INTRODUCTION

Some familial forms of Alzheimer's disease (FAD)¹ are caused by autosomal dominant mutations (1) in the gene for the -amyloid precursor protein (APP). Analyses of -amyloid (A) in genetically engineered cell lines expressing these mutants have shown that expression of the "Swedish" mutant of APP causes increased overall secretion of A (2, 3), whereas expression of the 717 mutations causes increased secretion of a "long" (42-43-amino acid) form of A relative to the shorter 40-amino acid form (4). Consequently, a leading hypothesis for the etiology of AD is that increased A42(43) is a shared molecular correlate of FAD mutations and that it represents a gain of deleterious function that can cause FAD (5).

This gain-of-function hypothesis is attractive; however, molecular mechanisms other than those mediated by extracellular A could also constitute a deleterious gain of function that leads to AD neurodegeneration. In particular, intracellular events mediated by APP metabolites may cause neuronal death. For example, amyloidogenic C-terminal fragments of APP are neurotoxic in vitro and cause AD-like neuropathology in vivo (6-8). Moreover, whereas previous studies showing increased secretion of A42(43) by cells expressing FAD APP mutants in vitro have utilized cell lines, the metabolism of APP in neurons is of particular relevance to this neurodegenerative disorder. To identify metabolic fragments of APP that are generated by neurons in FAD, we used a herpes simplex virus (HSV) vector to express five different FAD mutants of APP in primary neurons in culture. All of the mutants caused abnormal intracellular increases in the level of a C-terminal APP fragment encompassing the A sequence and cytoplasmic domain, as well as increased secretion of A.

EXPERIMENTAL PROCEDURES

Trans-polymerase chain reaction mutagenesis (9) was used to make the Swedish mutation, APP_K595N,M596L (numbering based on the APP-695 spliced form) at the N-terminal end of A, the three mutations APP_V642(F/G/I) C-terminal to A, and the mutation APP_A617G, which can cause AD or "Dutch" hereditary cerebral hemorrhage with amyloidosis (HCHWA) in both the APP-695 and APP-751 cDNAs.

We prepared replication-defective HSV vectors expressing human APP-695 and -751, the FAD mutants of each, and APP-C100 (6) in pHSVPrpUC as described (10). The titer of the helper virus component of each stock was 1-1.2 × 10⁶ plaque forming units/ml on 2-2 cells. The titer of the recombinant virus component of each stock, as assayed by expression of the exogenous gene in PC12 cells, was consistently 3 × 10⁷ infectious units/ml; the HSVlac negative control was 9 × 10⁷ infectious units/ml.

Primary cortical cultures from E21 rat embryos were plated at a density of 5 × 10⁶ viable cells per 10-cm poly-D-lysine-coated dish in neurobasal medium supplemented with B27 (Life Technologies, Inc.), 10% fetal bovine serum, and 5% horse serum. 6-8 days after plating, each dish was infected with 20 µl of the indicated virus stock (multiplicity of infection, approximately 1) as described (10). The complete set of infections was performed six times and utilized recombinant viruses from at least two independently generated stocks. Primary astrocyte cultures were generated as described (11).

16 h after infection of the cultures, media were removed and frozen in a dry ice-ethanol bath. The infected cells were homogenized in buffer A (20 mM Tris, pH 7.5, 1 mM MgCl₂, 125 mM NaCl, 1% Triton X-100) + 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 50 mM NaF, 100 µM Na₃VO₄, boiled, sonicated, and centrifuged at 13,000 × g for 10 min at 4 °C. 5 µg of each sample were separated by SDS-polyacrylamide gel electrophoresis (10-20% Tris-glycine gradient gels (Bio-Rad) or 10-20% Tris-Tricine gradient gels (Integrated Separation Systems)) and transferred to polyvinylidene difluoride (Millipore) membranes for immunoblot analysis (Western Star immunodetection system, Tropix) with antibody C8 (gift of D. Selkoe). The affinity-purified monoclonal antibody 6E10 (Senetek, Maryland Heights, MO) directed against residues 5-10 of the A sequence was used in some experiments. Blots were also probed with a monoclonal antibody to tubulin (TUB2.1, Sigma) to confirm equal loading of protein in the lanes.

Three sets of infections were each analyzed in triplicate for quantification. Controls included mock infections and infections with HSVlac. Relative optical densities with background subtraction were quantified on a film-based imaging system (MCID, Imaging Research, St. Catherine's, ON, Canada).

16 h post-infection, rat cortical cultures (7 days in vitro) were harvested for biochemical fractionation (12). 5 µg each of the fractions were blotted with the C8 antibody.

Metabolic labeling followed by immunoprecipitation was performed as described (13) to assay for C-terminal APP metabolites in the conditioned media.

A_(1-40) and A_(1-42) were quantified in samples of conditioned media as described (4), except that in the experiment (see Table II) evaluating A in the media of cells infected with HSV/C100 and HSV/lac, and mock-infected, the BNT77 capture antibody, directed against A residues 11-28, was used instead of the BAN50 capture antibody directed against the N terminus of A. The BAN50 capture system detected A in the HSV/C100-infected cultures but not in the mock or HSV/lac-infected cultures. A secreted in these latter cultures may be N-terminally truncated and/or modified.

Table II. Values (mean ± S.E. of triplicate samples) are pM concentrations. A40 A42 Ratio 42/40 Mock 750.8  ± 68.6 99.3  ± 9.0 13.2  ± 0.1 Lac-Z 492.4  ± 22.7 55.7  ± 4.3 11.3  ± 0.4 C100 2402.0  ± 80.7 353.9  ± 12.1 14.7  ± 0.3

RESULTS

Primary rat cortical cultures were infected with HSV recombinant vectors expressing cDNAs for wild type APP-695/751, for five different FAD mutations of each spliced form, and for APP-C100. 16 h later (prior to the appearance of C100-induced neurotoxicity), the infected cells were harvested and immunoblot analysis was performed. Representative samples are shown in Fig. 1. Amounts of APP expressed from the HSV vectors in the neurons are severalfold greater than endogenous levels, as determined by comparison with mock-transfected or HSVlac-transfected neurons (ranging from a 3.2-fold average increase over endogenous levels for V642G-751 to a 17-fold average increase for wild type APP-695; data not shown). The higher levels of APP observed in neurons infected with HSV vectors expressing wild type APP transgenes compared with those seen in neurons infected with HSV/FAD-APP vectors are consistent with results obtained by Cai et al. (7) with APP-transfected cell lines. A 12-kDa fragment that co-migrates with a fragment representing the C-terminal 100 amino acids of APP (APP-C100, expressed from a HSV/C100 vector) is detected at low levels in cell lysates from neurons infected with APP-695 or -751. Below it (P10) is the fragment presumably generated by the constitutive -secretase cleavage of APP (14). In the figure shown, the amount of the fragment co-migrating with APP-C100 appears to be increased severalfold in specific cell lysates from neurons infected with HSV/APP FAD mutant recombinants relative to those infected with wild type recombinants.

The data were quantified (Fig. 2) by measuring the intensities of the bands of the fragments co-migrating with APP-C100 and normalizing each value to the intensity of the APP band in the same sample. The Swedish mutation caused a 7-fold increased accumulation of the C100-sized fragment over wild type in APP-695 and an 8-fold increase in APP-751. Each of the other mutations, except for the V642I mutation in APP-695, caused increased levels of the C100-sized fragment in neurons, with the increases achieving significance for V642I in APP-751, V642F in APP-695, V642G in APP-695, and A617G (HCHWA) in APP-751 and near significance for the remaining mutations.

The FAD mutations that we analyzed had differential effects on the processing of APP-695 compared with APP-751. In particular, the V642I mutation, when expressed in APP-751, caused a very large increase in the accumulation of a C100-sized fragment in neurons, whereas when it was expressed in APP-695 it caused no detectable alteration in the levels of this same C-terminal fragment. Levels of the presumed P10 fragment were increased as well, but the increase was not as pronounced as that of the C100-sized fragment.

The C100-sized Fragment Contains the -Amyloid Sequence

To determine whether the accumulated C-terminal fragment was a product of - or -secretase cleavage of APP, we probed blots of the lysates with the antibody 6E10 (Fig. 3). This antibody immunodetected a band that co-migrated with the APP-C100 fragment in all lysates from neurons infected with FAD APP HSV vectors (V642G and HCHWA samples not shown) and that was not detectable in mock- or HSV/lac-infected cultures while only faintly detected in neurons infected with wild type APP HSV vectors. The lower P10 band seen in Fig. 1 with the C8 antibody was not immunopositive with 6E10. These data demonstrate that the band co-migrating with APP-C100 is not derived from -secretase cleavage of APP, which occurs between amino acids 16 and 17 of the A sequence and therefore is likely to be a product of -secretase cleavage of APP, which occurs at the beginning of the A sequence.

Immunoblot analysis of lysates from neurons infected with HSV vectors expressing wild type and FAD mutants of APP, using the monoclonal antibody 6E10, directed toward amino acids 5-10 of the A sequence.

On the basis of its mobility on SDS-polyacrylamide gels, the C-terminal fragment of APP that accumulates in neurons infected with HSV vectors expressing FAD mutants of APP appears to be very similar to, if not identical to, APP-C100. To determine whether the C100-sized fragment is present in the same cellular compartments that APP-C100 is, we harvested HSV/FAD-APP-infected cultures for biochemical fractionation (Fig. 4). The C100-sized fragments that accumulated in cultures infected with HSV/V642I-751, HSV/SWE-751, and HSV/C100 were all enriched in membrane fractions, particularly in P2-0.8 M (synaptosomes), P2-1.2 M (lysosomes and mitochondria), and P3 (microsomes).

The primary cultures used contain glial as well as neuronal cells. To test whether the abnormal processing of APP observed in primary neuronal cultures infected with HSV/FAD-APP vectors occurs in neurons or the glia, we generated primary astrocyte cultures, which were infected with the HSV vectors after 7 days in vitro. Although infection of these cells by the HSV vectors was efficient and resulted in the production of APP and P10, C100-sized fragments were not detectable in astrocyte cultures infected with the HSV/FAD-APP vectors (data not shown). These data suggest that the abnormal accumulation of C100-sized fragments in neuronal cultures infected with HSV/FAD-APP vectors is due to abnormal processing that occurs in neurons rather than in astrocytes.

Increased Levels of A Are Secreted by Neurons Infected Either with HSV/FAD-APP Vectors or with HSV/C100

Metabolic labeling followed by immunoprecipitation was performed as described (21, 22) to assay for C-terminal APP metabolites in the conditioned medium of neurons infected with HSV/FAD-APP, HSV/C100, and control HSV vectors. Neither P10 nor C100-sized fragments were detected in any of the experimental or control cultures (data not shown), suggesting that these polypeptides are not secreted or are secreted in amounts below the level of detection. These data are consistent with those obtained by Dyrks et al. (15) and Citron et al. (16).

It has been reported that cell cultures transfected with vectors expressing FAD mutants of APP display increased secretion of A_(1-42) (2-4). To ascertain whether the same holds true for neurons infected with FAD APP vectors, levels of A_(1-40) and A_(1-42) were quantified in conditioned media from cells infected with HSV/FAD-APP, HSV/C100, and control HSV vectors. As shown in Table I, the Swedish mutation in both APP-695 and APP-751 caused an approximately 20-fold increase in the secretion of A relative to that caused by vectors expressing wild type APP, whereas the V642I mutation in APP-751 increased secretion of A over 30-fold. The ratio of secreted A_(1-42) to A_(1-40) was not altered significantly in neurons infected with HSV/FAD-APP vectors except in the case of the Swedish mutation in APP-695.

Table I. Values (mean ± S.E. of triplicate samples) are nM concentrations normalized to the optical density of intracellular APP, as measured by immunoblot analysis, for each sample. A1-40 A1-42 Ratio 42/40 APP-695wt 2271.7  ± 195.5 200.5  ± 14.6 8.8  ± 0.2 APP-695swe 42407.9  ± 1268.2 4671.2  ± 116.0 11.0  ± 0.2 APP-751V642I 71687.0  ± 782.3 5820.5  ± 129.9 8.1  ± 0.1 APP-751wt 4378.4  ± 271.3 395.6  ± 30.1 9.0  ± 0.3 APP-751swe 49796.8  ± 2374.8 4956.0  ± 59.3 10.0  ± 0.4

In a separate experiment (Table II), A released from neurons infected with HSV/C100 was compared with that from mock- or HSV/lac-infected neurons. HSV/lac-infected neurons showed slightly less secretion of A than did mock-infected neurons. However, infection of neurons with HSV/C100 caused an approximately 3-fold increase in secretion of A relative to HSV/lac-infected controls. In addition, the ratio of A_(1-42) to A_(1-40) was significantly increased in HSV/C100-infected cultures compared with HSV/lac-infected cultures.

DISCUSSION

We have shown that expression of FAD APP mutants in primary rat neurons in vitro causes abnormal intracellular accumulation of a C-terminal fragment of APP that co-migrates and co-fractionates with APP-C100. This C-terminal fragment probably is generated by -secretase cleavage of APP, and the data suggest that the abnormal processing occurs predominantly in neurons rather than in glial cells. Neurons expressing these APP mutants or APP-C100 show increased secretion of both A_(1-40) and A_(1-42). Although a previous report has noted increased overall secretion of A by hippocampal neurons expressing the Swedish and Dutch mutants of APP-695 via the Semliki Forest Virus (17), the present paper is, to our knowledge, the first to analyze the effect of all five published FAD mutations of APP in both APP-695 and APP-751 on the generation of C-terminal metabolites of APP by neurons. Expression of V642 mutations in non-neuronal mammalian cells has not been observed to cause changes in APP-C100 levels (3, 18), suggesting that the presumed increased -secretase processing of these mutations observed in the present study occurs primarily or exclusively in neurons. In contrast to data from an earlier study showing that neuroblastoma cells expressing the V642I mutation secrete an altered ratio of A_(1-42) to A_(1-42) but show no change in the absolute level of A (4), our preliminary data suggest that the same mutation expressed in neurons causes an overall increase in A secretion but no change in the ratio of A_(1-42)/A_(1-40). The results may reflect a difference in APP processing in primary neurons relative to cell lines.

The electrophoretic mobility of the intracellular C-terminal fragment of APP that accumulates abnormally, together with its co-localization with APP-C100 in specific membrane fractions, suggest that it is very similar, if not identical, to APP-C100. We have proposed that APP-C100 is involved in the etiology of Alzheimer's disease (7). It is neurotoxic in vitro (6, 19, 20) and has been implicated in amyloidogenesis (15, 21, 22, 24-27). In addition, expression of APP-C100 in vivo can cause neuropathology that is similar in some ways to that in AD, including neurodegeneration and cognitive dysfunction (28-32, 7).²

APP-C100 is a normal metabolic product of APP in the human brain (33). Its catabolism may be altered in AD so that APP-C100 accumulates in the cell. The locations of the mutations in APP that cause AD are consistent not only with the possibility that they may alter APP processing to A but also with the possibility that they may alter APP processing to APP-C100. The Swedish mutation at the N terminus of A is also at the N terminus of APP-C100, and the mutations at the C terminus of A may enhance the production of APP-C100, e.g. by altering the requirements for "-secretase" cleavage of APP. These observations led us to test the hypothesis, in the present work, that FAD mutations of APP may have additional effects on APP metabolism in neurons besides increasing A release. Indeed, these mutations cause build-up of a C100-like APP fragment in the neurons. It is interesting that the FAD mutations that we analyzed had differential effects on the processing of APP-695 compared with APP-751.

Overexpression of APP-C100 alone in the neurons causes increased secretion of A_(1-42), suggesting that the increased secretion of A_(1-42) by neurons expressing FAD mutations of APP may be a consequence of accumulation of APP-C100 in the cells. This raises the question of whether APP-C100 is increased in neurons of individuals with the Swedish mutation, as has been shown for A_(1-42) (3) and whether these fragments are elevated in the neurons of patients with other FAD mutations of APP. Increased release of A_(1-42) from fibroblasts of AD patients with presenilin mutations, as well as increased levels of A_(1-42) in their plasma, have been demonstrated (23). It will be instructive to determine whether APP-C100 levels in neurons are increased not only by FAD mutations of APP but also by FAD mutations of the presenilins.