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differentially affected. PS-1 is reported to be expressed primarily in CNS neurons in the brain, suggesting that this
protein may perform a neuron-specific function [20]. In fact, in AD, neurons that express PS-1 antigen are less
vulnerable to the disease than are neurons that do not express it [30], and inhibition of PS-1 expression results in
apoptosis [88], suggesting a protective role for this protein. Although the precise role of PSs in regulation of apoptosis
in the neuron is still unclear, the evidence that they do play a role in this pathway is strong. These data implicate both
APP and PSs in the control of apoptotic death in the brain, and it is not unreasonable to suppose that FAD mutations
in these genes may cause dysfunction in this pathway.
It has been noted [82] that since the apoptotic process proceeds to completion within 16–24 h, the extent of
apoptosis reported in AD brain would predict a complete loss of neurons within a very brief period of time. Clearly,
this does not happen in AD. Cotman has suggested [15] that the induction of compensatory responses to apoptosis
in the AD brain protects the neurons from terminal apoptosis, and that a dynamic competition between cell
death processes and compensatory responses exists in AD brain.
5. Cell cycle abnormalities in Alzheimer’s disease
The implication of apoptosis in AD etiology is con-
Fig. 2. The yeast two-hybrid reporter assay reveals that APP-BP1
sistent with the numerous findings of cell cycle abnor-
interacts with hUba3. Top: schematic diagram showing the deletion and
malities in AD. Apoptosis and the cell cycle are closely
point mutants used for the assay. Below: a table indicating the strength of
tied together, and the reexpression of cell cycle markers
each interaction, based on length of time for the X-gal substrate to turn
has been linked with the occurrence of certain types of
blue.
neuronal cell death [40,39,22]. One interpretation of these findings [56] is that a neuron is committed to the perma-
APP, V642I, causes DNA fragmentation when expressed in nent cessation of cell division, so if for any reason it is
a neuronal cell line [113]. Luo et al. [61] showed that the forced to reenter the cell cycle after this commitment, it
same mutation, as well as two additional FAD APP dies. Notably, ectopic expression of cell cycle proteins and
mutations, induced apoptosis in differentiated PC12 cells. their associated kinases in AD brain have been reported
Barnes et al. [2] reported that levels of APP are increased [84,59,108,109]. More recently, Busser et al. [7] found
in motoneurons dying of apoptosis, and that APP is abnormal appearance of cell cycle markers in regions of
cleaved by caspase-3, a caspase activated in apoptotic AD brain where cell death is extensive, and Chow et al.
motoneurons. Interestingly, we [6] and others [77] have [11] found increases in expression of genes encoding cell
shown that overexpression of wild-type APP causes apo- cycle proteins in single neurons in late-stage relative to
ptotic death of neurons, although to a lesser degree than early-stage AD brain. The phosphoepitope S214 of the
does expression of FAD mutants of APP. microtubule associated protein tau, that appears in the
Approximately half of inherited AD cases are caused by neurofibrillary tangles in AD, is a prominent phosphoryla-
mutations in the presenilin genes PS1 and PS2. It has been tion site in metaphase but not in interphase of dividing
reported that overexpression of these genes in transfected cells expressing tau [44], supporting the view that reactiva-
cell lines can cause apoptosis [45] or result in an increased tion of the cell cycle machinery may be involved in tau
susceptibility to apoptosis [112,32–34,16]. On the other hyperphosphorylation in AD brain. The possibility that
hand, we have found [6] that expression of PS1 in primary phosphorylation-dependent events occurring during the cell
neurons does not cause or enhance apoptosis; rather, it cycle affect the normal function of APP is suggested by
protects neurons against experimentally-induced apoptosis. the finding that regulation of the phosphorylation and
Thus, the ability of PS-1 to induce apoptosis appears to be metabolism of this protein occurs in a cell-cycle dependent
cell type specific; and this may have important implica- manner [103,78].
tions for the pathogenesis in AD, in which neurons are We hypothesize that dysfunction of pathways mediated
R .L. Neve et al. Brain Research 886 2000 54 –66
59
by APP may be one cause of the reactivation of cell cycle appear to have much in common with those of ubiquitin,
proteins in AD brain. In particular, we have isolated a but the Ubls have novel regulatory functions not necessari-
binding protein for APP, termed APP-BP1 [10], and have ly linked to proteolysis. APP-BP1 is a member of one of
shown that it is a cell cycle protein that normally regulates these pathways. APP-BP1 is homologous to the ubiquitin
negatively the progression of cells into the S phase and activating enzyme E1, but lacks the catalytic site. It has
regulates positively their progression into mitosis [9]. Over been found, by our lab [9], and others, that APP-BP1 acts
the past few years, it has emerged that eukaryotes express in concert with a second protein that possesses an E1-like
a set of ubiquitin-like proteins Ubls that are significantly catalytic site, so that the two-molecule complex behaves
diverged from ubiquitin itself yet are also ligated to other like E1, except that this complex activates the ubiquitin-
proteins [35,46]. The reactions involving these variants like protein NEDD8 rather than ubiquitin itself:
Fig. 3. APP-BP1 rescues the ts41 cell phenotype at the nonpermissive temperature. A Time course of viability of ts41 cells transfected with the vector alone or with the APP-BP1 construct at the nonpermissive temperature 408C. Data for the vector-transfected cells at the permissive temperature 348C
are shown for comparison. Counts were performed at 32, 64, and 80 h except for the vector-transfected cells at the permissive temperature, which were counted at 32 and 64 h only. Note that cells transfected with a vector expressing human wild type APP-BP1 maintained a rate of growth at 408C that was
similar albeit slightly shifted to the right to that of cells transfected with the vector alone and maintained at 348C. Vector-transfected cells that were shifted to the nonpermissive temperature showed a decreased mitotic index and eventually died. B Quantification of viability of wild type and mutant
APP-BP1-transfected ts41 cells grown at 408C for 81 h. Note that the d401–479 deletion mutant of APP-BP1, which does not interact with hUba3, is unable to rescue the ts41 phenotype Scheffe post hoc t tests; [[ or indicates P ,0.01, and indicates P ,0.05; [ comparison with pcDNA3;
comparison with wild type APP-BP1. C Dominant negative mutants of hUba3 or hUbc12 inhibit the ability of APP-BP1 to rescue the ts41 mutant phenotype. Quantitation of the viability of ts41 cells transfected with APP-BP1 and wild type or dominant negative mutant hUba3 at 81 h after the shift to
the nonpermissive temperature 408C is shown. Co-expression of the dominant negative mutant of hUba3 C216S inhibits the ability of APP-BP1 to rescue the ts41 mutant phenotype. The hUba3 APP-BP1 sample is significantly different from the C216S APP-BP1 sample P ,0.02; t test assuming
unequal variance.
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temperature, after which they were neutralized in 0.1 M
E1-likeUb activating Ubiquitin-like
sodium borate buffer pH 8.2 for 10 min. After two 5-min
APP-BP1 1 hUBA3 activates NEDD8
→ targets e.g., cullin-4A
washes the neurons were incubated in 5 horse serum in NEDD8 then forms a thiol ester linkage with hUbc12, a
PBS plus 0.1 Triton-X 100 blocking buffer for 1 h at human protein that has a function parallel to that of the
room temperature, after which they were incubated over- ubiquitin-conjugating enzyme, prior to its modification of
night in a 1:1000 dilution of the anti-BrdU monoclonal target proteins such as cullin-4A. The functions of this
antibody BU-33 Sigma in blocking buffer. After three pathway reviewed in Ref. [41] are still unclear; but it
5-min washes in blocking buffer, the neurons were incu- does in some cases lead to modification of ubiquitin-like
bated for 1.5 h at room temperature in a 1:200 dilution of proteins that are linked to cell cycle regulation [46,51].
biotinylated secondary anti-mouse antibody Vector Lab- We have shown that APP-BP1 plays a role in the cell
oratories in blocking buffer, washed, and visualized using cycle [9]. APP-BP1 co-immunoprecipitates with hUba3
DAB. Stained cells were visualized under a 403 objective. from mammalian cells and binds to a region between
Ten random fields were counted from each condition, and amino acids 443 and 479 in hUba3 Fig. 2. Wild type
the data were expressed as the percentage ratio of stained APP-BP1 rescues the cell cycle S–M checkpoint defect in
cells to total cells. ts41 hamster cells [9] and this rescue is dependent on the
The results Fig. 7 indicated that expression of FAD binding of APP-BP1 to hUba3 Fig. 3. Dominant negative
mutants of APP in cortical neurons caused a significant mutants of hUba3 and Ubc12 prevent the rescue. Notably,
increase over controls in the number of cells undergoing overexpression of APP-BP1 in primary neurons Fig. 4
DNA synthesis. Overexpression of wild type APP in the causes apoptosis by a pathway that also involves hUba3
cultures also caused an increase in DNA synthesis, al- and hUbc12 Fig. 5. We hypothesize that overexpression
though to a lesser extent. of APP-BP1 pushes neurons into the S phase of the cell
cycle Fig. 6, causing DNA synthesis and, for example, aberrant expression of mitotic cdc2 cyclin B1 kinase, as is
6. Conclusions
