta APP cDNA, bafA1 caused a stabilization of mature beta APP and its 10-kDa COOH-terminal fragment. Moreover, it caused a 2-3-fold increase in secretion of soluble APP and amyloid-beta protein (A beta). Interestingly, bafA1 treatment of cells transfected with a mutant beta APP isoform that occurs in a Swedish kindred with familial Alzheimer's disease resulted in a decrease of A beta production and no increase of soluble APP secretion. Identical results were obtained when the effect of bafA1 was analyzed on fibroblasts derived from affected versus unaffected members of the Swedish family. These data demonstrate a differential effect of bafA1 on the production of A beta derived from wild-type or Swedish mutant beta APP. Radiosequencing of A beta derived from bafA1-treated cells expressing wild-type beta APP revealed a marked increase of A beta peptides starting at amino acids phenylalanine 4 and valine -3 and a relative decrease of A beta molecules beginning at the typical NH2 terminus of aspartate 1. Cells transfected with the Swedish mutation and treated with bafA1 did not produce these alternative A beta peptides, so that bafA1 treatment resulted in a decrease of A beta starting at aspartate 1. Our data indicate that multiple proteases are able to cleave A beta at or near its NH2 terminus. Inhibition of the protease cleaving at aspartate 1 by bafA1 and perhaps other similar agents can result in an increase of alternatively cleaved peptides.

Secretory cleavage of β-amyloid precursor protein in the cerebral white matter produces amyloidogenic carboxyl-terminal fragments

To elucidate the metabolic process generating amyloid-β protein (Aβ) from β-amyloid precursor protein (APP) in human brain, we partially purified secretory forms and carboxyl-terminal fragments (CTFs) of APP from the white matter of a Down's syndrome brain. We obtained secretory forms of APP which lack the entire Aβ sequence and CTFs which contain the full-length Aβ from the cerebral white matter. Some Aβ-lacking secretory APP isoforms in the white matter were derived from APP695. These results suggest that amyloidogenic CTFs can be produced by secretory cleavage of APP which is anterogradely transported through the axon in human brain.

Beta-Amyloid precursor protein isoforms show correlations with neurones but not with glia of demented subjects.

Post-mortem cerebral cortex from 15 demented patients was specially collected to minimise autolysis and two membrane fractions and one soluble fraction were quantitatively examined for the major species of beta-amyloid precursor protein (APP) of high apparent molecular mass (> or = 80 kDa) together with the major mRNA species encoding APP isoforms. The number of pyramidal neurones and astrocytes, putative biochemical indices of interneurones and pyramidal neurones, and choline acetyl transferase activity were also determined. Multiple regression analysis has been used to investigate intercorrelations of APP species with biochemical and morphometric measures, free of any effects of confounding demographic variables. Subjects with Alzheimer's disease showed a loss of cholinergic activity and D-aspartate uptake compared with patients with other causes of dementia. The major finding of the study is that measures of neurones rather than astrocytes most closely correlate with the concentration of APP. Pyramidal cell numbers were positively correlated with mRNA for APP695. APP in the soluble fraction showed a negative correlation with pyramidal cell numbers and cholinergic activity. These results indicate that neurones within the cerebral cortex are the major source of APP, and that secretion of APP is dependent upon cortical pyramidal neuronal activity and cholinergic activity.

APP gene family: unique age-associated changes in splicing of Alzheimer's βA4-amyloid protein precursor

The βA4-amyloid protein precursor (APP) is a transmembrane glycoprotein that is the source of the characteristic βA4-amyloid deposits of Alzheimer brains. It exists in eight isoforms generated by alternative splicing of exons 7, 8 and 15, of which the L-APP mRNAs lacking exon 15 are significantly expressed in non-neuronal cells and tissues, but not in neurones. Recently, it was shown that APP is a member of a multigene family of which the amyloid precursor-like protein 2 (APLP2) is the nearest relative. Analysis of APLP2 expression revealed regulated alternative splicing of the Kunitz protease inhibitor domain (KPI, homologous to exon 7 of APP) and a non-homologous insert of 12 amino acids on the NH 2-terminal side of the transmembrane domain. While expression of the KPI encoding exon of APLP2 is abundant in neurones and thus differs from APP, L-APLP2 mRNA isoforms lacking the latter, non-homologous insert show a tissue-specific expression pattern similar to that of exon 15 of APP. Comparison of alternatively spliced APP and APLP2 mRNA isoforms in rat brain regions from early post-natal and adult rats revealed significantly higher relative amounts of KPI-encoding APP isoforms in the adult rat brain and an even more pronounced augmentation of L-APP mRNAs. Both effects were not observed for APLP2. This indicates an APP-specific age-associated regulation pattern within the APP gene family which has intriguing implications for the development of Alzheimer's disease in humans.

Expression of β-amyloid precursor protein (APP) in human dorsal root ganglia

The present study reports the occurrence and localization of β-amyloid precursor protein (APP) immunoreactivity (IR) in human lumbar dorsal root ganglia of healthy adult subjects (age range 25–43 years). To ascertain that ganglionic cells displayed APP IR, neurofilament (NFP) and S-100 proteins (S100P) were studied in parallel. Immunoblotting revealed four or five major proteins with apparent molecular masses between 100–125 kDa, which corresponded with the different full-length APP isoforms. Moreover, an additional protein of approximately 55 kDa was detected. Selective APP IR was observed restricted to the satellite glial cell cytoplasms whereas neuron cell bodies resulted unlabeled. Moreover, some intraganglionic nerve fibers also displayed APP IR, apparently labelling Schwann cells. No individual differences among subjects were observed neither in the pattern of APP IR distribution, nor in the intensity of APP IR. Although it remains to be demonstrated whether or not human primary sensory neurons express APP, present results strongly suggest that supporting glial cells may be a primary source of APP or any related peptide, at least in adult healthy people. The functional and clinical relevance of these findings, if any, remain to be clarified.

Secretory form of β-amyloid precursor protein is much abundantly contained in the cerebral white matter in human brain

To elucidate the metabolic process of β-amyloid precursor proteins (APP) in cerebral gray and white matter of the human brain, we compared the content and characteristics of secretory APP between these two parts. The white matter contained much more secretory APP than the gray matter in both a control and a Down's syndrome brain, but no difference in the characteristics of the APP isoforms was detected. Our results suggest that, in the human brain, a considerable amount of APP is carried by axonal transport, during which some of the APP isoforms are processed to their secretory forms.