Rachael L. Neve
Department of Genetics,
Harvard Medical School and Molecular Neurogenetics Laboratory,
McLean Hospital, Belmont, MA 02178

Introduction

One of the most popular hypotheses that invokes the participation of APP in the neuronal cell death in AD is the amyloid hypothesis, which posits that plaque amyloid depositions or partially aggregated soluble Ab trigger a neurotoxic cascade, thereby causing neurodegeneration and AD (reviewed in [2,3]). This theory is based on in vitro studies suggesting that Ab is toxic to neurons [4] and on the measurements of increased release of Ab by cells carrying familial AD (FAD) mutant genes (reviewed in [2,3,5]).

While this amyloid hypothesis is attractive, molecular mechanisms other than those mediated by extracellular Ab could also lead to neuronal death and impaired cognition in AD. We and others have proposed a modified version of the amyloid hypothesis, which postulates that the primary contributor to the etiology of AD is the C-terminal 100-amino acid fragment of the amyloid precursor protein (APP-C100; Fig. 1), which includes the 42-amino acid Ab peptide and 58 adjacent amino acids in the carboxyl-terminus of APP. The genetic and biochemical data that support this hypothesis and that will be discussed in this presentation are that (1) APP-C100 is both neurotoxic and amyloidogenic, (2) neurons expressing any of the known FAD mutant APP cDNAs show significant intracellular accumulation of APP-C100 and (3) expression of APP-C100 in vivo can cause neuropathology, including neurodegeneration and cognitive dysfunction, that is similar in many ways to that in AD,

Figure 1

Scheme suggesting the relative roles of APP-C100 and Ab in Alzheimer's disease. In this scenario, the lysosomal processing pathway for APP catabolism becomes overloaded with APP-C100 due to any of a number of possible insults. APP-C100 is directly toxic to neurons. Further processing of APP-C100 produces Ab, which may product secondary toxicity.

1. In Vitro Neurotoxicity and Amyloidogenicity of APP-C100

The first indication that fragments of APP could be toxic to neurons was the discovery [6] that pheochromocytoma (PC12) cells genetically engineered to express APP-C100 died when induced by nerve growth factor (NGF) to differentiate into neuronal cells. Fig. 2A shows that when two independent APP-C100 PC12 transfectants (open vs. closed symbols) are exposed to NGF, they die gradually over days. The degeneration occurs more rapidly in serum-containing medium (circles) than in serum-free medium (triangles). In panel 2B, cultures of vector-transfected PC12 cells (DOJ-PC12) and of APP-C100-transfected PC12 cells (bAPP-C104-PC12) are shown after 6 days of NGF treatment. Note that the C100-transfected cells show retraction of processes, after which they die. Notably, PC12 transfectants expressing Ab1-42 differentiated normally without dying when exposed to NGF. The APP-C100 toxicity probably was not a specific consequence of NGF, but rather was due to the acquisition by the cells of a neuronal phenotype, because neuronal differentiation by retinoic acid of a human neuroblastoma line (LAN-5) expressing APP-C100 caused the death of these transfectants as well (unpublished data of R.L.N.). Subsequently the neurotoxicity of APP-C100 in neuronally differentiating mouse embryonic stem cells [7] and in neuroblastoma cells [8] was reported, and the neurotoxicity of APP-C100 to neurons and to PC12 cells treated with NGF was confirmed independently [9,10].

Figure 2

Coincidentally with the finding that APP-C100 was neurotoxic, several groups reported that it was amyloidogenic [11-13]. In a cell-free assay, APP-C100 was shown to aggregate, and to yield peptides the size of Ab when treated with proteinase K [11]. Its amyloidogenicity in this system was later found to be dependent on metal-catalyzed oxidation [14]. Bacterial expression of a 109-amino acid fragment of APP yielded a product that spontaneously formed fibrils [15]. When overexpressed in mammalian cells, the APP C-terminus aggregated into deposits in CV-1 cells [12] and formed amyloid-like fibrils in COS cells [13]. Its expression in human neuroblastoma cells resulted in secretion of Ab, establishing a precursor-product relationship between the two molecules [16]. Further correlation between the intracellular accumulation of APP-C100 and its toxicity to neural cells was established with the reports that overproduction of normal full-length APP in neuroblastoma cells [17] or in neuronally differentially mouse stem cells [18] resulted in the overproduction of C-terminal fragments of APP and concomitant degeneration of the cells.

The mechanism behind the amyloidogenecity and the neurotoxicity of APP-100 is not known. However, both Martin and colleagues [19] and Yoshikawa [20] have suggested a model in which intracellular amyloidogenic fragments such as APP-C100 kill neurons "from inside", in contrast to the popular hypothesis that extracellular Ab causes neurodegeneration "from outside". APP-C100 is a normal metabolic product of APP in the human brain [21]. We propose that its catabolism may be altered in AD so that APP-C100 accumulates in the cell.

2. Accumulation of C-terminal fragments of APP in neurons expressing FAD mutants of APP

To test this hypothesis using known causes of AD, we expressed (22) five different Alzheimer mutations of the b-amyloid precursor protein (APP) in neurons via recombinant herpes simplex virus (HSV) vectors and quantified the levels of APP metabolites. The predominant intracellular accumulation product was a carboxyl-terminal (C-terminal) fragment of APP that co-migrated with the protein product of an HSV recombinant expressing the C-terminal 100 amino acids (C100) of APP. Fig. 3 shows immunoblot analysis of C-terminal fragments of APP present in neurons infected with representative HSV vectors expressing wild type and FAD mutants of APP. The blot was probed with C8 (gift of D. Selkoe), a polyclonal antibody directed to the C-terminal 20 amino acids of APP. A fragment that co-migrates with C100 (as expressed in the rat neurons infected with HSV/C100; see lane labeled "C100"), just below the 14.3 kDa marker, is detected at low levels in cell lysates from neurons infected with APP-695 or -751. Below it (p10) is the fragment that is presumably generated by the cleavage of APP between lys16 and leu17 in the Ab sequence. It is clear that the amount of the fragment co-migrating with C100 is increased several-fold in cell lysates from neurons infected with HSV/APP-751 expressing the Swedish mutation (SWE-751) and from neurons infected with HSV/APP-751 expressing the V642I mutation (V642I-751). This fragment is detected also in lysates from neurons infected with the other FAD APP recombinants (data not shown).

Figure 3

We quantified the level of the C100-sized fragment relative to APP in neurons infected with HSV vectors expressing wild type and FAD mutants of APP (Fig. 4) by measuring the intensities of the bands of the fragments co-migrating with APP-C100 and normalizing each of these values to the intensity of the APP band in the same sample. The Swedish mutation, APPK595N,M595L, 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.

Figure 4

We carried out immunoblot analysis (Fig. 5) of lysates from neurons infected with HSV vectors expressing wild type and FAD mutants of APP, using the monoclonal antibody 6E10, directed towards amino acids 5-10 of the Ab sequence. Immunoreactive bands that co-migrate with APP-C100 are seen in all lanes containing lysates of neurons infected with HSV/FAD-APP vectors. These data demonstrate that the band co-migrating with APP-C100 is not derived from a-secretase cleavage of APP, which occurs between amino acids 16 and 17 of the Ab sequence, and therefore is likely to be a product of b-secretase cleavage of APP, which occurs at the beginning of the Ab sequence.

Figure 5

Fractionation studies revealed that the C-terminal fragment generated by expression of the Alzheimer mutations, like C100, partitioned into membrane fractions and was particularly enriched in synaptosomes (data not shown). 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 and infected them with the HSV vectors (Fig. 6). Immunoblot analysis of the lysates with the C-terminal antibody C8 revealed that P10 but not C100-sized fragments accumulate in astrocytes infected with FAD mutants of APP, whereas infection of mixed neuronal-glial cortical cultures with the Swedish mutant of APP (leftmost lane), for example, resulted in the accumulation of large amounts of both P10 and C100-sized fragments. These data indicate that the processing abnormality caused by expression of the Alzheimer mutations occurs predominantly in neurons.

Figure 6

Expression of these mutations (Table 1) or of C100 alone in neurons (Table 2) caused increased secretion of Ab relative to that of neurons infected with wild type APP recombinant and control vectors. Expression of V642 mutations in non-neuronal mammalian cells has not been observed to cause changes in APP-C100 levels (23), suggesting that the presumed increased b-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 Ab(1-42) to Ab(1-42) but show no change in the absolute level of Ab (24), our preliminary data suggest that the same mutation expressed in neurons causes an overall increase in Ab secretion but no change in the ratio of Ab(1-42)/Ab(1-40). The results may reflect a difference in APP processing in primary neurons relative to cell lines, but need to be expanded before such a conclusion can be made.

3. AD-like neuropathology in mice expressing APP-C100 in the brain

To carry out in vivo tests of the hypothesis that the neurotoxic APP-C100 fragment may play a role both in amyloidogenesis and in the development of the progressive neurodegneration and loss of cognitive functions in AD, we made transgenic mice [25,26] expressing APP-C100 in the brain under the control of the dystrophin brain promoter (Fig. 7). At the age of 4 1/2 months, these mice evinced abnormal Ab deposits in neurons and their processes, amyloid deposition in the cerebrovasculature, and accumulation of C-terminal fragments of APP in secondary lysosomes. The last observation in particular was of interest because it replicated our previous discovery [27] that C-terminal fragments of APP build up in secondary lysosomes in affected regions of AD brains, and because both findings supported the notion that accumulation of APP-C100 in cells may play a role in AD neurodegeneration.

Figure 7

When these animals reached the age of 18 months, profound neurodegeneration and synaptic loss was evident in the hippocampus, accompanied by proliferation of microglia and cytoskeletal changes characteristic of AD. Second- and third-generation C100 transgenics have been made in which expression of the transgene is higher than it was in the original transgenics; neurodegeneration is seen as early as 4 months in these mice (unpublished data). Furthermore, the death of neurons in the C100 transgenic mice correlates with impairment in spatial learning [28]. Figure 8 shows that the percent time spent during a 60 second probe trial in the quadrant of the pool where the platform had formerly been located during spatial trials is less for the C100 transgenic mice than it is for control mice (* indicates statistical significance at p<0.01 [ANOVA]), This learning impairment is associated with hippocampal neurodegeneration. Recently, Nalbantoglu et al. [29] replicated this phenotype in transgenic mice expressing APP-C100 under the control of the neurofilament promoter. As these mice age, their short-term spatial memory becomes impaired. Also, the normal strengthening of synapses that occurs when the synapses are stimulated artifically did not occur in the transgenic mice. Therefore, these new APP-C100 transgenic mice independently replicate the correlations between deposition of Ab, neuronal degeneration, synaptic loss and impaired short-memory that were observed in the transgenic mice that we have described [25,26,28].

Figure 8

4. A new signal transduction pathway in Alzheimer's disease?

If C-terminal amyloidogenic APP fragments are neurotoxic, what are the cellular pathways by which they cause the destruction of cells in the brain? In searching for an answer to this question, it is instructive to recall that synaptic loss and neuronal cell death correlate more strongly with the degree of cognitive impairment in Alzheimer disease than does amyloid deposition [30,31]. If APP-C100 does, as we hypothesize, play a significant role in AD neurodegeneration, it must activate (or at least interact with) intracellular pathways that cause the neuronal death, disruption of the neuronal cytoskeleton, loss of cortico-cortical connectivity, synaptic damage, and lysosomal abnormalities [32] that are hallmarks of AD neuropathology. Work by other groups (reviewed in [33]) has suggested that APP-C100 disrupts the cell membrane, perhaps by forming nonselective ion channels. We have proposed that APP-C100 is linked to an intracellular signal transduction pathway [34], and that abnormal accumulation of APP-C100 may disrupt this normal pathway.

This proposal is based on our work in which we sought binding proteins for the intracellular carboxyl-terminal (C-terminal) tail of APP by screening brain expression cDNA libraries with a radiolabeled C-terminal fragment of APP synthesized in vitro. We thereby cloned cDNAs encoding APP-BP1 (APP-binding protein 1, a protein of unknown function; ref. 34) and N-Pak, a novel neural-specific p21 activated kinase that is a binding protein for the C-terminus of APP (35). The n-pak cDNA represents an ~11-kilobase mRNA encoding a novel 61-kilodalton putative serine/threonine kinase. RNA blot analyses and in situ hybridization histochemistry have been used to show that n-pak mRNA is nervous system-specific and is expressed at highest levels in regions of the adult human brain that are most at risk for AD pathology. APP was specifically co-precipitated with N-Pak from transfected neurons; and N-Pak immunoprecipitated from transfected neurons phosphorylated a myelin basic protein substrate in the presence of the activated p21 proteins Rac1 and Cdc42. These two members of the Ras-related Rho subfamily of small GTPases regulate cytoskeletal organization and neuronal polarity. A schematic showing the domain structure of N-Pak and the binding sites for APP/APP-C100 and Rac1/Cdc42 is depicted in Fig. 9. The interaction of the C-terminal domain of APP with N-Pak is likely to be involved in neuronal signal transduction mediated by Rac1 and Cdc42 (Fig. 10). Disruption of this pathway may play a part in the disassembly of the cytoskeleton that occurs in Alzheimer's disease. One such scenario is shown in Figure 11. Whatever the mechanism by which APP-C100 kills neurons in AD, preventing its intracellular accumulation in these cells is likely to be an important target for drug development for the treatment of Alzheimer's disease.

Figure 9

Figure 10

Figure 11

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Transcript of Live Discussion