Down-regulation of the amyloid protein precursor of Alzheimer's disease by antisense oligonucleotides reduces neuronal adhesion to specific substrata

The hallmark of Alzheimer's disease is the cerebral deposition of amyloid which is derived from the amyloid precursor protein (APP). The function of APP is unknown but there is increasing evidence for the role of APP in cell-cell and/or cell-matrix interactions. Primary cultures of murine neurons were treated with antisense oligonucleotides to down-regulate APP. This paper presents evidence that APP mediates a substrate-specific interaction between neurons and extracellular matrix components collagen type I, laminin and heparan sulphate proteoglycan but not fibronectin or poly-

Full-size image

-lysine. It remains to be determined whether this effect is the direct result of APP-matrix interactions, or whether an intermediary pathway is involved.     

The binding of basic fibroblast growth factor to ischaernic neurons in the rat

Transient occlusion of the right middle cerebral artery for 15 min produced a small ischaemic lesion in the dorsal portion of the right striatum in rats as seen on days 3, 7 and 14 post-operatively. The lesions consisted mainly of reactive astrocytes and 'ischaemic neuron's with chromatin-condensed (pyknotic) nuclei and homogenously eosinophilic cytoplasm. The incubation of tissue sections with basic fibroblast growth factor (bFGF) followed by anti-bFGF, or with biotinylated bFGF without anti-bFGF, labelled virtually all ischaemic neurons, indicating that bFGF had bound to the latter. The pretreatment of sections with heparitinase prevented the binding of bFGF to these cells, suggesting that the chemical substrate for the bFGF binding was heparan sulphate. In light of the findings that many normal-looking neurons were observed in the corresponding portion of the right striatum in most rats on post-operative days 28 and 90, the appearance of bFGF-binding sites in ischaemic neurons may contribute to the repair process of injured neurons.     

Association and release of the amyloid protein precursor of Alzheimer's disease from chick brain extracellular matrix.

The amyloid protein precursor (APP) of Alzheimer's disease was found to bind saturably (Kd = 60 nM) to embryonic chick brain extracellular matrix (ECM). The binding of APP to ECM was not inhibited by 10 micrograms/ml heparin or heparan sulfate. However, pretreatment of cells with 1 mM 4-methylumbelliferyl-beta-D-xyloside, an inhibitor of proteoglycan biosynthesis, reduced the number of APP binding sites on the ECM by 80%. The binding of APP to ECM was also inhibited by pretreatment with chlorate, an inhibitor of glycan sulfation, and heparitinase, which digests the carbohydrate component of heparan sulfate proteoglycans. These results suggest that APP binds with high affinity to one or more heparan sulfate proteoglycans. Acidic and basic fibroblasts growth factor (FGF) also bound to chick ECM. When ECM was incubated with a protease associated with the enzyme AChE (AChE-AP), APP and acidic FGF were released intact from the matrix. The AChE-AP was at least 100-fold more potent in releasing APP from ECM than other trypsin-like proteases (trypsin, plasmin, thrombin). The action of the AChE-AP was inhibited by glia-derived nexin (protease nexin I) and by human brain APP at low nanomolar concentrations. These results suggest that in vivo an AChE-AP may cleave ECM proteins to regulate the availability of soluble APP or other factors bound to the ECM.     

Influence of glial-derived matrix molecules, especially chondroitin sulfates, on neurite growth and survival of cultured mouse embryonic motoneurons

Mechanisms controlling neuronal survival and regeneration play an important role during development, after birth, and under lesion conditions. Isolated embryonic mouse motoneurons have been a useful tool for studying such basic mechanisms. These cultured motoneurons depend on extracellular matrix (ECM) molecules, which are potent mediators of survival and axonal growth and guidance in the CNS and in vitro, exhibiting either attractive or repellent guidance cues. Additionally, ECM proteoglycans and glycoproteins are components of the glial scar acting as a growth barrier for regenerating axons. Compared with CNS axon outgrowth, less is known about the cues that guide motoneurons toward their peripheral targets. Because we are interested in the effects of glial-derived chondroitin sulfate proteoglycans (CSPGs), we have worked out a model system for investigating the influences of glial-derived matrix molecules on motoneuron outgrowth and survival. We used cultured embryonic mouse motoneurons to investigate axon growth effects of matrix molecules produced by the glial-derived cell lines A7, Neu7, and Oli-neu primary astrocytes as well as the immortalized Schwann cell line IMS32. The results indicate that molecules of the ECM, especially chondroitin sulfates, play an important role as axon growth-promoting cues. We could demonstrate a modifying effect of the matrix components on motoneuron survival and caspase3-induced apoptosis.     

Association between vascular basement membrane components and the lesions of Alzheimer's disease.

A relationship between the microvasculature and Alzheimer senile plaques has been suggested by several lines of evidence. Besides close anatomic and biochemical relationships, both extrinsic (fibronectin) and intrinsic [heparan sulfate proteoglycan (HSPG)] components of the vascular basement membrane (VBM) have been colonized with amyloid plaques. The present study was designed to examine the association between three intrinsic components of the VBM [HSPG, collagen type IV (CIV), and laminin] and the histopathologic lesions of Alzheimer's disease (AD). Six cases with neuropathologically confirmed AD were immunocytochemically labeled for the presence of HSPG, CIV, laminin, or tau-2 (a marker for degenerating neurites) and examined at the light and electron microscopic levels. For light microscopic analyses, sections were counterstained with a fluorescent marker for amyloid. The present study illustrates an involvement of VBM components in the lesions associated with AD. First, we replicate our previous finding that HSPG antibodies immunolabel a subset of neurons; ultrastructural analyses indicate that at least some of these are actually extracellular neurofibrillary tangles. Second, we report that CIV and laminin immunoreaction product was not associated with neurons but did label several perivascular cells with the morphologic characteristics of microglia. Finally, we demonstrate that all three intrinsic VBM components, CIV and laminin as well as HSPG, are localized to senile plaques. Both light and electron microscopic studies indicate that the VBM components are associated with amyloid rather than degenerating neurites. These findings suggest that the VBM or its components may play a role in the AD pathogenetic cascade.     

Individual isoforms of the amyloid βprecursor protein demonstrate differential adhesive potentials to constituents of the extracellular matrix

The amyloid β precursor protein (AβPP) can exist as a membrane-bound glycoprotein which modulates neural cell adhesion. The adhesion of clones of the AtT20 mouse pituitary cell line, transfected with cDNA coding for the 695 (AβPP₆₉₅) and 751 (AβPP₇₅₁) amino acid forms of the protein, to individual components of the extracellular matrix was determined using a centrifugal shear assay. On laminin, poly-L-lysine, fibronectin, and uncoated glass substrata, the cells transfected with AβPP₆₉₅ (6A1 cells) demonstrated a 50% increase in adhesivity over non-transfected cells, while those transfected with AβPP₇₅₁ (7A1 cells) showed a significant decrease in adhesion. There was, however, a significant increase in the adhesive strength of the 7A1 cells to collagen type IV with no change in the adhesivity of the 6A1 cells when compared with control. These changes in adhesivity could be attributed to changes in the levels of the membrane-bound protein and were not due to the interaction of soluble AβPP with elements of the extracellular matrix. These studies provide evidence for differential adhesivities of the constituent AβPP isoforms and the possible role of the Kunitz protease inhibitor (KPI) domain in influencing the adhesive properties of the protein backbone. J. Neurosci. Res. 49:154–160, 1997. © 1997 Wiley-Liss, Inc.     

The amyloid precursor protein of Alzheimer's disease and the Abeta peptide

Alzheimer's disease is characterized by the accumulation of beta amyloid peptides in plaques and vessel walls and by the intraneuronal accumulation of paired helical filaments composed of hyperphosphorylated tau. In this review, we concentrate on the biology of amyloid precursor protein, and on the central role of amyloid in the pathogenesis of Alzheimer's disease. Amyloid precursor protein (APP) is part of a super-family of transmembrane and secreted proteins. It appears to have a number of roles, including regulation of haemostasis and mediation of neuroprotection. APP also has potentially important metal and heparin-binding properties, and the current challenge is to synthesize all these varied activities into a coherent view of its function. Cleavage of amyloid precursor protein by beta-and gamma-secretases results in the generation of the Abeta (betaA4) peptide, whereas alpha-secretase cleaves within the Abeta sequence and prevents formation from APP. Recent findings indicate that the site of gamma-secretase cleavage is critical to the development of amyloid deposits; Abeta1-42 is much more amyloidogenic than Abeta1-40. Abeta1-42 formation is favoured by mutations in the two presenilin genes (PS1 and PS2), and by the commonest amyloid precursor protein mutations. Transgenic mouse models of Alzheimer's disease incorporating various mutations in the presenilin gene now exist, and have shown amyloid accumulation and cognitive impairment. Neurofibrillary tangles have not been reproduced in these models, however. While aggregated Abeta is neurotoxic, perhaps via an oxidative mechanism, the relationship between such toxicity and neurofibrillary tangle formation remains a subject of ongoing research.     

Inhibition of neurite outgrowth following intracellular delivery of anti-GAP-43 antibodies depends upon culture conditions and method of neurite induction

NB2a/dl neuroblastoma cells acquire a neuronal phenotype in response to several differentiating agents, including dibutyryl cAMP (dbcAMP) and the withdrawal of serum. As shown previously, antibodies to the growth-associated protein, GAP-43, introduced intracellularly using a lipid carrier, blocked the differentiation induced by dbcAMP. Antibodies to GAP43, at a low concentration, also blocked neurite out-growth induced by serum withdrawal when cells were grown on a relatively unadhesive substrate. On more adhesive substrates such as poly-L-lysine and laminin, however, anti-GAP-43 antibodies had less of an effect on neurite outgrowth. Previous studies have shown that the increased adhesivity of laminin allows a small but significant population of neurites to grow from serum-deprived cells, even in the presence of the microtubule-depolymerizing drug, colchicine. The outgrowth of this population of neurites was blocked by antibodies to GAP-43. These results are in conformity with recent studies showing that the requirement for GAP-43 in neuritogenesis may be related to membrane adhesiveness, and may contribute to an understanding of some of the apparent discrepancies in the literature concerning the involvement of GAP-43 in neuronal differentiation. © 1995 Wiley-Liss, Inc.     

Distribution of amyloid beta protein precursor in the Alzheimer's disease brain

In order to clarify the distribution and pathological changes of the amyloid beta protein precursor (betaAPP), 10 Alzheimer's disease (AD) brains and seven normal control brains were examined by immunocytochemistry and in situ hybridization histochemistry. All betaAPP isoforms were distributed evenly in neuronal cell bodies and their axons and dendrites. The betaAPP-positive neuronal processes showed mesh-like networks. In AD brains, betaAPP-positive neurons and mesh-like networks were generally decreased in spite of some intensely labeled neurons. All betaAPP isoforms accumulated in neuronal processes, dystrophic neurites and senile plaques. In situ hybridization histochemistry confirmed that all isoforms of betaAPP were expressed in neurons in control brains. In AD brains, the betaAPP mRNA signal was generally decreased besides some intense signal neurons corresponding to immunostaining findings. Few astrocytes expressed betaAPP. Thus, uniform expression and distribution of betaAPP were disturbed in AD brains showing uneven decreases or increases of neuronal betaAPP expression in individual neurons and betaAPP accumulation in neurons, neuronal processes and abnormal structures including dystrophic neurites, senile plaques and neurofibrillary tangles.     

Differentiation of PC12 cells with nerve growth factor is associated with induction of transin synthesis and release.

We have identified and characterized a calcium-dependent metalloproteinase which is induced in rat pheochromocytoma cells (PC12 cells) during differentiation with nerve growth factor (NGF). Assays of proteolytic activity in media from differentiated PC12 cell cultures revealed a NGF-dependent increase in the activity of a proteinase which has a molecular weight of 62 kDa. Studies using serine, thiol, and metalloproteinase inhibitors demonstrated that the secreted enzyme is a metalloproteinase. Treatment of culture supernatants with aminophenylmercuric acid (APMA), a known activator of metalloproteinases, resulted in a decrease in the molecular weight of the proteinase. Western blot analysis of culture media from NGF-treated PC12 cells using an antibody directed against a synthetic peptide of rat transin identified this metalloproteinase as transin. Treatment of PC12 cells with acidic and basic fibroblast growth factor (FGF) resulted in distinct morphological changes as well as transin release. Incubation with epidermal growth factor (EGF) did not induce transin release. Dexamethasone inhibited the induction of transin release by NGF. 35S-methionine labeling and immunoprecipitation of newly synthesized proteins from culture supernatants confirmed that NGF induced the synthesis of this enzyme 8 hr after NGF treatment. The NGF-dependent induction of transin, a calcium-dependent metalloproteinase which degrades type IV collagen, laminin, and fibronectin suggests that transin may function to degrade the surrounding extracellular matrix during the invasive process of axonal elongation in neuronal development thereby allowing the movement of growth cones and axons toward specific targets.     

Studies on the action of nerve growth factor. II. Neurotubule protein levels during neurite outgrowth.

The neurotubule protein content of chick embryo 8-day dorsal root and 14-day sympathetic ganglia, induced to extend neurites in the presence of Nerve Growth Factor, was determined by the time-decay colchicine binding assay procedure and by two independent polyacrylamide gel electrophoresis systems. The initial level of neurotubule protein in dorsal root ganglia was approximately 16% of the total soluble protein. This value was constant during Nerve Growth Factor-mediated neurite outgrowth. The initial level of neurotubule protein in sympathetic ganglia was also approximately 16%, and was unchanged during neurite outgrowth. In addition, C1300 mouse neuroblastoma cells, induced to extend neurites in 0.1% serum, also did not exhibit a change in neurotubule protein concentration, which remained approximately 9% of the total soluble protein.     

Effect of cell density on intracellular levels of the Alzheimer's amyloid precursor protein

The precise function of APP (Alzheimer's amyloid precursor protein) remains to be fully elucidated, but various lines of evidence suggest that it may be involved in cell adhesion processes. Because APP is a transmembrane glycoprotein, variations in its expression level may have direct bearing on its putative role in cell adhesion. Our results revealed that although APP levels did not change markedly with increasing cell density (ICD), there was a small but reproducible increase in APP expression at subconfluent conditions. Higher expression APP levels led to corresponding increases in the amount of APP processed and secreted APP (sAPP) released into the cell media. Given that phorbol esters stimulate the non-amyloidogenic pathway at the expense of reducing production of Abeta (the peptide found deposited as neuritic plaques in the brains of patients with Alzheimer's disease), thus providing an interesting therapeutic focus, we tested the effect of the phorbol 12-myristate 13-acetate (PMA) on APP processing at ICD. PMA not only stimulated sAPP release at all densities tested, but also produced a corresponding decrease in the intracellular levels of APP. Further experimentation revealed that increased APP expression with ICD was dependent on factors present in conditioned medium. Interestingly, exposing cells to the Abeta peptide itself could mimic these results, thus providing evidence for a potential positive feedback mechanism between Abeta production and intracellular APP levels.     

Proteolytic processing of the Alzheimer's disease amyloid precursor protein in brain and platelets

Proteolytic processing of the amyloid precursor protein by beta -and gamma-secretases results in the production of Alzheimer's disease (AD) Abeta amyloid peptides. Modulation of secretase activity is being investigated as a potential therapeutic approach. Recent studies with human brain have revealed that the beta-secretase protein, BACE, is increased in cortex of AD patients. Analysis of betaCTF (or C99), the amyloid precursor protein (APP) product of BACE cleavage that is the direct precursor to Abeta, shows it is also elevated in AD, underlying the importance of beta-secretase cleavage in AD pathogenesis. The C-terminal product of gamma-secretase cleavage of APP, epsilonCTF (or AICD), is enriched in human brain cortical nuclear fractions, a subcellular distribution appropriate for a putative involvement of APP cytosolic domain in signal transduction. Analysis of AD cortex samples, particularly that of a carrier of a familial APP mutation, suggests that processing of APP transmembrane domain generates an alternative CTF product. All these particularities observed in the AD brain demonstrate that APP processing is altered in AD. The transgenic mouse model Tg2576 seems to be a promising laboratory tool to test potential modulators of Abeta formation. Indeed, C-terminal products of alpha-, beta-, and gamma-secretase cleavage are readily detectable in the brain of these transgenic mice. Finally, the finding of the same secretase products in platelets and neurons make platelets a potentially useful and easily accessible clinical tool to monitor effects of novel therapies based on inhibition of beta- or gamma-secretase.     

The precursor pro-nerve growth factor is the predominant form of nerve growth factor in brain and is increased in Alzheimer's disease

Nerve growth factor (NGF) is important for regulation, differentiation, and survival of peripheral and central nervous system neurons, including basal forebrain cholinergic neurons (BFCN) which degenerate in Alzheimer's disease (AD). Mature NGF protein is processed from a larger precursor, proNGF. We demonstrate that proNGF is the predominant form of NGF in mouse, rat, and human brain tissue, whereas little or no mature NGF is detected. Previous reports showed NGF protein, measured by ELISA, is increased in AD BFCN target regions such as hippocampus and cortex. Using Western blotting, we demonstrate a twofold increase in proNGF in AD parietal cortex compared to controls, indicating that it is this precursor form, proNGF, that accumulates in AD. This increase may reflect either a role for biologically active proNGF or posttranslational disturbances in NGF biosynthesis that decrease the processing of proNGF to mature NGF in AD.     

Induction of neurite outgrowth in the IMR-32 human neuroblastoma cell line by nerve growth factor

The nerve growth factor protein (NGF) stimulates neurite outgrowth from embryonic sensory ganglia and sympathetic ganglia at all stages of development. In addition, NGF is required for the maintenance of the differentiated state in adult sympathetic ganglia. A clonal cell line, IMR-32, derived from a human neuroblastoma was found to contain a population of cells that respond to NGF by exhibiting morphological differentiation. The effect of NGF on these cells is compared with that of other agents known to induce differentiation of IMR-32, including glioma-conditioned media.     

Effects of insulin, insulin-like growth factor-II and nerve growth factor on neurite outgrowth in cultured human neuroblastoma cells

The identification of biologically important and chemically well-defined substances that can promote axon and dendrite formation would improve present understanding of the development of the nervous system. Physiological concentrations of insulin and insulin-like growth factor-II (IGF-II) reversibly enhanced neurite outgrowth (NTO) in human neuroblastoma SH-SY5Y cells cultured in media with and without serum. Nerve growth factor (NGF), in contrast, did not enhance NTO in serum-free media. Furthermore, anti-NGF antiserum inhibited NGF but not insulin-enhanced NTO. Insulin increased [3H]leucine and [3H]uridine uptake. These increases, together with increased NTO, were inhibited by cycloheximide and actinomycin D, respectively. The inhibition of NTO by cycloheximide was reversible. Human neuroblastoma cell lines that were responsive by NTO to NGF were also responsive to insulin, with the exception of line CHP-270. Moreover, cell lines unresponsive by NTO to NGF, and to tumor promoters, were uniformly unresponsive to insulin. These findings suggest that there are common defects in distal sites, because specific NGF and tumor promotor receptors are present in these lines. Insulin increased [3H]thymidine uptake in SH-SY5Y and CHP-100 cells. However, the enhancement of NTO by insulin and IGF-II in SH-SY5Y cells was independent of the cellular proliferation rate. Our results, together with the observations of others, suggest that insulin and IGF-II may modulate NTO in the nervous system.     

Screening for mutations at codon 717 of the amyloid precursor protein gene in Alzheimer's disease

Abstract Three kinds of missense mutation at codon 717 of amyloid precursor protein (APP) gene (Val→Ile; Val→Gly; Val→Phe) were screened in 114 patients with familial and sporadic Alzheimer's disease (AD), using a rapid testing method for each Val→Gly and Val→Phe mutation and Goate's method for Val→Ile mutation based on the polymerase chain reaction. Mutations were not found in the subjects, confirming earlier suggestions that these three mutations at codon 717 of APP gene account for only a small proportion of cases of not only familial AD but also sporadic AD.     

Laminin and growth factor receptor activation stimulates differential growth responses in subpopulations of adult DRG neurons

Neurons in the adult rat dorsal root ganglion (DRG) can be classified into at least three separate subpopulations based on morphologic and phenotypic differences. In this study we have focused on the growth response of these specific subpopulations in vitro with respect to laminin (LN) and growth factor receptor activation. Using a cell selection approach we show that LN-induced neurite growth occurs in the absence of added trophic factors only in heavy-chain neurofilament-positive and calcitonin gene-related peptide-positive DRG neurons [nerve growth factor (NGF)-responsive population]. In contrast, LN alone is not sufficient to stimulate significant neurite growth from lectin Griffonia simplicifolia IB4-positive neurons (IB4+ve), although it is still required to elicit a growth response from these cells in the presence of glial-derived neurotrophic factor (GDNF, e.g. neurite growth occurred only when cells were plated on LN in the presence of GDNF). By using chemical inhibitors we demonstrate that only the phosphatidylinositol 3 kinase (PI 3-K)/Akt pathway is required for neurite growth from the NGF-responsive cell population. However, both the PI 3-K/Akt and MEK/mitogen-activated protein kinase signaling pathways are required for neurite growth from the IB4+ve cell population. Thus, we have identified specific signaling events and environmental requirements associated with neurite growth for different subpopulations of adult DRG neurons, pointing to potential therapeutic targets while identifying an inability for any one treatment alone to repair peripheral nerve damage.     

Activity requires soluble amyloid precursor protein alpha to promote neurite outgrowth in neural stem cell-derived neurons via activation of the MAPK pathway

It is known that activity modulates neuronal differentiation in the adult brain but the signalling mechanisms underlying this process remain to be identified. We show here that activity requires soluble amyloid precursor protein (sAPP) to enhance neurite outgrowth of young neurons differentiating from neural stem cells. Inhibition of sAPP secretion and anti-APP antibodies both abolished the effect of depolarization on neurite outgrowth, whereas exogenous sAPPα, similar to depolarization, induced neurite elongation. Depolarization and sAPPα both required active N -methyl- d -aspartic acid receptor (NMDAR) and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) recruitment to induce neurite outgrowth. However, depolarization and sAPPα played different roles in modulating this signalling cascade. Depolarization induced ERK phosphorylation with fast kinetics via activation of NMDAR. By contrast, acute application of sAPPα did not lead to ERK activation. However, continuous generation of sAPPα was necessary for depolarization-induced ERK phosphorylation, indicating that sAPPα promotes MAPK/ERK recruitment by an indirect mechanism. In addition, we found that blockade of NMDAR down-regulated APP expression, whereas depolarization increased sAPPα, suggesting that activity may also act upstream of sAPP signalling by regulating the amount of cellular APP and extracellular sAPPα. Finally, we show that soluble amyloid precursor-like protein 2 (sAPLP2), but not sAPLP1, is functionally redundant to sAPP in promoting neurite outgrowth and that soluble members of the APP family require membrane-bound APP to enhance neurite outgrowth. In summary, these experiments indicate a novel role of APP family members in activity-dependent neuronal differentiation.     

Subcellular trafficking of the amyloid precursor protein gene family and its pathogenic role in Alzheimer's disease

Changes in the intracellular transport of amyloid precursor protein (APP) affect the extent to which APP is exposed to alpha- or beta-secretase in a common subcellular compartment and therefore directly influence the degree to which APP undergoes the amyloidogenic pathway leading to generation of beta-amyloid. As the presynaptic regions of neurons are thought to be the main source of beta-amyloid in the brain, attention has been focused on axonal APP trafficking. APP is transported along axons by a fast, kinesin-dependent anterograde transport mechanism. Despite the wealth of in vivo and in vitro data that have accumulated regarding the connection of APP to kinesin transport, it is not yet clear if APP is coupled to its specific motor protein via an intracellular interaction partner, such as the c-Jun N-terminal kinase-interacting protein, or by yet another unknown molecular mechanism. The cargo proteins that form a functional complex with APP are also unknown. Due to the long lifespan, and vast extent, of neurons, in particular axons, neurons are highly sensitive to changes in subcellular transport. Recent in vitro and in vivo studies have shown that variations in APP or tau affect mitochondrial and synaptic vesicle transport. Further, it was shown that this axonal dysfunction might lead to impaired synaptic plasticity, which is crucial for neuronal viability and function. Thus, changes in APP and tau expression may cause perturbed axonal transport and changes in APP processing, contributing to cognitive decline and neurodegeneration in Alzheimer's disease.