Abstract
The molecules that mediate neuron death in Alzheimer's disease (AD) are largely unknown. We report that β-amyloid (Aβ), a death-promoting peptide implicated in the pathophysiology of AD, induces the proapoptotic protein Bcl-2 interacting mediator of cell death (Bim) in cultured hippocampal and cortical neurons. We further find that Bim is an essential mediator of Aβ-induced neurotoxicity. Our examination of postmortem AD human brains additionally reveals upregulation of Bim in vulnerable entorhinal cortical neurons, but not in cerebellum, a region usually unaffected by AD. Accumulating evidence links inappropriate induction/activation of cell cycle-related proteins to AD, but their roles in the disease have been unclear. We find that the cell cycle molecule cyclin-dependent kinase 4 (cdk4) and its downstream effector B-myb, are required for Aβ-dependent Bim induction and death in cultured neurons. Moreover, neurons that overexpress Bim in AD brains also show elevated levels of the cell cycle-related proteins cdk4 and phospho-Rb. Our observations indicate that Bim is a proapoptotic effector of Aβ and of dysregulated cell cycle proteins in AD and identify both Bim and cell cycle elements as potential therapeutic targets.
Introduction
Alzheimer's disease (AD) is a progressive disorder characterized by selective neuron loss and formation of neurofibrillary tangles and of plaques containing amyloid-β peptide (Aβ) (Masters et al., 1985; Wisniewski and Wegiel, 1995; Yankner, 1996; Hardy, 1997; Selkoe, 1999, 2001). Accumulating data indicate that Aβ plays a central role in AD (Cotman et al., 1994; Cotman and Su, 1996; Yankner, 1996; Stadelmann et al., 1999; Selkoe, 2001). In addition to genetic evidence that Aβ promotes neuron degeneration and death in vivo, in vitro studies show that aggregated Aβ rapidly induces neuron death (Pike et al., 1991; Li et al., 1996; Estus et al., 1997; Troy et al., 2000; Hartman et al., 2005). However, the mechanisms by which neuron degeneration and death occur in AD and whereby these are induced by Aβ are incompletely understood.
One focus regarding the mechanism of neuron death in AD has been the aberrant neuronal expression of cell cycle-related proteins (for review, see Greene et al., 2004; Herrup et al., 2004; Webber et al., 2005). Although such observations in postmortem neurons have been correlative with susceptibility to death, in vitro studies have provided evidence that cycle-related proteins mediate neuron death in AD. It was reported that Aβ activates cell cycle components in cultured neurons and that interference with such molecules by pharmacologic agents and dominant-negative constructs [cyclin-dependent kinases 4/6 (cdk4/6), in particular] protects from Aβ toxicity (Giovanni et al., 1999, 2000).
Past studies have indicated similarities between the mechanisms by which trophic factor deprivation and Aβ induce neuron death (Park et al., 1998; Giovanni et al., 1999, 2000; Troy et al., 2000, 2001; Liu and Greene, 2001; Greene et al., 2004; Liu et al., 2004, 2005; Biswas et al., 2005). This includes activation of cell cycle molecules and an apparent requirement for cdk4 activity. In the case of neurotrophic factor deprivation, a multistep pathway requiring cdk4 has been described that leads to induction of Bcl-2 interacting mediator of cell death (Bim), a proapoptotic Bcl-2 family member (Puthalakath and Strasser, 2002; Biswas et al., 2005). Bim induction in turn plays an important role in neuron death evoked by trophic deprivation (Putcha et al., 2001; Linseman et al., 2002; Biswas et al., 2005; Ham et al., 2005). Because of the shared involvement of cell cycle molecules in death associated with trophic factor deprivation and Aβ exposure, we hypothesized that the latter may also involve induction of Bim through a cdk4-dependent mechanism.
The present work identifies critical regulators of neuron death that act distally to Aβ and that are relevant to AD. We report that Bim is induced by Aβ in cultured neurons and that this is essential for Aβ-dependent neuron death. Bim appears directly pertinent to AD in that its expression is selectively elevated in susceptible neurons in postmortem AD brains. We further show that Bim induction by Aβ is mediated by cdk4 and plays a required role in Aβ-dependent apoptosis. Our observations thus outline an AD-relevant pathway that sequentially links exposure to Aβ, activation of cell cycle components, Bim induction, and promotion of neuron death.
Materials and Methods
Preparation of amyloid.
Lyophilized, HPLC-purified Aβ1–42 was purchased from American Peptide (Sunnyvale, CA). Aβ1–42 was reconstituted in sterile water at a concentration of 400 μm as described previously (Troy et al., 2000). Aliquots of stocks were incubated at 37°C for 3 d to form aggregated amyloid. Oligomeric Aβ was prepared as described previously (Barghorn et al., 2005).
Primary neuron culture.
Embryonic rat cortical, and hippocampal neurons were cultured as previously described (Park et al., 1998; Troy et al., 2000).
Western immunoblotting.
Human brain tissues were dissolved in 2× sample buffer by sonication. Bim protein expression was analyzed by Western immunoblotting as described previously (Biswas and Greene, 2002). The extracellular signal-regulated kinase 1 (ERK1) antibody was from Santa Cruz Biotechnology (Santa Cruz, CA) and the Bim antibody was from StressGen (Victoria, British Columbia, Canada). Western blots were scanned and analyzed by NIH Image J to provide quantitative values for relative expression of Bim protein (normalized to ERK protein). The statistical significance of changes was tested by the paired t test.
Reverse transcription-quantitative PCR.
Each sample of total RNA was isolated from cultured neurons by using TRI reagent (Molecular Research Center, Cincinnati, OH) and was from human brain samples by using Trizol reagent (Invitrogen, San Diego, CA). cDNA was transcribed from total RNA with Superscript RT II (Invitrogen). The primers used for PCR amplification of rat Bim were 5′-GCCCCTACCTCCCTACAGAC-3′ and 5′-CCTTATGGAAGCCATTGCAC-3′. The primers used for PCR amplification of human Bim were 5′-CACATGAGCACATTTCCCTCT-3′ and 5′-AAGGCACAAAACCTGCAGTAA-3′. Equal amounts of cDNA template were used for each PCR analysis of Bim, α-tubulin, Eef1a, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Quantitative PCR was performed using a Cepheid (Sunnyvale, CA) SmartCycler following the manufacturer's specifications. GAPDH was used for normalization of human Bim transcripts and α-tubulin or Eef1a was used for rat Bim transcript normalization. cDNA was added to a 25 μl volume reaction mix containing Ready-to-Go Beads (GE Healthcare, Piscataway, NJ) or OmniMix HS master mix (Cepheid) and SYBR Green I (Invitrogen) together with appropriate primers at 0.2 μm each. Analyses of growth curves of real-time fluorescence and of melting curves were performed as described previously (Troy et al., 2000).
Staining of human AD and control brain sections.
Alzheimer's disease and control brain samples (supplemental Table 1, available at www.jneurosci.org as supplemental material) were obtained at autopsy by the New York Brain Bank at Columbia University (Dr. J.-P. Vonsattel, Director) as described in their website (http://nybb.hs.columbia.edu/index.htm) (Vonsattel et al., 1995) under a protocol approved by the institutional review board. The criteria applied for assigning the diagnosis of Alzheimer's disease were that of “The National Institute on Aging, and Reagan Institute Working Group on Diagnostic Criteria for the Neuropathological Assessment of Alzheimer's Disease”; and that of CERAD (Consortium to Establish a Registry for Alzheimer's Disease). Two blocks were harvested fresh from one-half of each brain. The blocks were frozen in liquid nitrogen vapor at −160°C. One block included the rostral entorhinal area with gyrus ambient and amygdaloid nucleus. The other block included the cerebellar cortex and dentate nucleus. Contralateral mirror image blocks were fixed in 10% buffered formalin phosphate, and then paraffin embedded and processed for evaluation of the tissue using conventional neuropathology methods. A set of sections from each paraffin-embedded block was stained according the Bielschowsky protocol. In addition, sections were subjected to antibodies directed against β-amyloid, ubiquitinated proteins, α-synuclein, and hyperphosphorylated tau (AT8). Frozen sections were prepared from the rostral entorhinal area with gyrus ambient and amygdaloid nucleus and cerebellum of AD (moderate to severe) and control patients (for information such as patient age, gender, postmortem interval, and neuropathological diagnosis, see supplemental Table 1, available at www.jneurosci.org as supplemental material). Sections of the rostral entorhinal area with gyrus ambient and amygdaloid nucleus included three clearly discernable layers of the six present, and nearby amygdala. Staining of sections on the contralateral side for tau and by the Bielschowsky method confirmed the presence of Alzheimer changes including amyloid plaques and tangles. Tissue sections were 10 μm thick and immunostained as described in Angelastro et al. (2003). Briefly, sections were blocked in 3% nonimmune goat serum and 0.3% Triton X-100 for 1 h. The sections were then incubated with rabbit anti-Bim (1:500; Calbiochem, La Jolla, CA) antiserum and mouse anti-NeuN (1:100; Chemicon) antibody or mouse anti-cdk4 (1:500; Cell Signaling) antibody or mouse phospho-Rb (1:500; Cell Signaling) antibody in blocking solution overnight at 4°C, followed by secondary labeling with goat anti-rabbit (1:4000; Alexa Fluor 568; Invitrogen) and goat anti-mouse antibodies (1:4000; Alexa Fluor 488; Invitrogen) in blocking solution for 1 h. Finally, slides were covered with glass coverslips using mounting solution. No staining was observed when primary antisera/antibodies were omitted. As another indication of the staining specificity of the anti-Bim antiserum, no staining of cultured cells was observed after transfection with Bim small hairpin RNA (shRNA).
Immunostaining of cultured neurons.
Cortical neurons were transfected as indicated with appropriate constructs of shRNA in 500 μl of serum free medium per well in 24-well dishes using LipofectAMINE 2000. Six hours later, medium with LipofectAMINE 2000 was replaced with fresh complete medium. Forty-eight hours later, cells were exposed to aggregated 10 μm Aβ1–42 for 6 h or 1 μm oligomeric Aβ for 18 h, and then immunostained as described in Angelastro et al. (2003). Briefly, neurons were fixed with 4% paraformaldehyde for 10 min. After three washes in PBS, cells were blocked in 3% nonimmune goat serum for 2 h. The cultures were immunolabeled with rabbit anti-Bim (1:500; StressGen) antibody in 3% nonimmune goat serum overnight at 4°C, followed by secondary labeling with goat anti-rabbit antibody (1:4000; Alexa Fluor 568; Invitrogen) for 1 h.
Survival assays.
Cortical neurons were cotransfected with either pSIREN-cdk4-shRNA, pSIREN-Luc-shRNA, pSIREN-Bim-shRNA, or a control pSIREN-ZsGreen (to see the transfected cells) and empty pU6 or pU6-B-myb-shRNA, and then 48 h later treated with aggregated 10 μm Aβ1–42 or 1 μm oligomeric Aβ. The numbers of surviving transfected (green) cells per well were assessed by a blinded observer just before or after treatment and at 24 and 48 h after Aβ exposure as described previously (Liu and Greene, 2001). There was a 13–28% death of cells attributable to transfection alone without any treatment, and data were normalized taking this into consideration. Data represent means ± SEM of three experiments performed in triplicate.
Statistics.
All experiments were performed at least in triplicate, and results are presented as means ± SEM with an exception in Figure 1 in which the result is presented as mean ± SD of two experiments. Student's t test was used to evaluate the significance of differences between means and was performed as unpaired, two-tailed sets of arrays and presented as p values. In the case of multiple comparisons as in Figure 2 in which two t tests were used, Bonferroni's adjustment was applied and a value of p < 0.025 was considered to be a significant difference.
Results
β-Amyloid upregulates Bim in cultured hippocampal and cortical neurons
Hippocampal and cortical neurons are severely affected in AD brains and both undergo death after exposure to aggregated or oligomeric Aβ protein in vitro (Pike et al., 1991; Estus et al., 1997; Troy et al., 2000; Barghorn et al., 2005). A time course revealed that Bim is upregulated by aggregated Aβ1–42 in both neuron types in culture. Bim transcript levels increased by threefold to fourfold within 6 h of exposure (Fig. 1a). Bim extra large (BimEL) protein expression was also elevated by 3–4 h and was maximally induced by 8–9 h (Fig. 1b,c). Under the conditions of our experiment, neuron death first becomes apparent by ∼12–16 h of Aβ exposure and ∼50% of neurons die within 24 h. Thus, maximal Bim induction by Aβ precedes overt signs of neuron death.
Bim is elevated in entorhinal cortical neurons of AD brains
We next determined whether Bim transcripts and protein are elevated in postmortem brains derived from AD patients. Immunohistochemical staining of 10 AD and 9 control non-AD brains revealed that Bim levels were consistently elevated in the entorhinal cortex of AD brains where its localization appeared to be exclusively in neurons (identified by costaining with the neuronal marker NeuN) (Fig. 2a). The sections used in our study contained three clearly distinct cortical layers (of the six present in this area) of the rostral entorhinal area with gyrus ambient and amygdaloid nucleus. Examination of pathological changes in contralateral tissue revealed deposition of amyloid plaques and tangles in layers 2 and 3 as well as in nearby amygdala and it was in these areas that Bim-stained neurons were present. In contrast, layer 1 was relatively preserved with little presence of Alzheimer changes or Bim-positive cells (data not shown). Quantitation of Bim-positive neurons in layers 2 and 3 revealed that on average 85 ± 2.2% (n = 10) of NeuN-positive cells were Bim positive in AD brains, whereas only 18.4 ± 1.5% (n = 9) of neurons were Bim positive in the same areas of control non-AD brains (Fig. 2b). The difference between the means of the two populations was highly significant (p < 0.0001). Additionally, brains of two aged patients with mild AD-like changes in morphology, but no known cognitive deficits (supplemental Table 1, available at www.jneurosci.org as supplemental material) and of one patient with moderate Parkinson's disease (PD) showed an average 30.7 ± 3.8% Bim-positive neurons, which is significantly lower than in brains from AD patients (p < 0.0001). Even when detected in control non-AD and mild AD or PD brains, Bim staining was generally far less intense than in AD brains (Fig. 2a). We also examined cerebellar granule neurons a population apparently unaffected in AD. In this case, Bim was not elevated in either control or AD material (Fig. 3a). It has been reported that Aβ25–35 induces Bim and Bim-dependent death in cultured cerebral endothelial cells (Yin et al., 2002). However, we did not detect specific staining of cell types other than neurons in our sections.
Western immunoblots performed on isolated entorhinal cortex from a portion of the same brains confirmed BimEL protein expression and indicated (relative to ERK protein) increased BimEL levels in four of the five AD samples (exception, brain AD293) compared with controls (Fig. 3b) (data not shown). Quantification revealed that there was a significant difference in relative levels of Bim in entorhinal cortex derived from non-AD control and AD patients (1.0 ± 0.02 vs 1.5 ± 0.06, relative to ERK1; mean ± SEM; p < 0.05). This is consistent with a previous report in which Western immunoblotting indicated an increase of Bim protein in extracts of AD cerebral cortex, but not of cerebellum (Engidawork et al., 2001). Finally, as determined by quantitative real-time PCR, there was a significant difference in relative levels of Bim transcripts in entorhinal cortex isolated from AD and non-AD controls (38 ± 14 vs 6 ± 4, relative to GAPDH transcripts; mean ± SEM; p < 0.05) (Fig. 3c). The degree of variability in protein and mRNA levels in the AD material could reflect in part differences in the numbers of surviving neurons (and the consequent ratios of neuronal to non-neuronal tissue and of affected to nonaffected neurons) as well as the relative stabilities of these molecules in postmortem tissues. The loss of neurons in AD would tend to decrease the apparent changes of Bim expression detected in bulk tissue. Together, these findings indicate that BimEL protein levels are elevated in both a cellular model of AD and in relevant neurons of AD patients and that these changes reflect increases in Bim mRNA.
Bim plays a required role in neuron death evoked by Aβ in a cellular model of AD
Because Bim has been shown to have proapoptotic activity and plays a role in neuron death evoked by apoptotic signals such as NGF deprivation and seizure, we next assessed whether Bim induction is required for neuron death evoked by Aβ. To achieve this, we used a shRNA construct specifically targeted to Bim, based on a previously described similar human target sequence (Reginato et al., 2003) and the efficacy of which has been described previously in our system (Biswas et al., 2005) (see also Fig. 5). As shown in Figure 4, a and b, compared with an irrelevant shRNA (shLuc), the Bim shRNA significantly protected cultured cortical neurons from aggregated Aβ1–42. There was more than a twofold increase in surviving neurons after 24 and 48 h of Aβ exposure (Fig. 4b) with good preservation of overall neuron morphology (Fig. 4a). The specificity of Bim shRNA silencing was confirmed by overexpressing a silencing-resistant Bim (short isoform of Bim) that restored Aβ-induced death of cultured cortical neurons (supplemental Fig. 1, available at www.jneurosci.org as supplemental material). No protection was additionally seen with an empty U6 vector (see Fig. 6) or with a shRNA targeting no known sequence (supplemental Fig. 1, available at www.jneurosci.org as supplemental material). Thus, in a cellular model of AD, Bim plays an essential role in neuron death.
Cell cycle-related proteins are required for Bim induction by Aβ
It has been observed that cell cycle-related proteins are elevated in susceptible neurons of AD patients and that such molecules play essential roles in neuron death associated with a variety of paradigms of neuron death (Greene et al., 2004; Herrup et al., 2004). Of particular relevance, there is pharmacological and molecular evidence that cdk4 is required for death of cultured cortical neurons evoked by Aβ (Giovanni et al., 1999), and it is reported that cdk4 levels are elevated in AD neurons (Busser et al., 1998). In addition, we recently found that, in NGF-deprived cells, Bim is a target of a neuronal apoptotic pathway dependent on elevated cdk4 activity and consequent de-repression of E2F-regulated Myb genes (Biswas et al., 2005). We therefore assessed the possibility that Bim induction in a cellular model of AD requires cdk4 and Myb. As an initial test, we applied Aβ to cultured cortical neurons in presence of the cdk inhibitors flavopiridol and roscovitine and measured Bim expression by Western immunoblotting. Past work has shown that these inhibitors protect neurons from apoptotic death evoked by Aβ (Giovanni et al., 1999), and we also confirmed these results in our paradigm (supplemental Fig. 2, available at www.jneurosci.org as supplemental material). As shown in Figure 4c, these fully blocked Aβ-evoked Bim induction. In contrast, anthra[1,9-cd]pyrazol-6(2H)-one (SP600125), an inhibitor of c-Jun N-terminal protein kinase activity (Bennett et al., 2001), only partially inhibited induction of Bim. These findings are similar to those we have previously reported for Bim induction evoked by NGF deprivation (Biswas et al., 2005). Because enzymatic inhibitors may lack specificity, we additionally used a previously characterized shRNA that targets cdk4 (Biswas et al., 2005). As shown in Figure 4, a and b, the cdk4 shRNA effectively protects cultured cortical neurons from death evoked by Aβ. Moreover, the shRNA substantially blocked the induction of endogenous Bim in cortical neurons after Aβ exposure (Fig. 5).
To test the potential role of Myb proteins in Bim induction and death promoted by Aβ, we made use of a previously characterized B-myb shRNA (Liu et al., 2004; Biswas et al., 2005). In our past work, we found that B- and C-myb regulated each another so that interfering with synthesis of either one was sufficient to protect from NGF deprivation (Liu et al., 2004). Here, we observed that B-myb shRNA provided significant protection of cultured cortical neurons from death evoked by Aβ and blocked the induction of endogenous Bim in cortical neurons after Aβ exposure (Fig. 6). Together, these findings support the conclusion that cell cycle-related proteins are required for Bim induction and neuron death in response to Aβ.
Bim is coexpressed with cell cycle-related proteins in AD brains
As noted above, dysregulated levels of cell cycle-related proteins have been reported in vulnerable neuronal populations of AD brains. If, as found in our cell culture studies, Bim is a target of such molecules, then it should be present in neurons that express such proteins. To test this, we costained AD and control non-AD entorhinal cortex for expression of Bim and either cdk4 or phospho-Rb. Rb is a target of cdk4 that undergoes enhanced phosphorylation in AD neurons (Busser et al., 1998) and that, along with other members of the Rb family, regulates expression of Mybs in neurons (Liu et al., 2004). As shown in Figure 7, high levels of Bim immunoreactivity appeared to be present exclusively in AD neurons that were also highly positive for cdk4 and phospho-Rb (and vice versa). Moreover, like Bim, cdk4 and phospho-Rb expression within the same sets of brains (control and AD) was not elevated in cerebellar granule neurons (data not shown). These findings thus indicate that elevated Bim is coexpressed in vulnerable AD neurons that show activation of cell cycle-related proteins.
Discussion
The molecular chain of events that lead to death of neurons in AD is incompletely understood and any progress in this regard has the possibility to identify potential targets for therapeutic intervention. Our findings now implicate the proapoptotic Bcl-2 family member Bim in neuron death associated with AD. We show that Bim transcripts and protein are elevated in the entorhinal cortex of postmortem AD brains and that the elevation of Bim expression is limited to vulnerable populations within layers of this structure that possess plaques and tangles. In contrast, neurons in layer 1 of the entorhinal cortex and in the cerebellum that are spared in AD, did not exhibit elevated Bim protein. Additional complementary studies using cultures of cortical and hippocampal neurons (which are neuron types that are vulnerable in AD) further support a role for Bim in AD and link induction of this molecule to Aβ and to Aβ-dependent neuron death. That is, exposure of such cultures to Aβ induced Bim mRNA and protein, whereas downregulation of Bim using shRNA protected the neurons from Aβ-dependent toxicity.
Our immunostaining results indicate that a large proportion of vulnerable neurons in the entorhinal cortex of AD brains have elevated expression of Bim. Given that overexpression of Bim in cultured neurons induces a rapid apoptotic death (Whitfield et al., 2001), this raises the potential question as to why neuron death in AD is progressive and relatively prolonged despite apparent widespread Bim induction. One potential explanation is that, in addition to being induced, Bim must be appropriately modified to promote neuron death. There is evidence that Bim must be activated by c-Jun N-terminal kinase (JNK)-dependent phosphorylation to induce death of neurons (Putcha et al., 2003; Becker et al., 2004). Moreover, the proapoptotic activity of Bim appears to be suppressed by growth factor-stimulated phosphorylation via the ERK pathway (Biswas and Greene, 2002). An additional possibility is that a second event must occur to permit Bim-evoked neuron death. For example, it was recently reported that Aβ25–35 downregulates the antiapoptotic molecule bcl-w in cultured cortical neurons and that this effect plays a role in death evoked by the peptide (Yao et al., 2005). Thus, Bim induction may play a necessary, but not sufficient role in neuron death associated with AD. Suitable genetic animal models of AD that include neuron death will be helpful to further evaluate the in vivo role of Bim in this disorder.
The elevated expression of cell cycle-related proteins including cdk4 in AD neurons has been widely recognized (cf. Busser et al., 1998; Greene et al., 2004; Herrup et al., 2004; Webber et al., 2005). However, despite much speculation, the potential role of such molecules in the pathophysiology of AD has been less clear as have been the mechanisms by which they might trigger neuron death. In vitro experiments using pharmacologic cdk inhibitors and dominant-negative constructs have previously implicated cdk4 in neuron death evoked by Aβ (Giovanni et al., 1999, 2000). However, such reagents can possess nonspecific activities and it is significant that our use of shRNA directed to cdk4 corroborates this molecule as a required mediator of Aβ-dependent neuron toxicity.
Our cell culture findings along with observations in AD brains begin to outline a sequential molecular pathway of events by which cell cycle-related molecules link Aβ exposure with neuron death. cdk4 is elevated in AD neurons and past and present culture studies show that this molecule and its activity are essential for Aβ-induced neuron death. Among the major targets for activated cdk4 are members of the Rb family and these are hyperphosphorylated both in AD neurons (Ranganathan et al., 2001) and in Aβ-treated cultured neurons (Giovanni et al., 1999). Although the mechanism by which Rb family hyperphosphorylation might promote neuron death in response to Aβ has not been studied in detail, our recent findings on the role of cell cycle molecules in neuron death evoked by loss of trophic support (Liu and Greene, 2001; Liu et al., 2004, 2005; Biswas et al., 2005) may provide significant insight in this regard. NGF deprivation leads to activation of cdk4 and hyperphosphorylation of Rb family proteins. Once hyperphosphorylated in response to NGF deprivation, Rb proteins dissociate from DNA-bound transcription-silencing complexes comprising the transcription factor E2F and the chromatin modifiers Suv39H1 and histone deacetylase. This dissociation results in gene derepression. Among the genes that are de-repressed under such circumstances are the transcription factors B- and C-myb. The promoter for Bim contains a myb binding site and, as a consequence of the chain of events triggered by trophic factor deprivation, this becomes occupied by mybs and results in Bim induction and neuron death. Our present study now reveals that B-myb is also a required mediator of Bim induction as well as of neuron toxicity after Aβ exposure. Thus, it appears that a similar cdk4-dependent apoptotic pathway plays an essential role in Bim induction after Aβ exposure as in neurotrophic deprivation of neurons. Thus, we propose that Aβ sequentially leads to cdk4 activation, hyperphosphorylation of Rb family members, de-repression of B- and C-myb, and induction of the death-promoting molecule Bim.
In the present study, we found that essentially all AD neurons that we examined with elevated cdk4 or phospho-Rb also exhibit elevated Bim expression. This is consistent with and supports the possibility that the cell cycle molecule pathway outlined above also occurs in AD neurons and culminates in induction of proapoptotic Bim.
Although we have used acute Aβ exposure to link cdk4, Bim induction, and neuron death, there are other potential means by which Bim may be elevated in AD. For example, loss of synaptic connections and axonal degeneration seen in this disorder (Spires and Hyman, 2004) (whether triggered by Aβ or other causes) could result in failure of transsynaptic supply and/or retrograde transport of trophic molecules such as NGF. Such loss of trophic support would also lead to cdk4-dependent Bim elevation and consequent neuron death by the mechanisms discussed above.
In summary, our findings show that expression of the proapoptotic protein Bim is significantly elevated in a vulnerable population of neurons in AD brains. Additionally, we find that Bim is induced by the AD-associated Aβ peptide in cultured neurons and plays an essential role in Aβ-dependent neuron death. Finally, induction of Bim and of neuron death by Aβ appear to be mediated by pathway that includes the cell cycle molecule cdk4 and myb. These observations thus suggest that both the product of this pathway (Bim) and the cell cycle-related proteins involved in its induction appear to be potential targets for therapeutic intervention in AD.
Footnotes
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This work was supported in part by grants from the National Institutes of Health–National Institute of Neurological Disorders and Stroke (L.A.G., C.M.T.) and the Alzheimer's Disease Research Center at Columbia University AG-08702 (M. Shelanski). We thank Katerina Mancevska for aid in preparing and providing brain tissue samples. We thank Dr. Michael L. Shelanski for critical reading of this manuscript and Drs. Giselle F. Prunell and Peter Canoll for helpful discussions.
- Correspondence should be addressed to Subhas C. Biswas, Department of Pathology, Columbia University College of Physicians and Surgeons, P&S 15-401, 630 West 168th Street, New York, NY 10032. scb34{at}columbia.edu