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Cell Reports, Volume 3, Issue 1, 42-51, 03 January 2013
γ-Secretase Modulators and Presenilin 1 Mutants Act Differently on Presenilin/γ-Secretase Function to Cleave Aβ42 and Aβ43
Masayasu Okochi1, , , Shinji Tagami1, Kanta Yanagida1, Mako Takami2, 3, Takashi S. Kodama1, Kohji Mori1, Taisuke Nakayama1, Yasuo Ihara2, 3 and Masatoshi Takeda1
1 Neuropsychiatry and Neurochemistry, Department of Integrated Medicine, Division of Internal Medicine, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan
2 Department of Neuropathology, Faculty of Life and Medical Sciences, Doshisha University, Kizugawa 619-0225, Japan
3 JST, CREST, Kawaguchi, 332-0012, Japan
Graphical Abstract
► A concept to explain the production of AD-associated Aβ42(43) is proposed ► Aβ42(43) is not only a product of, but also a substrate for, γ-secretase in cells ► The mechanism underlying the action of γ-secretase modulators is proposed ► Enhancing γ-secretase function to cleave Aβ42(43) is a strategy for anti-AD drugs
- Deciphering the mechanism by which the relative Aβ42(43) to total Aβ ratio is regulated is central to understanding Alzheimer disease (AD) etiology; however, the mechanisms underlying changes in the Aβ42(43) ratio caused by familial mutations and γ-secretase modulators (GSMs) are unclear. Here, we show in vitro and in living cells that presenilin (PS)/γ-secretase cleaves Aβ42 into Aβ38, and Aβ43 into Aβ40 or Aβ38. Approximately 40% of Aβ38 is derived from Aβ43. Aβ42(43) cleavage is involved in the regulation of the Aβ42(43) ratio in living cells. GSMs increase the cleavage of PS/γ-secretase-bound Aβ42 (increase kcat) and slow its dissociation from the enzyme (decrease kb), whereas PS1 mutants and inverse GSMs show the opposite effects. Therefore, we suggest a concept to describe the Aβ42(43) production process and propose how GSMs act, and we suggest that a loss of PS/γ-secretase function to cleave Aβ42(43) may initiate AD and might represent a therapeutic target.
Alzheimer disease (AD) amyloid-β42 peptide (Aβ42) and Aβ43 are generated from β-amyloid protein precursor (βAPP) and accumulate in senile plaques (Gravina et al., 1995). Because Aβ is secreted as multiple peptide species with different C termini, intramembrane proteolysis of Aβ by presenilin (PS)/γ-secretase (De Strooper et al., 1998, De Strooper et al., 1999; Sherrington et al., 1995; Struhl and Greenwald, 1999; Wolfe et al., 1999) does not occur at a unique site. However, because even small elevations in the ratio of Aβ42(43) to total Aβ (Aβ42[43] ratio) in secreted Aβ by PS or βAPP mutations trigger familial AD (Kuperstein et al., 2010; Scheuner et al., 1996; Suzuki et al., 1994), the proteolysis is regulated strictly in this aspect. How the variation in Aβ is generated remains unclear.
How βAPP-CTF is cleaved into Aβ42 is controversial because of conflicting findings. First, cleavage at the ε-site generates primarily long fragments, namely Aβ48 and Aβ49, and is followed by stepwise cleavage of every three amino acid residues starting at the C terminus (Qi-Takahara et al., 2005). Two distinct lines have been proposed for Aβ40 and Aβ42 production. Second, cleavage at the γ-site does not correlate directly with cleavage at the ε-site (He et al., 2010).
The ratio of APL1β28, a surrogate marker of Aβ42, to total APL1β is elevated in the cerebrospinal fluid of patients with sporadic AD, including those in the mild cognitive impairment stage and in those with familial AD (Yanagida et al., 2009). Thus, an increase in the Aβ42 ratio in the brain may play a role in the etiology of most AD cases, and the mechanism underlying the regulation of the Aβ42 ratio is a central issue in understanding AD.
γ-Secretase modulators (GSMs) are disease-modifying drugs that specifically reduce Aβ42 generation (Kounnas et al., 2010; Weggen et al., 2001); some GSMs are being studied in clinical trials. Despite the development of GSMs (Wolfe, 2012), their mechanism of action (Chávez-Gutiérrez et al., 2012) remains unclear.
Given that the VVIA peptide located between the γ38 and γ42 cleavage sites was detected in an in vitro βAPP-CTF cleavage assay (Takami et al., 2009), we asked whether Aβ42(43), formerly considered a product of PS/γ-secretase, could also be a substrate for PS/γ-secretase. Here, we show that Aβ42(43) is an intermediate of PS/γ-secretase, a finding that provides important insight into the regulation of the Aβ42(43) ratio. Our results will help elucidate the mechanism underlying the actions of GSMs and PS mutants.
AD-Associated Aβ42 Is a Substrate of PS/γ-Secretase
Initially, we performed a modified in vitro γ-secretase assay in which we used CHAPSO (3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxypropanesulfonate) at its critical micelle concentration of 0.5% instead of 0.25%, the concentration used in the original method (Li et al., 2000). We experimented with using Aβ42 as a substrate instead of the βAPP-C-terminal fragment (βAPP-CTF) (Chávez-Gutiérrez et al., 2012). Surprisingly, Aβ38 was generated de novo from Aβ42 by PS/γ-secretase, a process that we refer to as "Aβ42 cleavage" (Figure 1). MALDI-TOF mass spectroscopy (MS) showed a de novo product of 4132.6 Da, which matches ionized Aβ38 (Figure 1A). Liquid-chromatography tandem MS (LC-MS/MS) (Takami et al., 2009) showed high levels of VVIA tetrapeptide between Aβ38 and Aβ42 (Figure 1B). No further cleavage of Aβ38 was observed, indicating that Aβ38 was the final product (Figure S1A). We also observed a small amount of VIA (∼1/40th the amount of VVIA), the tripeptide between Aβ39 and Aβ42, indicating very low-level production of Aβ39 (Figure 1B). Cleavage of Aβ42 was eliminated by γ-secretase inhibitors (Figures 1A and 1B). We also found that the N terminus of the substrate Aβ42 was not necessarily the first residue, because Aβ11–42 was also cleaved (Figure S1B). Collectively, these results indicate that Aβ42 is cleaved into Aβ38 and Aβ39. Irrespective of how cleavage at the γ42-site occurs (Qi-Takahara et al., 2005; He et al., 2010), our current results demonstrate that Aβ42, the pathological PS/γ-secretase product, is also a substrate for PS/γ-secretase.
We next studied Aβ42 cleavage, when βAPP-CTF is the substrate. Cleavage of Aβ42 in the new assay was optimal at ∼0.5% CHAPSO, whereas cleavage of βAPP-CTF in the conventional assay was optimal at ∼0.2% CHAPSO (Li et al., 2000) (Figure 1C). Given this result, we considered the possibility that the Aβ42 ratio in the de novo Aβ generation in the βAPP-CTF cleavage assay might decrease at a higher CHAPSO concentration (∼0.5%). As the CHAPSO concentration increased, the relative ratio of Aβ42(43) production decreased and the ratio of Aβ38 production increased (Figure 1D; Figure S2A). Surprisingly, the relative ratio of Aβ42(43) to Aβ40 was ∼0.1 at ∼0.5% CHAPSO. Thus, we suspected that the much higher Aβ42(43) ratio produced in the conventional βAPP-CTF cleavage assay than that secreted from living cells may be because the assay was performed in the presence of ∼0.25% CHAPSO. In this regard, the in vitro γ-secretase assay in the presence of ∼0.5% CHAPSO may be a better model.
Because Aβ42 levels are determined by the balance between its rate of production and its rate of degradation, we measured further the levels of small peptides produced by βAPP-CTF cleavage (Figure 1E). The relative amount of VVIA produced by βAPP-CTF cleavage increased as the Aβ38 ratio increased. These data suggest that the relative levels of Aβ42 are also a function of Aβ42 cleavage. Moreover, CTFγ38/40/42/43, the counterparts of direct cleavage at the Aβ38/40/42/43 sites, have never been observed (Gu et al., 2001). Collectively, our results indicate that Aβ42 is an intermediate stopover product of the cleavage of βAPP-CTF by PS/γ-secretase.
GSMs and Mutant PS1/γ-Secretases Increase and Decrease Aβ42 Cleavage in Living Cells, Respectively
Next, we asked whether the Aβ42 cleavage process plays a role in conditions where the Aβ42 ratio in secreted Aβ changes in vitro and in living cells. First, we examined whether GSMs and inverse GSMs (iGSMs) (Kukar et al., 2005) change the rate of Aβ42 cleavage in vitro when the enzyme activity is low (0.25% CHAPSO) or high (0.5% CHAPSO). Strikingly, in the presence of 0.25% CHAPSO, all GSMs tested (GSM1, Eisai, Compound W, and Sulindac sulfide) increased the relative rate of Aβ42 cleavage (Figure 2A; Document S1. Tables S1–S3A). In contrast, in the presence of 0.5% CHAPSO, the iGSMs tested (fenofibrate and S2474) decreased the relative rate of Aβ42 cleavage (Figure 2B; Document S1. Tables S1–S3B).
We do not know how CHAPSO affects the rate of Aβ42 cleavage in vitro. However, in the presence of 0.25% CHAPSO, when Aβ42 cleavage activity is low, iGSMs did not slow the reaction (Figure 2A). Similarly, GSMs did not increase Aβ42 cleavage in the presence of 0.5% CHAPSO, when the activity is high (Figure 2B). These findings suggest that the effects of CHAPSO and GSMs/iGSMs are related.
Next, we investigated whether an increase in Aβ42 cleavage by GSMs is responsible for the decrease in the Aβ42 ratio in living cells. We added GSMs and iGSMs to cultured HEK cells expressing wild-type (WT) and Swedish mutant (sw) βAPP stably, which were the same cell lines used for extracting PS1/γ-secretase for the in vitro experiments. We immediately boiled the treated cells for 2 min to inhibit completely the active degradation of tri-, tetra-, and pentapeptides in the living cells. We extracted the soluble fraction from the resultant cell lysate and measured the levels of the small peptides by LC-MS/MS (Figures 2C and 2G).
Hereafter, the mention of an increase or a decrease in the levels of each peptide associated with the generation of secreted forms of Aβ (e.g., VVIA, VVIAT, and IAT) implies that the relative ratio of a peptide was calculated in relation to that of the sum of peptides associated with Aβ generation (i.e., ITL, VIT, VIV, TVI, IAT, VVIA, and VVIAT) (Figures 2, 3, and 4). GSM-treated cells, which secreted a lower ratio of Aβ42 (Figure 2D; Document S1. Tables S1–S3D), contained a higher ratio of VVIA (produced by cleavage of Aβ42 into Aβ38) than did DMSO-treated cells (Figure 2C; Document S1. Tables S1–S3C). In contrast, iGSM-treated cells, which secreted a higher ratio of Aβ42 (Figure 2D), contained a lower ratio of VVIA (Figure 2C). These data indicate that GSMs and iGSMs increase and decrease, respectively, the rate of Aβ42 cleavage in living cells.
Next, because the Aβ42 ratio in secreted Aβ is increased in most PS1 pathological mutants (Scheuner et al., 1996), we examined whether PS1 mutants integrated into the PS/γ-secretase complex exhibit a reduced rate of Aβ42 cleavage. We extracted the PS/γ-secretase fraction (Figure 2E; see also Figures S2F and S2B–S2D) (Winkler et al., 2009) from HEK cells stably expressing WT or mutant (L286V, L381V, or G384A) PS1 (Figures S2E and S2F). The same amount of enzyme complex was included in each reaction (Figure 2E). We found that the rate of Aβ42 cleavage was lower for mutant than for WT PS1/γ-secretase (Figure 2F; Document S1. Tables S1–S3E), which is consistent with a previous report (Chávez-Gutiérrez et al., 2012). The results are reminiscent of the fact that cells expressing mutant PS1/γ-secretases generally secrete less total Aβ than do WT cells (Shen and Kelleher, 2007). However, it is of note that the reduced rate of Aβ42 cleavage described here is different from the overall loss of function of PS/γ-secretase.
We investigated whether reducing Aβ42 cleavage by mutant PS/γ-secretases could increase the Aβ42 ratio in living cells. We cultured WT or mutant PS1-expressing cells coexpressing sw βAPP (the same cell line used for the in vitro experiments) and analyzed the cell lysates by LC-MS/MS. The relative levels of VVIA were lower in the lysates of mutant cells than in WT cells (Figure 2G; Document S1. Tables S1–S3F). We confirmed the increased Aβ42 ratio in the conditioned medium of the mutant-expressing cells (Figure 2H; Document S1. Tables S1–S3F). These data demonstrate that mutant PS1/γ-secretases decrease the rate of Aβ42 cleavage in living cells. Collectively, our data suggest that the Aβ42 cleavage process is associated with the Aβ42 ratio in secreted Aβ.
A New Concept for the Production of Bona Fide Aβ42
To gain insight into the regulation of Aβ42 cleavage, we next examined how GSMs, iGSMs, and mutant PS1/γ-secretases alter Aβ42 cleavage activity in vitro. The conversion of Aβ42 into Aβ38 can be described by the following scheme and with the following rate constants:
One may think that once βAPP-CTF and PS/γ-secretase form a complex, intermediate long Aβ does not dissociate from the enzyme during the stepwise cleavages. According to the model, the kcat values of the Aβ42 cleavage should vary depending on the substrate. However, without any changes in the relative position of long Aβ species to PS/γ-secretase, the stepwise cleavages might be interrupted. Moreover, we showed clearly that Aβ42, Aβ43, Aβ45, and Aβ46 can bind to PS/γ-secretase and undergo cleavage (Figures 1 and 4). It is unknown whether all of the intracellular Aβ45 and Aβ46 (Qi-Takahara et al., 2005) bind to PS/γ-secretase. Based on these findings, we suggest that the long Aβ species undergoes association/dissociation events with PS/γ-secretase. Therefore, we revised the formulas describing the stepwise cleavage process proposed originally by Takami et al., 2009 (Figure 3B). We introduced the association/dissociation steps clearly for each cleavage step and did not consider that the cleavage at every three amino acid residues is an essential part of the cleavage process. Aβ42 and Aβ43 correspond to AβXn in the revised scheme. Aβ45 and Aβ46 correspond to AβX(n-1). Whether the various free AβXn products produced in each step remain at the membrane depends on their physicochemical nature. According to our model, the kcat values of the Aβ42 cleavage should be the same, regardless of the substrate (e.g., βAPP-CTF and Aβ42).
GSMs and Mutant PS1 Increase and Decrease, Respectively, the Velocity at which Bound Aβ42 Is Cleaved to Aβ38 In Vitro
First, to obtain the relative kcat values for Aβ42 cleavage, we performed enzyme kinetic analysis of Aβ42 cleavage over a range of Aβ42 concentrations (Figures 3C–3E; see also Document S1. Tables S1–S3A–S2C). The Aβ42 cleavage reaction conformed to Michaelis-Menten kinetics (Figures S3A–S3D). Next, we examined whether GSM or iGSM alters the kcat for Aβ42 cleavage. Hanes-Woolf plots were used to determine the Vmax values (Figures 3C, 3D, and 3F). Because Aβ42 was in excess in the reactions, we assumed that Vmax = kcat [PS/γ-secretase]. The relative kcat value was larger in the presence of GSM1 or Eisai than with WT PS/γ-secretase alone, whereas the value was smaller in the presence of S2474 than with WT PS/γ-secretase (Figure 3E; Document S1. Tables S1–S3C). Depending on CHAPSO concentration (0.25% or 0.5%), the y intercepts of the Hanes-Woolf plots differed, indicating that CHAPSO noncompetitively modified the action of PS/γ-secretase. We next performed reactions using equal amounts of each mutant and WT PS1/γ-secretase (Figure 3F; Document S1. Tables S1–S3D). The relative kcat values were smaller for PS1 L286V, L381V, and G384A/γ-secretase than for WT PS/γ-secretase (Figure 3G; Document S1. Tables S1–S3E). Thus, in the assay conditions used, GSMs increased the velocity at which bound Aβ42 was cleaved to Aβ38, whereas both PS1 mutants and iGSM reduced the velocity of the cleavage.
A GSM Reduces the Velocity at which Bound Aβ42 Dissociates from WT PS1/γ-Secretase, but the Complex of Mutant PS1/γ-Secretases and Aβ42 Dissociates Faster
We also used Biacore binding analysis to measure the relative dissociation rate constant kb for the complex of Aβ42 bound to PS1/γ-secretase. Aβ42 was immobilized to the sensor tip, and purified PS1/γ-secretases were injected as the analytes. We tried to measure the kb values to show how fast Aβ42 dissociates from the active center of PS/γ-secretase. We performed the Biacore assay with PS/γ-secretase preincubated in the presence or absence of L685,458, a transition state mimic that blocks the active site of PS/γ-secretase (see Figures S3E–S3J). We assumed that subtracting the resonance unit (RU) value for the L685,458–PS/γ-secretase complex binding to Aβ42 from the RU value for PS/γ-secretase alone (without mixing with L685,458) binding to Aβ42 would give the RU value for PS/γ-secretase, which holds Aβ42 in its active center (see Figures 3H and 3J). Using the RU values, we calculated kb values for the dissociation of the bound Aβ42 from the active center.
We studied whether GSM1 or S2474 affects the kb value. During the period from 5 s to 155 s in the dissociation phase, the washout curves of compounds tested with Aβ42 showed simple one-step dissociation with the single exponential rate expected from the model (Figure 3H; Document S1. Tables S1–S3F). The relative kb values of dissociation in the presence of GSM1 and S2474 were smaller and larger, respectively, than the value for WT PS/γ-secretase alone (Figure 3I; Document S1. Tables S1–S3G). The results indicate that GSM1 and S2474 decreased and increased the rate of dissociation of Aβ42 from the active center of PS/γ-secretase by 0.36 and 2.7 times, respectively, compared with the DMSO control in the assay condition. We also performed similar experiments to measure the relative kb values for the complex of Aβ42 with WT and mutant PS1/γ-secretase (Figure 3J; Document S1. Tables S1–S3H). The dissociation rates of Aβ42 from L286V and G384A mutant PS1/γ-secretases were 2.4 and 2.0 times larger, respectively, than the rate for WT PS1/γ-secretase (Figure 3K; Document S1. Tables S1–S3I). These data suggest that the velocity at which bound Aβ42 dissociates from PS1/γ-secretase contributes to the changes in the Aβ42 ratio in secreted Aβ caused by the compounds and some mutants. However, we are not yet able to show the extent of the relative effects of the two factors (i.e., kcat and kb) when the Aβ42 ratio changes in living cells.
Aβ43, Another Long Aβ Species, Is Cleaved into Aβ40 or Aβ38 by PS/γ-Secretase in Living Cells
Aβ43 is another long species of Aβ (Saito et al., 2011). We also investigated whether Aβ43 undergoes further proteolysis in a manner similar to Aβ42. MALDI-TOF MS (Figure 4A) and LC-MS/MS (Figure 4B) showed that Aβ43 was cleaved to Aβ40 or Aβ38 by PS/γ-secretase in vitro. Thus, Aβ38 has multiple precursors. This was confirmed by the in vitro βAPP-CTF cleavage assay (Figure 4C). Why PS/γ-secretase cleaved the substrate at only one of two sites remains unclear. The production of Aβ37 and GVV indicates minor but further cleavage of de novo Aβ40 (Figures 4A and 4B; Figures S1C and S1D).
We measured the amounts of tri-, tetra-, and pentapeptides produced during the stepwise processing of βAPP in living cells (Figure 4D; Figure S4A). Approximately 40% of Aβ38 was derived from Aβ43 (Document S1. Tables S1–S3A). GSMs increased the relative rate of Aβ43 cleavage into Aβ38 (i.e., the level of VVIAT relative to that of total Aβ-related small peptides) (Figure 4E; Document S1. Tables S1–S3B), whereas iGSM and mutant PS1/γ-secretases decreased the rate (Figure 4E and 4F; Document S1. Tables S1–S3C). These results are very similar to the effects of the compounds and the mutants on Aβ42 cleavage (see Figures 2C and 2G; summarized in Figures 4I and 4J). However, both the tested GSM/iGSM (Figure 4G; Document S1. Tables S1–S3D) and some mutant PS1/γ-secretases (i.e., PS1 L381V and G384A) (Figure 4H; Document S1. Tables S1–S3E) decreased the rate of Aβ43 cleavage into Aβ40 (i.e., IAT). Thus, the cleavage of Aβ43 into Aβ38 and that into Aβ40 were affected differently by GSMs.
Next, we asked whether the relative level of Aβ38 derived from Aβ42 and that derived from Aβ43 (i.e., VVIA and VVIAT) in living cells could be changed by the compounds and mutants. GSMs drastically increased the relative levels of both VVIA and VVIAT, whereas an iGSMs decreased the relative levels of the two peptides (Figure 4I; Document S1. Tables S1–S3F). Notably, because of the GSM effect, more than half of the Aβ38 was derived from Aβ43. The PS1 mutants decreased the relative levels of VVIA and VVIAT; however, the degree of the changes was much smaller than that observed for GSM (Figure 4J; Document S1. Tables S1–S3G). The PS1 G384A mutant did not decrease the relative levels of VVIAT. Very similar data were obtained when the levels of VVIA and VVIAT were normalized to the level of each major cleavage in the previous step (i.e., TVI for VVIA and VIV for VVIAT; Figures S4B and S4C). Collectively, these results indicate that GSMs strongly affect Aβ42(43) cleavage, and the increase in Aβ38 production is largely attributed to the cleavage of Aβ43 into Aβ38.
We also performed an Aβ45 and Aβ46 cleavage assay. MALDI-TOF MS showed that both Aβ45 and Aβ46 were cleaved by PS/γ-secretase. Interestingly, Aβ41 and Aβ40, in addition to Aβ42, were produced from Aβ45. Aβ42, in addition to Aβ43, was produced from Aβ46 (Figures 4K and 4L). Combined with the results showing that considerable amounts of Aβ38 were derived from Aβ43, the data indicate that the proposed "Aβ38 product line" (from βAPP-CTF via Aβ48 and Aβ42) and the proposed "Aβ40 product line" (from βAPP-CTF via Aβ49 and Aβ43) (Takami et al., 2009) cross each other.
We studied how the CHAPSO concentration affects the rates of Aβ42(43) cleavage, when βAPP-CTF is cleaved in vitro (Figures S4D and S4E). Interestingly, CHAPSO affected the rates in a dose-dependent manner and in a similar way to the effect of GSMs.
Long Aβ-like Peptides Other Than Aβ Are Also Substrates of PS/γ-Secretase
Aβ-like peptides (Okochi et al., 2002), secreted by a process similar to that for Aβ secretion, include mNotch-1-derived Nβ (Okochi et al., 2006) and APLP1-derived APL1β (Yanagida et al., 2009). We found that PS/γ-secretase cleaved Nβ25 into Nβ21 (Figure 4M) and APL1β28 into APL1β25 (Figure 4N), suggesting that long Aβ-like peptides are generally intermediate products. This may explain why the relative levels of some longer secreted Aβ-like peptides, including Aβ42, change in parallel (Okochi et al., 2006; Yanagida et al., 2009). This finding also indicates that APL1β28 cleavage to APL1β25 is impaired in the sporadic AD brain (Yanagida et al., 2009).
In this study, we show that de novo Aβ42(43), a secreted species, is an intermediate of PS/γ-secretase in living cells, and this discovery affects the understanding of the nature of Aβ42(43) production. We suggest that Aβ42 production does not directly reflect the level of cleavage at the C terminus of Aβ42, but rather depends on how much newly produced Aβ42 dissociates from the PS/γ-secretase enzyme and thereby avoids further cleavage. Thus, competition between further cleavage and dissociation from the enzyme may be the key to determining the Aβ42(43) ratio. Importantly, our results also suggest that a new type of partial loss of function in PS/γ-secretase [e.g., reduction in Aβ42(43) cleavage or at the final step of PS/γ-secretase cleavage of βAPP] may cause a gain of function in AD [an increase in the Aβ42(43) ratio]. GSMs increase the relative kcat for the further cleavage of Aβ42 to Aβ38 and decrease the relative kb for the dissociation of Aβ42 from PS1/γ-secretase. This suggests a potential model to explain how GSMs can lower Aβ42 production.
Chávez-Gutiérrez et al., 2012 showed that PS1 mutations lower the relative levels of Aβ38 to Aβ42 and Aβ40 to Aβ43 compared with WT PS1. The GSMs tested increased both the level of Aβ38 relative to Aβ42 (Weggen et al., 2001) and the level of Aβ40 relative to Aβ43. Based on the hypothetical model proposed by Ihara and colleagues (Takami et al., 2009), those authors speculated that the PS1 mutants and GSMs decrease and increase, respectively, the rate of the fourth cleavage (i.e., Aβ43 cleavage to Aβ40 and Aβ42 cleavage to Aβ38, respectively), possibly because of the premature release of the Aβ42/Aβ43 peptides.
We found that, in living cells, ∼40% of Aβ38 was derived from Aβ43. Moreover, Aβ40 and Aβ41 were produced in the Aβ45 cleavage assay, and Aβ42 was produced in the Aβ46 cleavage assay. Thus, the putative Aβ38 and Aβ40 product lines (Takami et al., 2009) turn out to overlap at several points.
Takami et al., 2009 showed that Sulindac sulfide decreased the levels of Aβ42 and Aβ43 in a βAPP-CTF cleavage assay, but did not significantly increase the levels of VVIA and IAT. This may be because Sulindac sulfide exerts a weaker GSM action than GSM1 and Eisai, which were used in this study.
We also found that Notch-1 and APLP1 transmembrane domains are cleaved in a similar way, which should help clarify the physiological process of intramembrane proteolysis by PS/γ-secretase. We showed previously that Aβ42 ratio changed in parallel with APL1β28 ratio, and that the APL1β28 ratio increases in the cerebrospinal fluid of AD patients (Yanagida et al., 2009). In this article, we demonstrated that both Aβ42 and APL1β28 were cleaved similarly into the shorter species (i.e., Aβ38 and APL1β25). Therefore, we suggest that Aβ42 cleavage may also decrease in AD brains.
Collectively, we speculate that the increase in the Aβ42 ratio simply reflects the accelerated dissociation of membrane-bound long Aβs (ie, Aβ44∼49) from PS/γ-secretase. Because long Aβs on the membrane may perturb the physiological function of neuronal cells, further studies are necessary to investigate whether the prolonged stay of long Aβ at the membrane is pathologically relevant.
At present, inhibition of PS/γ-secretase activity using agents such as Notch-sparing inhibitors is the central approach to decreasing Aβ production specifically. Our results may shift the nature of new drugs for treating AD to repair or increase the ability of PS/γ-secretase to cleave Aβ42(43).
Experimental Procedures
Aβ and Aβ-like Peptide Cleavage Assays
In vitro γ-secretase assays (Li et al., 2000; Osawa et al., 2008) using Aβ and Aβ-like peptides (Aβ42, 43, 45, 46, Nβ25, and APL1β28) were performed under the modified conditions described here with a modified reaction buffer (150 mM citrate buffer [pH 6.0], 0.25 M sucrose, 0.04%–1.5% CHAPSO, 0.1% phosphatidylcholine, 10 μM bestatin, 10 μM amastatin, 5 μM phenanthroline, 10 μM captopril, and 5× Roche protease inhibitor mix).
Extraction of Tri-, Tetra-, and Pentapeptides from Living Cultured Cells
HEK cells stably expressing sw βAPP and PS1 derivatives were cultured to confluence in 10 cm dishes, and 24 hr before collection, the cells were treated with GSM or iGSM. Proteasome inhibitors (1 μM lactacystin, 100 nM MG262, and 1 μM epoxomicin) were added, with or without GSM/iGSM, to the conditioned medium for the last 4 hr. The cells were washed quickly with ice-cold PBS and then immediately boiled for 2 min. The boiled samples were sonicated for 5 s three times and ultracentrifuged. The resultant supernatant was subjected to LC-MS/MS analysis to measure the tri-, tetra-, and pentapeptides.
For additional details, please refer to the Extended Experimental Procedures.
Aβ38, Aβ40, Aβ42, Aβ43, Aβ45, Aβ46, Aβ11–42, APL1β28, and Nβ25 were synthesized by Peptide Institute, Inc., JAPAN. FLAG-tagged βAPP-CTF were generated as described previously (Kakuda et al., 2006). GSM (Winkler et al., 2009), Eisai ((3E)-1-[(1S)-1-(4-fluorophenyl)ethyl]-3-[3-methoxy-4-(4-methyl-1H-imidazol-1-yl)benzylidene]piperidine-2-one), and S2474 were generated synthetically. Compound W was synthesized as described previously (Okochi et al., 2006). Sulindac sulfide and fenofibrate were from Sigma, and L685,458 and DAPT were from Peptide Institute. Anti-Nβ antibody 6521 (Okochi et al., 2006) and anti-APL1β antibody OA858 (Yanagida et al., 2009) were produced as described previously. WGA-agarose was from Calbiochem. Anti-PEN-2 antibody 6510 was raised against ERVSNEEKLNL. Anti-nicastrin antibody N1660 was from Sigma. Anti-Aβ antibody 4G8 and anti-Aph1-aL C-terminal antibody were from Covance. Antibody 82E1 was from IBL. Anti-PS1-N-terminal fragment (NTF) antibody were from Millipore. Anti-PS2-CTF antiserum was from Calbiochem.
Tris-Tricine-Urea Gel Electrophoresis
Tris-tricine-urea polyacrylamide gel electrophoresis was performed essentially as described previously (Qi-Takahara et al., 2005).
Immunoprecipitation and Immunoblotting
Immunoprecipitation was performed essentially as described previously (Okochi et al., 2002). PS and Aβ derivatives were detected by immunoblotting essentially as described previously (Yanagida et al., 2009; Qi-Takahara et al., 2005). Aβ species were immunoprecipitated with antibody 4G8 and analyzed by immunoblotting with antibody 82E1.
Immunoprecipitation MALDI-TOF MS Analysis
Immunoprecipitation-MS experiments were performed essentially as described previously (Okochi et al., 2002).
Quantitative LC-MS/MS Analysis
Small Aβ-derived peptides were measured by LC/MS/MS as described previously (Takami et al., 2009). VIAT, ATVI, and VVIAT were examined. The m/z values for these peptides were as follows: 403.5 and 184.9 for VIAT; 403.5 and 254.1 for ATVI; and 502.7 and 199.1 for VVIAT.
Aβ ELISA
The levels of Aβ species (Aβ38, Aβ40, Aβ42, and Aβ1–x) were measured by ELISA according to the manufacturer's instructions (IBL). To exclude the effects of the bulk of substrates (Aβ42, Aβ43, and βAPP-CTF), each assay solution was diluted with EIA dilution buffer (IBL) to a substrate concentration of 1 nM.
Immunoprecipitation Purification of γ-Secretase Complexes from Cultured Cells
PS1/γ-secretases were purified by immunoprecipitation from HEK293 cells overexpressing the corresponding PS1 derivatives as described previously (Takami et al., 2009).
Purification of the PS1/γ-Secretase Complex from Cultured Cells
PS1/γ-secretase was purified as described (Winkler et al., 2009) with some modifications. EDTA and glycerol were omitted from the buffers in the Biacore analysis. Briefly, cell membranes were solubilized in buffer A containing 0.75% n-dodecyl β-d-maltoside (DDM) and 1.5% digitonin, and the soluble fraction was precipitated with 50% ammonium sulfate. The resulting supernatant was precipitated with 70% ammonium sulfate, and the precipitated PS1/γ-secretase-rich fraction was resuspended in 55% ammonium sulfate. This extract was purified further with WGA-agarose in modified buffer B (50 mM bis-Tris-HCl, 66 mM ε-aminocaproic acid, 5% glycerol, 10 mM dithiothreitol, 0.1% DDM, and 0.5% digitonin). The incubation time in batch mode was extended from 90 min to 3 hr. Beads were collected and washed four times with modified buffer C (5 mM sodium citrate, pH 6.4, 5% glycerol, 1% CHAPSO, 10 mM dithiothreitol, 300 mM methyl α-d-mannopyranoside). The PS1/γ-secretase complex was eluted with modified buffer D (5 mM sodium citrate, pH 6.4, 1 mM EDTA, 1% CHAPSO, 10 mM dithiothreitol, 300 mM N-acetylglucosamine). The eluate from the WGA-agarose was used as purified PS1/γ-secretase for the in vitro γ-secretase assays. Each reaction was performed using the same amount of the purified PS1/γ-secretase complex containing familial AD-associated PS1 mutants (L85P (Ataka et al., 2004), L286V (Sherrington et al., 1995), L381V (Dintchov Traykov et al., 2009), or G384A (Cruts et al., 1995)). Briefly, the purified fractions were subjected to SDS-PAGE and immunoblotting with anti-PS1-NTF antibody. Equal loading was checked by immunoblotting of the PS1-NTF band. Loading and immunoblotting were repeated until the same amount of PS1-NTF band per reaction was confirmed for all enzyme preparations. Equal amounts of PS1-NTF-containing enzyme preparations were analyzed further by SDS-PAGE and immunoblotting with anti-nicastrin, anti-PEN-2, and anti-Aph-1aL antibodies. Only the enzyme preparations showing about the same band densities for these proteins were used for further analysis. The same relative immunoblot band density of purified enzyme was added to each reaction. The protein concentration of each purified PS1/γ-secretase was measured using a Pierce 660 nm protein assay (Thermo Scientific).
Enzyme Kinetic Analysis
Aβ42 cleavage assays were performed using purified PS1/γ-secretases and in the presence of the indicated concentrations of the GSMs and iGSMs. An aliquot was removed from each reaction 5 and 25 min after the reaction was started. The aliquots were diluted immediately in EIA dilution buffer (IBL) containing 10 μM L685,458 and 0.5% Triton-X, and the amount was measured using the Aβ38 ELISA. The difference between the levels of de novo-synthesized Aβ38 after 5 and 25 min of incubation was used to calculate the relative velocity of Aβ38 production at each Aβ42 concentration.
Biacore Analysis
Aβ42 (1 μM Aβ42 in 100 mM sodium acetate) was injected into a Biacore T200 (GE Healthcare) and immobilized to a CM5 sensor tip up to about 1000 RUs using an Amine Coupling Kit (GE Healthcare) according to the manufacturer's instructions. The system was equilibrated with running buffer (150 mM sodium citrate, 1 mM EDTA, 2.5 mM dithiothreitol, 50 mM N-acetylglucosamine, 0.5% CHAPSO, and 0.1% phosphatidylcholine, pH 6.4) at 30°C. The purified WT or mutant PS/γ-secretase fraction eluted from WGA-agarose in running buffer was preincubated with the indicated compounds and phosphatidylcholine for 10 min. The resultant analyte was injected for 2 min (binding phase), followed by a 3 min injection of running buffer alone (dissociation phase) at a flow rate of 30 μl/min. The kb values were calculated from the dissociation phase (5–155 s) of PS1/γ-secretases from Aβ42 in the active center in the presence or absence of compounds. The dissociation curves and kb values were evaluated by BIAevaluation version 3.1 software.
We thank Drs. Satoru Funamoto and Junji Takeda for critical discussions, Dr. Edward H. Koo for a 7WD10 cell, and Ms. Kana Fujii-Horinouchi, Yukiko Sumi, Mika Nobuhara, and Mr. Yuto Satake for technical help. M.O. and coworkers are funded by KAKEN-C, the National Institute of Biomedical Innovation (05-26), and the National Center for Geriatrics and Gerontology (23-5). A part of this study is the result of "Integrated Research on Neuropsychiatric Disorders" carried out under the Strategic Research Program for Brain Sciences by the Ministry of Education, Culture, Sports, Science and Technology of Japan. S.T. and M.O. discovered Aβ42 cleavage by PS/γ-secretase. K.Y., S.T., M.T., T.S.K., T.N., and M.O. performed the experiments. S.T., Y.I., and M.T. participated in the study design. M.O. conceived and designed the study, analyzed the data, and wrote the manuscript with contributions from S.T. and Y.I. All authors discussed the results and commented on the manuscript.
Chávez-Gutiérrez et al., 2012 (2012). The mechanism of γ-Secretase dysfunction in familial Alzheimer disease. EMBO J. 31, 2261–2274. CrossRef | PubMed
De Strooper et al., 1998 (1998). Deficiency of presenilin-1 inhibits the normal cleavage of amyloid precursor protein. Nature 391, 387–390. CrossRef | PubMed
De Strooper et al., 1999 (1999). A presenilin-1-dependent gamma-secretase-like protease mediates release of Notch intracellular domain. Nature 398, 518–522. CrossRef | PubMed
Gravina et al., 1995 (1995). Amyloid beta protein (A beta) in Alzheimer's disease brain. Biochemical and immunocytochemical analysis with antibodies specific for forms ending at A beta 40 or A beta 42(43). J. Biol. Chem. 270, 7013–7016. CrossRef | PubMed
Gu et al., 2001 (2001). Distinct intramembrane cleavage of the beta-amyloid precursor protein family resembling gamma-secretase-like cleavage of Notch. J. Biol. Chem. 276, 35235–35238. CrossRef | PubMed
He et al., 2010 (2010). Gamma-secretase activating protein is a therapeutic target for Alzheimer's disease. Nature 467, 95–98. CrossRef | PubMed
Kounnas et al., 2010 (2010). Modulation of gamma-secretase reduces beta-amyloid deposition in a transgenic mouse model of Alzheimer's disease. Neuron 67, 769–780. Abstract | Full Text | PDF (1805 kb) | CrossRef | PubMed
Kukar et al., 2005 (2005). Diverse compounds mimic Alzheimer disease-causing mutations by augmenting Abeta42 production. Nat. Med. 11, 545–550. CrossRef | PubMed
Kuperstein et al., 2010 (2010). Neurotoxicity of Alzheimer's disease Aβ peptides is induced by small changes in the Aβ42 to Aβ40 ratio. EMBO J. 29, 3408–3420. CrossRef | PubMed
Li et al., 2000 (2000). Presenilin 1 is linked with gamma-secretase activity in the detergent solubilized state. Proc. Natl. Acad. Sci. USA 97, 6138–6143. CrossRef | PubMed
Okochi et al., 2002 (2002). Presenilins mediate a dual intramembranous gamma-secretase cleavage of Notch-1. EMBO J. 21, 5408–5416. CrossRef | PubMed
Okochi et al., 2006 (2006). Secretion of the Notch-1 Abeta-like peptide during Notch signaling. J. Biol. Chem. 281, 7890–7898. CrossRef | PubMed
Qi-Takahara et al., 2005 (2005). Longer forms of amyloid beta protein: implications for the mechanism of intramembrane cleavage by gamma-secretase. J. Neurosci. 25, 436–445. CrossRef | PubMed
Saito et al., 2011 (2011). Potent amyloidogenicity and pathogenicity of Aβ43. Nat. Neurosci. 14, 1023–1032. CrossRef | PubMed
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Shen and Kelleher, 2007 (2007). The presenilin hypothesis of Alzheimer's disease: evidence for a loss-of-function pathogenic mechanism. Proc. Natl. Acad. Sci. USA 104, 403–409. CrossRef | PubMed
Sherrington et al., 1995 (1995). Cloning of a gene bearing missense mutations in early-onset familial Alzheimer's disease. Nature 375, 754–760. CrossRef | PubMed
Struhl and Greenwald, 1999 (1999). Presenilin is required for activity and nuclear access of Notch in Drosophila. Nature 398, 522–525. CrossRef | PubMed
Suzuki et al., 1994 (1994). An increased percentage of long amyloid beta protein secreted by familial amyloid beta protein precursor (beta APP717) mutants. Science 264, 1336–1340. PubMed
Takami et al., 2009 (2009). gamma-Secretase: successive tripeptide and tetrapeptide release from the transmembrane domain of beta-carboxyl terminal fragment. J. Neurosci. 29, 13042–13052. CrossRef | PubMed
Weggen et al., 2001 (2001). A subset of NSAIDs lower amyloidogenic Abeta42 independently of cyclooxygenase activity. Nature 414, 212–216. CrossRef | PubMed
Winkler et al., 2009 (2009). Purification, pharmacological modulation, and biochemical characterization of interactors of endogenous human gamma-secretase. Biochemistry 48, 1183–1197. PubMed
Wolfe, 2012 (2012). γ-Secretase inhibitors and modulators for Alzheimer's disease. J. Neurochem. 120, (Suppl 1) 89–98. CrossRef | PubMed
Wolfe et al., 1999 (1999). Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and gamma-secretase activity. Nature 398, 513–517. CrossRef | PubMed
Yanagida et al., 2009 (2009). The 28-amino acid form of an APLP1-derived Abeta-like peptide is a surrogate marker for Abeta42 production in the central nervous system. EMBO Mol. Med. 1, 223–235. PubMed
Supplemental References
Ataka et al., 2004 Ataka, S., Tomiyama, T., Takuma, H., Yamashita, T., Shimada, H., Tsutada, T., Kawabata, K., Mori, H., and Miki, T. (2004). A novel presenilin-1 mutation (Leu85Pro) in early-onset Alzheimer disease with spastic paraparesis. Arch. Neurol. 61, 1773–1776..
Cruts et al., 1995 Cruts, M., Backhovens, H., Wang, S.Y., Van Gassen, G., Theuns, J., De Jonghe, C.D., Wehnert, A., De Voecht, J., De Winter, G., Cras, P., et al. (1995). Molecular genetic analysis of familial early-onset Alzheimer's disease linked to chromosome 14q24.3. Hum. Mol. Genet. 4, 2363–2371..
Dintchov Traykov et al., 2009 Dintchov Traykov, L., Mehrabian, S., Van den Broeck, M., Radoslavova Raycheva, M., Cruts, M., Kirilova Jordanova, A., and Van Broeckhoven, C. (2009). Novel PSEN1 mutation in a Bulgarian patient with very early-onset Alzheimer's disease, spastic paraparesis, and extrapyramidal signs. Am. J. Alzheimers Dis. Other Demen. 24, 404–407..
Kakuda et al., 2006 Kakuda, N., Funamoto, S., Yagishita, S., Takami, M., Osawa, S., Dohmae, N., and Ihara, Y. (2006). Equimolar production of amyloid beta-protein and amyloid precursor protein intracellular domain from beta-carboxyl-terminal fragment by gamma-secretase. J. Biol. Chem. 281, 14776–14786..
Koo and Squazzo, 1994 Koo, E.H., and Squazzo, S.L. (1994). Evidence that production and release of amyloid beta-protein involves the endocytic pathway. J. Biol. Chem. 269, 17386–17389..
Osawa et al., 2008 Osawa, S., Funamoto, S., Nobuhara, M., Wada-Kakuda, S., Shimojo, M., Yagishita, S., and Ihara, Y. (2008). Phosphoinositides suppress gamma-secretase in both the detergent-soluble and -insoluble states. J. Biol. Chem. 283, 19283–19292..
Thinakaran et al., 1997 Thinakaran, G., Harris, C.L., Ratovitski, T., Davenport, F., Slunt, H.H., Price, D.L., Borchelt, D.R., and Sisodia, S.S. (1997). Evidence that levels of presenilins (PS1 and PS2) are coordinately regulated by competition for limiting cellular factors. J. Biol. Chem. 272, 28415–28422..
Publication Information
Received: July 9, 2012
Revised: October 5, 2012
Accepted: November 6, 2012
Published: January 3, 2013


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Eunice M. from Tonopah, Arizona sent in this photo of her dog meeting a chicken for the first time! It looks like it won't be the last. See more unlikely pairs here!
Justin found this incredible star and was here to introduce her for her American television debut! Watch Carly perform her catchy song, "Call Me Maybe," right here!
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Here's your chance to show your love of Ellen and your support for breast cancer research, all in one step! Ellen's breast cancer awareness items are on sale in the Ellen Shop!

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Store Information
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A Ray Harryhausen Classic Double Feature
20 Million Miles to Earth (1957) and The 7th Voyage of Sinbad (1958)
Two classic Harryhausen films, presented in conjunction with the exhibition "The Fantastical Worlds of Ray Harryhausen."
Special late gallery hours from 6–7:30 p.m. and after the screening.
One of Harryhausen's favorite monsters is the star of "20 Million Miles to Earth," the Ymir, who survives the crash of a spaceship returning from the first human exploration of Venus, and ultimately wreaks havoc on the city of Rome.
In "The 7th Voyage of Sinbad," the famed sailor and his fiancée, Princess Parisa, land on an island in search of provisions while on their way to Baghdad to be married. On the island, they encounter an evil magician, a genie and many Harryhausen creations including a man-eating Cyclops. This action-packed adventure was Harryhausen's first Dynamation film in color and is considered one of his best.
20 Million Miles to Earth (1957)
(in Glorious Black and White)
With William Hopper, Joan Taylor, Frank Puglia, John Zaremba. Directed by Nathan Juran. Produced by Charles H. Schneer. Screenplay by Bob Williams, Christopher Knopf. Story by Charlott Knight. Technical effects created by Ray Harryhausen. Columbia Pictures. 35mm. 82 mins. Print courtesy of Sony Pictures.
The 7th Voyage of Sinbad (1958)
(in Vivid Technicolor)
With Kerwin Mathews, Kathryn Grant, Richard Eyer, Torin Thatcher. Directed by Nathan Juran. Produced by Charles H. Schneer. Screenplay by Kenneth Kolb. Story by Ray Harryhausen. Visual effects by Ray Harryhausen. Columbia Pictures. 35mm. 88 mins. Print courtesy of Sony Pictures.


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665 Club LN Lamar, SC 29069
Though most of the well-conditioned fairways are comfortably wide, a few, such as the dogleg par-4 sixth, require precise tee shots to stay in play. Best Hole: No. 5, a 175-yard par-3 guarded by water on the left side, a front bunkers and a densely wooded hazard on the right, making accuracy a must.


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Welcome to the This Is Gandara!.
The Mists are a complicated and fragile beast, throwing together adventurers to complete the simplest right through to the most impossible of tasks. Get to know your fellow companions, as Gandarans we pride ourselves on community spirit.
For anything else that doesn't fit in the categories below, this is where it goes!
Discuss: The Mighty Asura and the lesser long legger races, history and other lore here. Need to know about the departure of the human gods? The long legger plague on Tyria? or just a little upset about your
Please mark all related posts: [Elementalist], [Ranger], [Warrior], [Engineer], [Guardian], [Mesmer], [Necromancer] and [Thief] to allow users to find the threads relevant to their needs.
Anet aren't ones to shy away from content and the arrival of events, new dungeons and other wonderful PvE goodies aren't far from their minds but sometimes discussions need to be had especially when you're in AGONY!
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Invaders keep out! Full information on the Gandaran Alliance is available in the FAQ.
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Anything and everything not to do with Guild Wars 2 goes here. Movies, Music, Funny Kitten Pics and whatever you feel like talking about within reason.
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Darlene Kovacich and Edward Santangelo were united in marriage during a wedding ceremony held June 11, 2011, at LaPlaya Beach, Naples, Fla.
A dinner reception was held June 28, 2011, at Alberini's Restaurant in Niles.
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Edward and Darlene
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Business Buzz: Destination DC's Marketing Outlook Meeting – Opening Session, 8/28/12
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Sunday, October 28
6:30 am


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At the business meeting, Cody McMichael was elected as president-elect of South Dakota's Aspire program. His term will run from October 2009 to October 2010. Hupp also served as president from 2006-2007. The president of the South Dakota ASPIRE program also serves as a member of the regional ASPIRE Executive Board.
McMichael has served as the Chair of the ASPIRE Financial Aid Committee for three years and has also been selected as the Conference Chair for the Regional ASPIRE Conference that will be held in Spearfish in October 2009.
Gritts has served as the secretary/treasurer of South Dakota ASPIRE since 2002 and as the Development Chair for the six-state ASPIRE region for more than ten years. She has also served on the ASPIRE executive board and the Council for Opportunity in Education (COE) at the national level. The ASPIRE program for the region consists of South Dakota, North Dakota, Colorado, Wyoming, Montana, and Utah.
Madrid served as co-chair for the South Dakota ASPIRE meeting in Aberdeen in April, which is the same time that South Dakota also hosted TRIO staff from North Dakota. Larson and Feeney served on the conference planning committee as well. Larson has also served as the Chair of the ASPIRE Awards Committee for three years and now serves as chair of the ASPIRE Achievers Committee as well. Their involvement at the state, regional, and national level provides an additional opportunity to advocate for college access for first generation, low income students.
Black Hills State University faculty, staff, and students are transforming lives and making headlines with their achievements. Below you will find our most recent headlines. Articles are archived monthly and are available by following the links on the right. Email email@example.com or call 605.642.6215 to share your news item.
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Birmingham photographer Chuck St. John selected for inclusion in The Berg Fashion Library - The Authority on World Dress
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February 01, 2013, 2:18PM
(Birmingham, Ala.) -- Local photographer Chuck St. John is no stranger to national exposure as his glamorous images have graced the covers of fashion magazines around the world. With studios in Ft. Lauderdale, Fla., and Birmingham, Ala., and clients across the globe, St. John';s latest accomplishment is in his words "a very big deal." An image he originally shot for...
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Kirkwood By the River Retirement Community "Going Green"
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Birmingham, Alabama - Set on 120 acres of virgin forest above the Cahaba River in Birmingham, Alabama, Kirkwood by the River has served the continuing care needs of its residents since 1980 and has become a model community for retirement living. Known for finding innovative and healthy ways to serve its residents, Kirkwood recently launched a "Go Green Project". "The...
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Dried Herbs and Spices
A dazzling array of spices that we purchase in small quantities to ensure freshness. Each container is hand-packed for you by our staff. Unless otherwise noted, all spices are hand-packed at our shop in 4 ounce containers. Weight will vary depending upon the spice you choose - for example, dried oregano will be much lighter than ground mustard. We encourage you to purchase smaller quantities more frequently so that you can use the spices at their freshest.
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Well Edwina Currie, here it is, reports and evidence that yes, people really are going hungry. Infact children are going hungry. Kids Company in London, according to a recent article in the I, has reported a surge in children coming into their centres for foods. Food banks are being set up across the country at a rate of one a week.
Just in case you think these people are all scroungers who can't manage their affairs and are spending all their money on booze and fags some of the recent people at a food bank include a student who lost his job and is now homeless, and a nurse. A couple recently committed suicide because they had no money and no transport and couldn't cope with walking 12 miles to get food. That happened in this country. Kids Company operates in London – the 6th richest country in the world. Further evidence that the gap between rich and poor continues to get wider – it's the widest it has ever been.
To top it all many schools have seen the portions of their school meals reduced in an effort to save money meaning these hungry children can't even be guaranteed a filling meal at school.
Join www.kidscoplatepledge.org to help support children who are not sure where their next meal is coming from and then write to your MP and ask them what they are doing to sort this out.
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The best tree adventure parc in the Chamonix valley, children to expert loops.
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Over 70 adventure stations available, for all ages and all preferences; for children or beginners to outdoor thrill addicts !
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1986 Dodge 600 convertible in Graphic red with a maroon interior, new white wall tires and deluxe wheel covers. 2.5 litre 4 cyl, automatic,... More
Good body and paint, good top andbucket seat int, 4cyl, at, runs good... More
The sale will be held at The Palace at 301 Norwalk St. Greensboro, NC 27407. This facility offers 24/7 security during the sale... More
The Dodge Ramcharger was Mopar's answer to the K5 Blazer and the full-size Ford Bronco. Based on the Dodge pickup from the same era,... More
1986 Dodge Royal SE 150 Royal, This truck was owned and immaculately cared for by my father. 102,000 miles. Always Garaged, one owner, absolutely... More
New slim conv top, nice split bucket seat int, shows 31,xxxmi, newer tires, V8, AT, PS, PB, P seat, a/c, runs good. ... More
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Shelby 1986 Omni GLHS, intercooler, 500 made, 114K miles, rust-free, rebuilt at 100K $4,500, new DuPont paint, tires, brakes, Borla, tank, $3,000 upgrades, all... More


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After over ten years in existence, Heartstack.org will no longer be the main domain for Matthew Loiacono –>> MatthewCarefully.com will. Please add: http://feeds.feedburner.com/matthewcarefully to your RSS readers. If you are subscribed via email, everything should remain the same. (An update went out last night, safely, yes?) Ahhh. That feels much better.
Continue ReadingThis past weekend, the second annual Restoration Festival popped off at St. Joe's in Albany, N.Y.
Continue ReadingAs with last year's surprise St. Joseph's Oblivion album, I am bringing something special to the Merch Village at RestFest this year.
Continue ReadingLast year's inaugural Restoration Festival was a profoundly empowering and unforgettable event.
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My Memories of Mango
Come mango season and memories come alive of that first luscious bite, in fruit, drink, chutney, pickle, or dessert. Tune in to hear the stories of Indians and their mango nostalgia. As a special treat you'll find a range of timeless and experimental recipes too! Come join the fun. 'Tis after all, the season to mango! Share YOUR mango memory with us here: http://www.flickr.com/groups/yourpics/
1 - 4 of 5
For 20 years the mango tree that my father planted had stood its ground. It took …
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I am trying to transfer Maya 2013 and 3ds Max to new PC, LTU gives me this error when I try to login:
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Md. Sen. Mikulski becomes longest serving woman in Congress
ANNAPOLIS, Md. (AP) - Maryland Sen. Barbara Mikulski is now the longest-serving woman in the history of Congress.
Mikulski, a Democrat, reached the milestone on Saturday. She is scheduled to be honored on the Senate floor on Wednesday after serving more than 35 years in the House of Representatives and the Senate.
Mikulski, 75, won a U.S. House seat in 1976. The popular senator was re-elected four times in a heavily Democratic Maryland district.
She was elected to the Senate in 1986, and she was re-elected with big majorities in 1992, 1998, 2004 and 2010.
Mikulski became the longest serving female U.S. senator last year.
Now, she's passing the late Rep. Edith Nourse Rogers, who was a Massachusetts Republican, as the longest serving woman in Congress. Rogers served from 1925 until 1960.
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Women face health issues in their 30's
UPDATED 9:05 AM CST Feb 20, 2013Arkansas women face unique health issues as they move into their 30's.
The views expressed are not those of this site, this station or its affiliated companies. By posting your comments you agree to accept our terms of use.blog comments powered by Disqus


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N.Y.C. New York Color Liquid Eyeliner
N.Y.C. New York Color Liquid Eyeliner Reviews
Cheap and kind of effective
This was my first HG eye liner when I began using make up. I do not use it as much any more because it flakes by midday. Packaging is good but the brush is flimsy so you need a steady hand with this product. Great price and a really dark black though! — 3 years, 3 months agoThis review is: Helpful | Not helpful | Inappropriate | 2 of 2 people said helpful
Close to perfect
I'm really surprised to see so many bad reviews here, but perhaps they have changed the formula recently, because this eyeliner is really nice, and I'm very hard to please when it comes to eyeliners. I've got mine in black, and the color is gorgeous- great, intense pigmentation. The texture is good- not too liquid and not too thick, it goes on evenly and smoothly. The brush is one of those thinner...read more ones with longer flexible bristles, so if you like thick inflexible brushes, you might want to try something else. I find this brush easy to use, and I have no problem applying the liner evenly. I like that I can easily make a thin line close to my lashes. I haven't noticed it taking any longer to dry than other liquid eyeliners, so I don't know why other reviewers complained about this... I've been using the liner for about 2 months now, almost every day, in different weather and life situations, and it stayed put from the moment I put it on early in the morning to the moment I removed makeup at night. The color didn't fade, nothing flaked, nothing smudged- perfect line all day in all weather conditions despite it not being waterproof. A big plus for me is that it is easy to remove when it's time to do so. I will definitely buy this product again (hey, it's just 3$ for that beautiful black), and I highly recommend it, because they've made it right this time. — 1 year, 4 months agoThis review is: Helpful | Not helpful | Inappropriate | 1 of 3 people said helpful
this is the first beauty product i bought ever, when i was 11! it was only a dollar and let me tell you, best dollar ever spent. I have repurchased this every 6 months since i was 11. It is amazing long wearing, and dried matte and beautiful. its a little hard to get used to using because the brush is very tiny and holds a lot of product so its easy to over load your eyelid but once you get a hang of it it is amazing! pick one up! you might be repurchasing for 13 years too! — 1 year, 3 months agoThis review is: Helpful | Not helpful | Inappropriate | 1 of 1 people said helpful


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Tuesday, June 17, 2008
© Copyright 2014
Clayton News Daily
By Jason A. Smith
firstname.lastname@example.com Police have arrested a man accused of kidnapping his son and the boy's mother. According to Georgia Bureau of Investigation, four-year-old Colin Nathaniel Custer, along with his mother, were kidnapped in Florida, Monday morning at approximately 6:30 a.m. The mother, Rashell Rene Pennell, 36, was able to escape and alerted police to the alleged crime. The GBI issued a Levi's Call Monday afternoon for Colin Nathaniel Custer, who was reportedly last seen in Henry County. GBI Spokesman John Bankhead said the child and his mother were taken by the boy's father, 45-year-old Robert William Custer, II. Bankhead said the mother managed to escape from her abductor in South Carolina. According to the Associated Press, authorities in Lexington County, S.C., later found Pennell locked in a gas station bathroom. Pennell reportedly told police Robert Custer abducted her and her son Sunday, after a court-ordered visitation. In addition, the mother said the suspect placed a cloth over her nose when he abducted her, causing her to lose consciousness. Amber Alerts were then issued for Colin Custer, in South Carolina, North Carolina and Virginia. Unconfirmed reports, said Bankhead, indicate the suspect may have driven through Stockbridge. "A trucker spotted the vehicle on Interstate 75 at Exit 224," said the GBI spokesman. Robert Custer was reportedly driving an off-white, 1988 Chevrolet conversion van, with Florida tag: Q577ZE. Later, authorities recovered the van near the South Carolina and North Carolina line. Maj. Larry Shea, of the Lexington Sheriff's office, said U. S. Marshals, along with the Virgina State Police, apprehended Custer at a Bales Road residence in Blacksburg Va. "He was armed, but made no effort to resist arrest." Lexington Sheriff officers are scheduled to meet the Virginia State police at a "half-way point" on Monday to pick up Colin Custer and return the four-year-old to his mother. --The Associated Press contributed to this article.More like this story
- Police: Woman lied about child abduction ( November 4, 2009 )
- Police searching for missing 4-year-old boyBy Michael Davis ( December 5, 2003 )
- Police searching for missing 4-year-old boyBy Michael Davis ( December 6, 2003 )
- Child doesn't recall accused molester ( July 9, 2003 )
- Local gymnast makes performance debut ( July 5, 2011 )


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Bronco Stadium
Bronco Stadium | |
"The Blue" | |
South endzone exterior in 2012
Location | 1400 Bronco Lane Boise, ID 83725 |
Coordinates | 43°36′11″N 116°11′46″W / 43.603°N 116.196°WCoordinates: 43°36′11″N 116°11′46″W / 43.603°N 116.196°W |
Broke ground | November 1969 |
Opened | September 11, 1970 |
Expanded | 1975, 1997, 2009, 2012 |
Owner | Boise State University |
Operator | Boise State University |
Surface | Field Turf (blue) (2008–present) AstroPlay (blue) – (2002–2007) AstroTurf (blue) – (1986–2001) AstroTurf (green) – (1970–1985) |
Construction cost | $2.3 million ($14 million in 2014 dollars[1]) |
Architect | Sink Combs Dethlefs |
Capacity | 36,387 (2012–Present) 33,500 (2009–2011) 32,000 (2008) 30,000 (1997–2007) 20,000 (1975–1996) 14,500 (1970–1974) |
Tenants | |
Boise State Broncos (NCAA) (1970–present) Famous Idaho Potato Bowl (NCAA) (1997–present) |
Bronco Stadium is an outdoor athletic stadium in Boise, Idaho, the home field of the Boise State Broncos of the Mountain West Conference. Since 1997, it has hosted the Famous Idaho Potato Bowl (Humanitarian Bowl from 1997–Jan. 2004 & 2007–10; MPC Computers Bowl from 2004–06), the longest-running outdoor bowl game in a cold-weather venue.
Opened in 1970, Bronco Stadium was also a track & field stadium; it hosted the NCAA track & field championships twice, in 1994 and 1999.[2] The stadium was used extensively for local high school football for decades until August 2012, when games were transferred to the new Dona Larsen Park, which is also the new home venue of Boise State's track & field team.
Bronco Stadium is widely known for its unusual blue playing surface, installed in 1986 while in the Big Sky Conference. It was the first non-green playing surface (outside of painted end zones) in football history and remains the only one among NCAA Division I FBS schools.
Bronco Stadium is located at the east end of the BSU campus, bordered by Broadway Avenue to the east, University Drive to the south, and the Boise River to the north. The elevation of its playing field is 2,695 feet (821 m) above sea level.[3]
Bronco Stadium is the fourth venue and second of the same name at Boise State; the three on-campus stadiums were built in 1940, 1950, and 1970, respectively.[4]
Public School Field[edit]
During its first years at its original campus, BJC football was played at "Public School Field," located three blocks north-northeast of today's Bronco Stadium. The site was the home of East Junior High School from 1953 to 2009, and was remodeled and became Dona Larsen Park in 2012.
College Field[edit]
After the college moved to its present campus in 1940, "College Field" opened in September 1940 with lights and a seating capacity of 1,000. Also called "Chaffee Field," it was used through 1949 for junior college football (photo - 1940s). In the 1950s it became the baseball field, until right field was displaced by the construction of the Student Union Building, which opened in 1967. The baseball field migrated slightly east, then north, until it was eliminated in 1980 by the construction of the BSU Pavilion and the relocation of the tennis courts. (Baseball was dropped by both BSU and Idaho following the 1980 season;[5] the Broncos played their home games at Borah Field during their final season.)
Bronco Stadium (I)[edit]
The first "Bronco Stadium" was built in three months in 1950 at the east end of campus, with wooden grandstands, a natural grass playing field, lights, and a cinder running track; seating capacity was 10,000.[6] It was in approximately the same location as the present stadium, but aligned northwest to southeast. (photo - 1964) The 45-degree offset was designed to keep the mid-afternoon sun of mid-October out of the players' eyes (but put it into the eyes of half of the spectators). Through 1968, the University of Idaho Vandals usually played one home game per season in Boise, at the original Bronco Stadium.[7] After Boise State joined the Big Sky in 1970, Idaho discontinued its practice of scheduling home games in Boise. (Idaho did use the new Bronco Stadium for a "home" game in 1971, but it was against Boise State in the first football game ever played between the schools. Idaho's new stadium on campus in Moscow was behind schedule so the university rented Bronco Stadium for its opening game. The underdog "visitors" of Boise State built a 28-7 lead at halftime and won handily 42-14 and a rivalry game was born.)
The Boise College football program upgraded from junior college to four-year status in 1968 and competed as an NAIA independent for two seasons. The school became Boise State College in 1969 and the Broncos were accepted into the NCAA in October.[8] A month later they were voted into the Big Sky Conference, effective fall 1970.[9] Following the 1969 football season, the first Bronco Stadium was razed in November and the new concrete stadium was ready for play in less than ten months.[4][10]
Bronco Stadium (II)[edit]
Boise State began NCAA competition in 1970 in Division II ("College Division" prior to 1973) in a brand new venue.[10] The first game at the new Bronco Stadium was on September 11, a 49–14 victory over Chico State. The $2.2 million concrete stadium opened with a seating capacity of 14,500 and a green AstroTurf playing field, configured in the traditional north-south direction, and an all-weather running track. For its first five seasons, the stadium consisted of two sideline grandstands, the west side having an upper deck and the press box. (photo - 1971) Boise State became a charter member of NCAA Division II when the NCAA reorganized the former College Division in 1973.
Following the 1974 season, the school's first as Boise State University, an upper deck was added to the east side (photo - 1971) - (photo -1975), adding 5,500 seats as well as symmetry to the stadium. The permanent seating capacity grew to 20,000 for its Bronco Stadium's sixth season in 1975, with up to 2,600 temporary seats available in the north end zone seating for bigger games. The original green artificial turf was replaced with the same in 1978 as the Big Sky Conference and the Broncos moved up to the newly formed Division I-AA. (photo - mid 1980s)
The Broncos moved to the Big West and Division I-A in 1996, which resulted in another stadium expansion. The two-tier grandstands were extended around the corners of the south end zone, raising the permanent seating capacity to 30,000 in 1997.[2] The latest stadium expansion was completed in time for the 2008 season, with the addition of the Stueckle Sky Club press box, luxury suites, loge boxes, and club seating; raising the capacity to 32,000. In the summer of 2009, 1,500 additional bleacher seats were added to the south end zone to bring capacity up to 33,500.[11] Prior to the 2012 season, expanded bleacher sections were added to the north and south end zones, expanding capacity to a total of 36,387.[12]
Lyle Smith Field[edit]
During its eleventh season, the playing field at Bronco Stadium was named Lyle Smith Field during the I-AA national championship season of 1980. Ceremonies during halftime of the 14–3 victory over Nevada on November 8 marked the event. It honors Lyle H. Smith, the head coach from 1947–67 and athletic director from 1968–81, overseeing BSU's rise from the junior college ranks to Division I-AA champions in 1980.[13] Smith led Boise, as BJC, to multiple post-season bowls, including the 1958 national junior college championship, and compiled an overall record of 156–26–8 (.848), which included five undefeated seasons and 16 conference titles. He was also the baseball coach for 17 seasons and served as basketball coach for a season at the school. Smith hired Tony Knap to replace himself as football coach in 1968, and Jim Criner to replace Knap in 1976.[14][15]
Bronco Stadium's current attendance record is 36,864, set on September 20, 2012 against BYU.
Blue artificial turf[edit]
Bronco Stadium is best known for its distinctive blue playing surface, the only non-green football playing surface among Division I FBS programs.
Chris Berman of ESPN has also called Boise's turf "The Blue Plastic Tundra," a joking reference to "the frozen tundra" of Lambeau Field. Another nickname for the surface is "Smurf Turf." Players refer to it simply as "The Blue."
After 16 seasons of playing on standard green AstroTurf, athletic director Gene Bleymaier came up with the idea to install the blue turf. He decided that, if BSU was going to spend $750,000 on a new surface, he didn't want to see BSU install yet another green field, and that a blue field might provide some national notoriety for the school, then a member of the Big Sky Conference. Bleymaier gained the support of BSU President John Keiser, and on September 13, Bronco Stadium introduced its unique playing surface to the world with a 74-0 victory over Division II Humboldt State.[16] (BSU was 4-2 at home in 1986, but managed just one road victory[17] and posted their first losing record in four decades, resulting in the resignation of fourth-year head coach Lyle Setencich.)
BSU replaced the first blue AstroTurf with the same in 1995, then with blue Astroplay (a grass-like synthetic surface that is more forgiving than traditional AstroTurf) in 2002. The AstroPlay field lasted just six seasons and was replaced in the summer of 2008 with blue FieldTurf surface.[18] Due to complaints by fans that the reflection and glare off the field gave the new field a dull and uneven shade of blue, FieldTurf agreed to replace the field free of charge. The fifth blue turf was installed in the summer of 2010.[19]
The unique blue turf has spawned several myths. The most prevalent is that the NCAA subsequently banned playing surface colors other than green, but allowed Bronco Stadium's field to remain blue under a grandfather clause. In reality, the NCAA has never adopted such a rule. Any school may color its playing surface (or any part, mainly the end zones) any color it wishes. Indeed, other schools have non-green football fields including the University of New Haven (blue)[20] and Eastern Washington University (red).[21] On April 1, 2011, the University of Central Arkansas announced that they would install a purple and grey striped field to Estes Stadium.[22] In 2012, Lindenwood University in Belleville, Illinois, played their first football season, on a home field with alternating maroon and gray stripes. Ten high schools also have non-green fields: Barrow High School in Barrow, Alaska,[23] Lovington High School in Lovington, New Mexico, West Hills High School in Santee, California (alternating royal and sky blue every five yards), Hidalgo High School in Hidalgo, Texas (navy), Chaminade College Preparatory School in the Los Angeles neighborhood of West Hills, Oxford High School in Oxford, Michigan, New Braunfels (Texas) Canyon High (red),[24][25] Canutillo High School in El Paso, Texas, Ravenna High School (Ohio) in Ravenna, Ohio (blue), and West Salem High School in Salem, Oregon (black).[26]
Another myth is that, mistaking the blue field for a large body of water, birds have flown into the blue turf and to their deaths. Although Broncos coach Chris Petersen claimed to have found a dead duck on the field in 2007,[27] the origin of the duck on the field has never been confirmed.
BSU's blue turf has become such a highly visible icon for the Broncos that BSU obtained a U.S. trademark registration for a blue athletics field in November, 2009.[28]
In 2011, the NFL banned any playing surface color other than green, naming the rule the "Boise Rule" in reference to the university, though this was more a reaction to sponsor influence as no team had ever proposed a different turf color for their field.[29] Also in 2011, the Mountain West Conference banned Boise from wearing their all-blue uniforms during home conference games, after complaints from other Mountain West coaches that it was an unfair advantage.[30] However, the uniform restriction will be removed from the 2013 season forward, as part of the deal that kept Boise State in the MW after it had originally planned to leave the conference.[31]
Current/future expansion[edit]
As the Boise State football program rose to national prominence in the early 2000s, Bronco Stadium became increasingly insufficient. The school completed a 3-story complex on the stadium's west side called the Stueckle Sky Club (pronounced Stickle). Construction began on it on February 11, 2007, and it officially opened on August 27, 2008 with a gala for ticket holders prior to the first game on August 30.[32] It features levels for a new press box, luxury suites, loge boxes, and club seating and increased seating capacity to 32,000.
The practice facility, named the Caven-Williams Sports Complex, officially opened in February 2006, and it is located immediately northwest of Bronco Stadium.[33] Additional temporary seating of 1,500 was added prior to the 2009 season. The removable bleachers increased capacity to 33,500. Permanent bleachers were also added to the north and south end zones prior to the 2012 season, raising Bronco Stadium's capacity to a total of 37,000.
In late August 2010, new expansion plans were revealed for Bronco Stadium. The first stages were to include adding a new facility to the north endzone to house the football offices, weight room, training room, equipment room and locker room. Plans also included a 13,200 seat grandstand. The later stages of the expansion plan included removal of the track, lowering of the field, and adding 3,300 seats in front of the first deck of the stadium, completion of the south endzone horseshoe, building of an east side skybox, and renovation of the east side concourse. The total cost for all planned expansions was around $100 million. The total seating capacity for a fully expanded Bronco Stadium was estimated to be around 53,000.[34] The Boise State athletic director aims to have the north endzone complex open in time for the 2013 season and does not resemble the 2010 plans.[35]
Home dominance[edit]
During Boise State's recent streak of conference championships, Bronco Stadium has proven to be a tough place for opponents. As of November 30, 2013, the Broncos are 93–4 at home since the 1999 season with the only losses being to Washington State in 2001, AP #18 Boston College in the 2005 MPC Computers Bowl, TCU in 2011 and to San Diego State in 2012. The Broncos won 47 straight home conference games from 1999 to 2011 and were undefeated in conference during their 10 years in the WAC (40–0). The Broncos are 90–3 in regular season home games since 1999. They had a winning streak of 65 regular season games from the 2001 to 2011 seasons. Their current home winning streak stands at 7.
See also[edit]
- List of Boise State Broncos football seasons
- List of college football stadiums with non-traditional field colors
- ^ Consumer Price Index (estimate) 1800–2014. Federal Reserve Bank of Minneapolis. Retrieved February 27, 2014.
- ^ a b "Bronco Stadium "The Blue"". Boise State University Athletics. Retrieved December 28, 2007.
- ^ "Earth Explorer: 43 36 21 N 116 11 50 W". USGS. Retrieved December 28, 2007.
- ^ a b Bronco Sports.com - football - 2011 media guide (p.114-176) - p. 176 - accessed 2011-11-09
- ^ Goodwin, Dale (May 13, 1980). "Baseball's 'out' at Idaho". Spokesman-Review. p. 19.
- ^ Bronco Country.com - Lyle's Field: 1950-69 - 2010-08-12 - accessed 2012-03-05
- ^ CFB data warehouse - Idaho results - 1965-69 - accessed 2010-04-24
- ^ "Boise State joins NCAA". Spokane Daily Chronicle. Associated Press. October 15, 1969. p. 44.
- ^ "Boise State, Northern Arizona admitted to the Big Sky". Lewiston Morning Tribune. Associated Press. November 26, 1969. p. 13.
- ^ a b Ourada, Patricia K. (1994). "The Broncos: A History of Boise State University, 1932-1994". p. 131. Retrieved August 27, 2012.
- ^ http://www.idahostatesman.com/2010/08/22/4256/bronco-stadium-information.html
- ^ "2013 Weekly Release". Boise State Athletics. August 26, 2013. Archived from the original on August 26, 2013. Retrieved August 26, 2013.
- ^ BSU gameday program – Boise State vs. Nevada, Reno – November 8, 1980 – A Tribute to Lyle Smith, p. 8
- ^ "Lyle H. Smith collection". Boise State University Albertsons Library. Archived from the original on December 9, 2007. Retrieved December 28, 2007.
- ^ "Bronco Football: A Winning Tradition". Boise State University Athletics. Retrieved December 28, 2007.
- ^ "Boise's Blue Field Turns 20". September 14, 2006. Retrieved September 25, 2009.
- ^ CFB data warehouse - Boise State - 1985-89 - accessed 2011-11-09
- ^ "Board OKs Boise State and U of I Projects". Idaho Statesman (Boise). December 7, 2007. Retrieved December 28, 2007.[dead link]
- ^ http://www.idahostatesman.com/2010/04/17/1156098/new-blue-turf-to-be-installed.html
- ^ "Fast Facts". University of New Haven. Retrieved September 25, 2010.
- ^ Matt Hinton (January 28, 2010). "Eastern Washington literally seeing red over new turf". Retrieved September 25, 2010.
- ^ http://www.ucasports.com/news/2011/4/1/FB_0401113911.aspx
- ^ Kamm, Grayson (November 4, 2007). "First Coast Makes Frozen Football Dream Come True". WTLV. Retrieved December 28, 2007.
- ^ Perry, Daniel. "Districts pumping money into athletic facilities". Retrieved December 28, 2007.
- ^ [1][dead link]"Lovington High School Stadium". Retrieved December 28, 2007.
- ^ Chandler, Rick (August 21, 2012). "Oregon High School says 'The heck with everything, we're going with black turf'". Off the Bench (NBCSports.com). Retrieved October 23, 2013.
- ^ "Bronco Stadium BSU". Retrieved December 28, 2007.
- ^ "Boise State's Trademark Registration for Blue Stadium Field". Trademark Access. Retrieved June 22, 2012.
- ^ http://profootballtalk.nbcsports.com/2011/03/22/there-will-be-no-blue-nfl-fields/
- ^ Treadway, Daniel (July 27, 2011). "Boise State Banned From Wearing Blue Uniforms On Smurf Turf". Huffington Post.
- ^ "Boise State to stay in Mountain West". Sports Illustrated. December 31, 2012. Retrieved January 1, 2013.
- ^ idahostatesman.com/eyepiece/story/422281.html
- ^ broncosports.com/ViewArticle.dbml?DB_OEM_ID=9900&KEY=&ATCLID=530884
- ^ http://www.idahostatesman.com/2010/08/28/1318526/field-of-dreams.html
- ^ http://www.idahostatesman.com/2011/01/28/1505686/bsu-ad-aims-to-open-football-complex.html
External links[edit]
Wikimedia Commons has media related to Bronco Stadium. |
- Bronco Sports.com – History of Bronco Stadium
- World Stadiums.com – Bronco Stadium
- The Broncos: A History of Boise State University, 1932-1994 - Sports Complex


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March 12, 2012, is a date nearly eight months in the past. On that day, time seemed to stand still, while the life of the 7-year-old boy literally hung in the balance.
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Edward was right behind the driver on the school bus that crashed on the way to Lighthouse Charter Academy. That driver died, along with a little girl on the bus. Fifty children were passengers that morning, including Edward's sister. Dozens were hurt, but Edward's injuries were the worst. His leg had to be amputated just to get him free, then at the hospital, he was in a coma for weeks.
"I was in a coma?" Edward asked his mother while still recovering in the hospital last April.
"Yeah, you were in a medically-induced coma for three weeks, baby," said Dennis.
But somehow, when Edward came out of that coma, his recovery took on a life of his own. Just like Edward's personality, it wouldn't be held back very long.
Before long, our little man went from hospital bed, to a wheelchair, daily therapy and then -- with a prosthetic leg -- on his feet again. Slowly at first, and gradually with more confidence to that day when he walked out of Riley Hospital for Children at IU Health on his own.
Now, Edward's doing a lot more than just walking.
"[I'm] jumping, playing basketball and going down the slide!" Edward recently exclaimed while celebrating with classmates and friends at this 8th birthday party.
"It's really great," Dennis said. "I have to say this is more of a celebration of him making it to this birthday. It's wonderful that we're here, still."
His 8th birthday came after many difficult days and nights, after several surgeries to repair the breaks in his right leg, hip and stress on his spine.
"I remember when he got the new leg on when he came home, he said, 'Look Mommy, you can't even tell that I've been hurt,'" Nikki said. "And so just with him saying that was all I needed to know that he even feels more whole now."
In their backyard, 18 friends celebrated with Edward, putting his new leg to the test, in bounce houses.
"I love that all my friends are here, and that I'm playing with them." Edward chirped. "It's funner (sic) to be with my friends than to be alone."
Prayers, modern medicine, and strength of spirit, not just Edward's, but his family and friends-- it's all driving the little boy to a future not carried by a yellow school bus or speeding ambulance, but Edward's own two feet, one in front of the other, one step at a time.
"It's amazing, but he's always been a happy child. It's not unexpected, I'm just glad we went the positive direction, instead of the negative direction."
An unbelievable journey for a boy who wants everyone to know that nothing can keep him down.
As he grows, Edward will need new prosthetic legs. Doctors assume he'll need a new one every year. He's done with his surgeries after one more to remove a plate in his jaw in about six months. Then, no more hospitals.
With Nikki and his family pushing him every day, Edward's doctors say there shouldn't be anything standing in his way.


text:
March 11, 2014
AUBURN, Ala. (AP) - Quarterback Kiehl Frazier spent much of Auburn's open date watching film of passers such as Chad Henne and Tom Brady trying to improve footwork and timing.
Frazier says he thinks he'll "look a lot different from LSU to Arkansas." He's certainly facing a more vulnerable defense with the Razorbacks giving up nearly 350 passing yards a game entering Saturday.
Frazier has struggled just like the Razorbacks' passing defense. He's not among the 100 passers in yards per game or rating listed by the NCAA.
Frazier has thrown for two touchdown passes in four games and been intercepted seven times.
He'll be without one of his most reliable wide receivers with Quan Bray suspended.
Arkansas is 117th in pass defense and Auburn's passing offense is only a few spots better (114th).
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