Death Induced by Survival gene Elimination (DISE) correlates with neurotoxicity in Alzheimer's disease and aging.

Alzheimer's disease (AD) is characterized by progressive neurodegeneration, but the specific events that cause cell death remain poorly understood. Death Induced by Survival gene Elimination (DISE) is a cell death mechanism mediated by short (s) RNAs acting through the RNA-induced silencing complex (RISC). DISE is thus a form of RNA interference, in which G-rich 6mer seed sequences in the sRNAs (position 2-7) target hundreds of C-rich 6mer seed matches in genes essential for cell survival, resulting in the activation of cell death pathways. Here, using Argonaute precipitation and RNAseq (Ago-RP-Seq), we analyze RISC-bound sRNAs to quantify 6mer seed toxicity in several model systems. In mouse AD models and aging brain, in induced pluripotent stem cell-derived neurons from AD patients, and in cells exposed to Aß42 oligomers, RISC-bound sRNAs show a shift to more toxic 6mer seeds compared to controls. In contrast, in brains of "SuperAgers", humans over age 80 who have superior memory performance, RISC-bound sRNAs are shifted to more nontoxic 6mer seeds. Cells depleted of nontoxic sRNAs are sensitized to Aß42-induced cell death, and reintroducing nontoxic RNAs is protective. Altogether, the correlation between DISE and Aß42 toxicity suggests that increasing the levels of nontoxic miRNAs in the brain or blocking the activity of toxic RISC-bound sRNAs could ameliorate neurodegeneration.

Connections between ApoE, sleep, and Aß and tau pathologies in Alzheimer's disease.

In this issue of the JCI, Wang and colleagues investigate the relationship between sleep disturbances, an environmental risk factor for Alzheimer's disease (AD), and the apolipoprotein 4 (APOEε4) allele, a strong genetic risk factor for AD. The authors subjected an amyloid mouse model expressing human APOE3 or APOE4, with and without human AD-tau injection, to sleep deprivation and observed that amyloid and tau pathologies were worsened in the presence of APOE4. Moreover, decreased microglial clustering and increased dystrophic neurites around plaques were observed in sleep-deprived APOE4 mice. In addition, aquaporin 4, important for clearing amyloid-ß through the glymphatic system, was reduced and less polarized to astrocytic endfeet. These APOE4-induced changes caused alterations in sleep behavior during recovery from sleep deprivation, suggesting a feed-forward cycle of sleep disturbance and increased AD pathology that can further disrupt sleep in the presence of APOE4.

Aberrant glial activation and synaptic defects in CaMKIIα-iCre and nestin-Cre transgenic mouse models.

Current scientific research is driven by the ability to manipulate gene expression by utilizing the Cre/loxP system in transgenic mouse models. However, artifacts in Cre-driver mouse lines that introduce undesired effects and confound results are increasingly being reported. Here, we show aberrant neuroinflammation and synaptic changes in two widely used Cre-driver mouse models. Neuroinflammation in CaMKIIα-iCre mice was characterized by the activation and proliferation of microglia and astrocytes in synaptic layers of the hippocampus. Increased GFAP and Iba1 levels were observed in hippocampal brain regions of 4-, 8- and 22-month-old CaMKIIα-iCre mice compared to WT littermates. Synaptic changes in NMDAR, AMPAR, PSD95 and phosphorylated CaMKIIα became apparent in 8-month-old CaMKIIα-iCre mice but were not observed in 4-month-old CaMKIIα-iCre mice. Synaptophysin and synaptoporin were unchanged in CaMKIIα-iCre compared to WT mice, suggesting that synaptic alterations may occur in excitatory postsynaptic regions in which iCre is predominantly expressed. Finally, hippocampal volume was reduced in 22-month-old CaMKIIα-iCre mice compared to WT mice. We tested the brains of mice of additional common Cre-driver mouse models for neuroinflammation; the nestin-Cre mouse model showed synaptic changes and astrocytosis marked by increased GFAP+ astrocytes in cortical and hippocampal regions, while the original CaMKIIα-Cre T29-1 strain was comparable to WT mice. The mechanisms underlying abnormal neuroinflammation in nestin-Cre and CaMKIIα-iCre are unknown but may be associated with high levels of Cre expression. Our findings are critical to the scientific community and demonstrate that the correct Cre-driver controls must be included in all studies using these mice.

A conserved annexin A6-mediated membrane repair mechanism in muscle, heart, and nerve.

Membrane instability and disruption underlie myriad acute and chronic disorders. Anxa6 encodes the membrane-associated protein annexin A6 and was identified as a genetic modifier of muscle repair and muscular dystrophy. To evaluate annexin A6's role in membrane repair in vivo, we inserted sequences encoding green fluorescent protein (GFP) into the last coding exon of Anxa6. Heterozygous Anxa6gfp mice expressed a normal pattern of annexin A6 with reduced annexin A6GFP mRNA and protein. High-resolution imaging of wounded muscle fibers showed annexin A6GFP rapidly formed a repair cap at the site of injury. Injured cardiomyocytes and neurons also displayed repair caps after wounding, highlighting annexin A6-mediated repair caps as a feature in multiple cell types. Using surface plasmon resonance, we showed recombinant annexin A6 bound phosphatidylserine-containing lipids in a Ca2+- and dose-dependent fashion with appreciable binding at approximately 50 µM Ca2+. Exogenously added recombinant annexin A6 localized to repair caps and improved muscle membrane repair capacity in a dose-dependent fashion without disrupting endogenous annexin A6 localization, indicating annexin A6 promotes repair from both intracellular and extracellular compartments. Thus, annexin A6 orchestrates repair in multiple cell types, and recombinant annexin A6 may be useful in additional chronic disorders beyond skeletal muscle myopathies.

Oral nimodipine treatment has no effect on amyloid pathology or neuritic dystrophy in the 5XFAD mouse model of amyloidosis.

Dysregulation of calcium homeostasis has been hypothesized to play a role in Alzheimer's disease (AD) pathogenesis. Increased calcium levels can impair axonal transport, disrupt synaptic transmission, and ultimately lead to cell death. Given the potential role of calcium dyshomeostasis in AD, there is interest in testing the ability of already approved drugs targeting various calcium channels to affect amyloid pathology and other aspects of disease. The objective of this study was to test the effects of FDA-approved L-type calcium channel antagonist nimodipine on amyloid accumulation and dystrophic neurite formation in 5XFAD mice, a mouse model of amyloid pathology. 5XFAD transgenic mice and non-transgenic littermates were treated with vehicle or nimodipine-containing chow from two to eight months of age, then brains were harvested and amyloid pathology assessed by immunoblot and immunofluorescence microscopy analyses. Nimodipine was well tolerated and crossed the blood brain barrier, as expected, but there was no effect on Aß accumulation or on the relative amount of neuritic dystrophy, as assessed by either immunoblot, dot blot or immunofluorescence imaging of Aß42 and dystrophic neurite marker LAMP1. While we conclude that nimodipine treatment is not likely to improve amyloid pathology or decrease neuritic dystrophy in AD, it is worth noting that nimodipine did not worsen the phenotype suggesting its use is safe in AD patients.

Pregabalin Treatment does not Affect Amyloid Pathology in 5XFAD Mice.

BACKGROUND: Calcium dysregulation has been proposed to play a causative role in the development of Alzheimer's disease pathology. Pregabalin is a compound already approved for human use, marketed as the prescription drug Lyrica. It binds the α2-δ subunit of P/Q-type voltagegated calcium channels, lowering calcium influx and providing effective treatment for epilepsy and neuropathic pain. OBJECTIVE: We hypothesize that increased resting calcium in neuronal processes near amyloid plaques plays a role in the development of neuritic dystrophies and further progression of amyloid pathology. METHODS: 5XFAD mice were treated orally for 12 weeks with pregabalin, then immunoblotting and immunofluorescent imaging were used to quantify neuritic dystrophy and amyloid deposition in pregabalin compared to placebo-treated mice. RESULTS: The treatment did not decrease markers of neuritic dystrophy or amyloid deposition. The image analysis of neuritic dystrophy on a plaque-by-plaque basis showed a small non-significant increase in the relative proportion of LAMP1 to Aß42 in plaques with areas of 50-450 µm2 in the cortex of pregabalin-treated mice. In addition, there was a statistically significant positive correlation between the measured cerebral concentration of pregabalin and the relative levels of BACE1 and Aß in the cortex. This relationship was not observed in the hippocampus, and there was no increase in average Aß levels in pregabalin treated mice compared to placebo. We confirmed previous findings that smaller amyloid plaques are associated with a greater degree of neuritic dystrophy. CONCLUSION: Pregabalin may have an effect on Aß that merits further investigation, but our study does not suggest that pregabalin contributes substantially to amyloid pathology.

The innate immunity protein IFITM3 modulates γ-secretase in Alzheimer's disease.

Innate immunity is associated with Alzheimer's disease1, but the influence of immune activation on the production of amyloid-ß is unknown2,3. Here we identify interferon-induced transmembrane protein 3 (IFITM3) as a γ-secretase modulatory protein, and establish a mechanism by which inflammation affects the generation of amyloid-ß. Inflammatory cytokines induce the expression of IFITM3 in neurons and astrocytes, which binds to γ-secretase and upregulates its activity, thereby increasing the production of amyloid-ß. The expression of IFITM3 is increased with ageing and in mouse models that express familial Alzheimer's disease genes. Furthermore, knockout of IFITM3 reduces γ-secretase activity and the formation of amyloid plaques in a transgenic mouse model (5xFAD) of early amyloid deposition. IFITM3 protein is upregulated in tissue samples from a subset of patients with late-onset Alzheimer's disease that exhibit higher γ-secretase activity. The amount of IFITM3 in the γ-secretase complex has a strong and positive correlation with γ-secretase activity in samples from patients with late-onset Alzheimer's disease. These findings reveal a mechanism in which γ-secretase is modulated by neuroinflammation via IFITM3 and the risk of Alzheimer's disease is thereby increased.

ER stress is not elevated in the 5XFAD mouse model of Alzheimer's disease.

Alzheimer's disease mouse models that overexpress amyloid precursor protein (APP) and presenilin 1 (PS1) form ß-amyloid (Aß) plaques, a hallmark Alzheimer's disease lesion. It has been assumed that the neuroinflammation, synaptic dysfunction, neurodegeneration, and cognitive impairment observed in these mice are caused by cerebral Aß accumulation. However, it is also possible that accumulation of the overexpressed transmembrane proteins APP and PS1 in the endoplasmic reticulum (ER) triggers chronic ER stress and activation of the unfolded protein response (UPR). The 5XFAD mouse, a widely used amyloid pathology model, overexpresses APP and PS1, displays aggressive amyloid pathology, and has been reported to exhibit ER stress. To systematically evaluate whether 5XFAD mice have increased ER stress, here we used biochemical approaches to assess a comprehensive panel of UPR markers. We report that APP and PS1 levels are 1.8- and 1.5-fold, respectively, of those in 5XFAD compared with nontransgenic brains, indicating that transgenes are not massively overexpressed in 5XFAD mice. Using immunoblotting, we quantified UPR protein levels in nontransgenic, 5XFAD, and 5XFAD;BACE1-/- mice at 4, 6, and 9 months of age. Importantly, we did not observe elevation of the ER stress markers p-eIF2α, ATF4, CHOP, p-IRE1α, or BiP at any age in 5XFAD or 5XFAD;BACE1-/- compared with nontransgenic mice. Despite lacking Aß generation, 5XFAD;BACE1-/- mice still expressed APP and PS1 transgenes, indicating that their overexpression does not cause ER stress. These results reveal the absence of ER stress in 5XFAD mice, suggesting that artifactual phenotypes associated with overexpression-induced ER stress are not a concern in this model.

Presynaptic dystrophic neurites surrounding amyloid plaques are sites of microtubule disruption, BACE1 elevation, and increased Aß generation in Alzheimer's disease.

Alzheimer's disease (AD) is characterized by amyloid plaques composed of the ß-amyloid (Aß) peptide surrounded by swollen presynaptic dystrophic neurites consisting of dysfunctional axons and terminals that accumulate the ß-site amyloid precursor protein (APP) cleaving enzyme (BACE1) required for Aß generation. The cellular and molecular mechanisms that govern presynaptic dystrophic neurite formation are unclear, and elucidating these processes may lead to novel AD therapeutic strategies. Previous studies suggest Aß may disrupt microtubules, which we hypothesize have a critical role in the development of presynaptic dystrophies. To investigate this further, here we have assessed the effects of Aß, particularly neurotoxic Aß42, on microtubules during the formation of presynaptic dystrophic neurites in vitro and in vivo. Live-cell imaging of primary neurons revealed that exposure to Aß42 oligomers caused varicose and beaded neurites with extensive microtubule disruption, and inhibited anterograde and retrograde trafficking. In brain sections from AD patients and the 5XFAD transgenic mouse model of amyloid pathology, dystrophic neurite halos with BACE1 elevation around amyloid plaques exhibited aberrant tubulin accumulations or voids. At the ultrastructural level, peri-plaque dystrophies were strikingly devoid of microtubules and replete with multi-lamellar vesicles resembling autophagic intermediates. Proteins of the microtubule motors, kinesin and dynein, and other neuronal proteins were aberrantly localized in peri-plaque dystrophies. Inactive pro-cathepsin D also accumulated in peri-plaque dystrophies, indicating reduced lysosomal function. Most importantly, BACE1 accumulation in peri-plaque dystrophies caused increased BACE1 cleavage of APP and Aß generation. Our study supports the hypothesis that Aß induces microtubule disruption in presynaptic dystrophic neurites that surround plaques, thus impairing axonal transport and leading to accumulation of BACE1 and exacerbation of amyloid pathology in AD.

Aß reduction in BACE1 heterozygous null 5XFAD mice is associated with transgenic APP level.

BACKGROUND: The ß-secretase, BACE1, cleaves APP to initiate generation of the ß-amyloid peptide, Aß, that comprises amyloid plaques in Alzheimer's disease (AD). Reducing BACE1 activity is an attractive therapeutic approach to AD, but complete inhibition of BACE1 could have mechanism-based side-effects as BACE1-/- mice show deficits in axon guidance, myelination, memory, and other neurological processes. Since BACE1+/- mice appear normal there is interest in determining whether 50% reduction in BACE1 is potentially effective in preventing or treating AD. APP transgenic mice heterozygous for BACE1 have decreased Aß but the extent of reduction varies greatly from study to study. Here we assess the effects of 50% BACE1 reduction on the widely used 5XFAD mouse model of AD. RESULTS: 50% BACE1 reduction reduces Aß42, plaques, and BACE1-cleaved APP fragments in female, but not in male, 5XFAD/BACE1+/- mice. 5XFAD/BACE1+/+ females have higher levels of Aß42 and steady-state transgenic APP than males, likely caused by an estrogen response element in the transgene Thy-1 promoter. We hypothesize that higher transgenic APP level in female 5XFAD mice causes BACE1 to no longer be in excess over APP so that 50% BACE1 reduction has a significant Aß42 lowering effect. In contrast, the lower APP level in 5XFAD males allows BACE1 to be in excess over APP even at 50% BACE1 reduction, preventing lowering of Aß42 in 5XFAD/BACE1+/- males. We also developed and validated a dot blot assay with an Aß42-selective antibody as an accurate and cost-effective alternative to ELISA for measuring cerebral Aß42 levels. CONCLUSIONS: 50% BACE1 reduction lowers Aß42 in female 5XFAD mice only, potentially because BACE1 is not in excess over APP in 5XFAD females with higher transgene expression, while BACE1 is in excess over APP in 5XFAD males with lower transgene expression. Our results suggest that greater than 50% BACE1 inhibition might be necessary to significantly lower Aß, given that BACE1 is likely to be in excess over APP in the human brain. Additionally, in experiments using the 5XFAD mouse model, or other Thy-1 promoter transgenic mice, equal numbers of male and female mice should be used, in order to avoid artifactual gender-related differences.

Genetic inhibition of phosphorylation of the translation initiation factor eIF2α does not block Aß-dependent elevation of BACE1 and APP levels or reduce amyloid pathology in a mouse model of Alzheimer's disease.

ß-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) initiates the production of ß-amyloid (Aß), the major constituent of amyloid plaques in Alzheimer's disease (AD). BACE1 is elevated ∼2-3 fold in AD brain and is concentrated in dystrophic neurites near plaques, suggesting BACE1 elevation is Aß-dependent. Previously, we showed that phosphorylation of the translation initiation factor eIF2α de-represses translation of BACE1 mRNA following stress such as energy deprivation. We hypothesized that stress induced by Aß might increase BACE1 levels by the same translational mechanism involving eIF2α phosphorylation. To test this hypothesis, we used three different genetic strategies to determine the effects of reducing eIF2α phosphorylation on Aß-dependent BACE1 elevation in vitro and in vivo: 1) a two-vector adeno-associated virus (AAV) system to express constitutively active GADD34, the regulatory subunit of PP1c eIF2α phosphatase; 2) a non-phosphorylatable eIF2α S51A knockin mutation; 3) a BACE1-YFP transgene lacking the BACE1 mRNA 5' untranslated region (UTR) required for eIF2α translational regulation. The first two strategies were used in primary neurons and 5XFAD transgenic mice, while the third strategy was employed only in 5XFAD mice. Despite very effective reduction of eIF2α phosphorylation in both primary neurons and 5XFAD brains, or elimination of eIF2α-mediated regulation of BACE1-YFP mRNA translation in 5XFAD brains, Aß-dependent BACE1 elevation was not decreased. Additionally, robust inhibition of eIF2α phosphorylation did not block Aß-dependent APP elevation in primary neurons, nor did it reduce amyloid pathology in 5XFAD mice. We conclude that amyloid-associated BACE1 elevation is not caused by translational de-repression via eIF2α phosphorylation, but instead appears to involve a post-translational mechanism. These definitive genetic results exclude a role for eIF2α phosphorylation in Aß-dependent BACE1 and APP elevation. We suggest a vicious pathogenic cycle wherein Aß42 toxicity induces peri-plaque BACE1 and APP accumulation in dystrophic neurites leading to exacerbated Aß production and plaque progression.

Elevated Aß42 in aged, non-demented individuals with cerebral atherosclerosis.

The ß-secretase, BACE1, generates ß-amyloid (Aß), a major hallmark of Alzheimer's disease (AD) pathology. The elevation of BACE1 levels in brains of AD patients may play a role in initiating or propagating disease. BACE1 levels are increased under low energy or low oxygen conditions, which may occur in individuals with impaired circulation in the brain. We compared levels of BACE1 in the brains of aged, non-demented individuals with high or low levels of atherosclerosis in the circle of Willis, and found that while there is no change in BACE1, Aß42 levels are elevated in the high atherosclerosis group.

The Alzheimer's ß-secretase BACE1 localizes to normal presynaptic terminals and to dystrophic presynaptic terminals surrounding amyloid plaques.

ß-Site amyloid precursor protein (APP) cleaving enzyme-1 (BACE1) is the ß-secretase that initiates Aß production in Alzheimer's disease (AD). BACE1 levels are increased in AD, which could contribute to pathogenesis, yet the mechanism of BACE1 elevation is unclear. Furthermore, the normal function of BACE1 is poorly understood. We localized BACE1 in the brain at both the light and electron microscopic levels to gain insight into normal and pathophysiologic roles of BACE1 in health and AD, respectively. Our findings provide the first ultrastructural evidence that BACE1 localizes to vesicles (likely endosomes) in normal hippocampal mossy fiber terminals of both non-transgenic and APP transgenic (5XFAD) mouse brains. In some instances, BACE1-positive vesicles were located near active zones, implying a function for BACE1 at the synapse. In addition, BACE1 accumulated in swollen dystrophic autophagosome-poor presynaptic terminals surrounding amyloid plaques in 5XFAD cortex and hippocampus. Importantly, accumulations of BACE1 and APP co-localized in presynaptic dystrophies, implying increased BACE1 processing of APP in peri-plaque regions. In primary cortical neuron cultures, treatment with the lysosomal protease inhibitor leupeptin caused BACE1 levels to increase; however, exposure of neurons to the autophagy inducer trehalose did not reduce BACE1 levels. This suggests that BACE1 is degraded by lysosomes but not by autophagy. Our results imply that BACE1 elevation in AD could be linked to decreased lysosomal degradation of BACE1 within dystrophic presynaptic terminals. Elevated BACE1 and APP levels in plaque-associated presynaptic dystrophies could increase local peri-plaque Aß generation and accelerate amyloid plaque growth in AD.

Dimebon alters hippocampal amyloid pathology in 3xTg-AD mice.

A double blind, placebo-controlled phase II study revealed that the antihistamine, Dimebon® (dimebolin, latrepirdine) improved cognition in Alzheimer disease (AD) patients compared to placebo controls. However, the Phase III CONNECTION trial failed to demonstrate significant differences between dimebon and placebo treatments. Despite the controversial therapeutic outcomes in the treatment of AD, dimebon's mechanism(s) of action within the brain remain unclear. In the present study, we evaluated the effects of dimebon upon ß-amyloid (Aß), tau and astrocytes in the hippocampus of triple transgenic (3xTg-AD) mice, which develop AD-like pathology in an age-dependent manner. At age 6.5 months, prior to the development of Aß plaques in the hippocampus, male and female 3xTg-AD mice, received a daily intraperitoneal injection of 0.1 % dimebon or saline for 1.5 months. At 8 months, quantitative immunohistochemistry revealed a significant reduction in hippocampal/subicular APP/Aß in dimebon-treated mice, whereas protein bioassay found no change in full length APP, soluble Aß(1-40) and Aß(1-42), Aß oligomers, BACE1 and GFAP levels between groups. Interestingly, the number of the hippocampal APP/Aß plaques in female and male dimebon-treated mice was higher compared to gender-matched control mice. Dimebon did not alter hippocampal tau levels. Furthermore, dimebon protects SH-SY5Y neurons against Aß toxicity and promotes GFAP expression in primary mouse astrocyte cultures. Our findings demonstrate that dimebon in vivo modifies hippocampal APP/Aß pathology and in vitro protects against Aß toxicity promoting cell survival and activates astrocytes.

Cdk5 protein inhibition and Aß42 increase BACE1 protein level in primary neurons by a post-transcriptional mechanism: implications of CDK5 as a therapeutic target for Alzheimer disease.

The ß-secretase enzyme BACE1 initiates production of the amyloid-ß (Aß) peptide that comprises plaques in Alzheimer disease (AD) brain. BACE1 levels are increased in AD, potentially accelerating Aß generation, but the mechanisms of BACE1 elevation are not fully understood. Cdk5/p25 has been implicated in neurodegeneration and BACE1 regulation, suggesting therapeutic Cdk5 inhibition for AD. In addition, caspase 3 has been implicated in BACE1 elevation. Here, we show that the Cdk5 level and p25:p35 ratio were elevated and correlated with BACE1 level in brains of AD patients and 5XFAD transgenic mice. Mouse primary cortical neurons treated with Aß42 oligomers had increased BACE1 level and p25:p35 ratio. Surprisingly, the Aß42-induced BACE1 elevation was not blocked by Cdk5 inhibitors CP68130 and roscovitine, and instead the BACE1 level was increased greater than with Aß42 treatment alone. Moreover, Cdk5 inhibitors alone elevated BACE1 in a time- and dose-dependent manner that coincided with increased caspase 3 cleavage and decreased Cdk5 level. Caspase 3 inhibitor benzyloxycarbonyl-VAD failed to prevent the Aß42-induced BACE1 increase. Further experiments suggested that the Aß42-induced BACE1 elevation was the result of a post-transcriptional mechanism. We conclude that Aß42 may increase the BACE1 level independently of either Cdk5 or caspase 3 and that Cdk5 inhibition for AD may cause BACE1 elevation, a potentially negative therapeutic outcome.

Phosphorylation of the translation initiation factor eIF2alpha increases BACE1 levels and promotes amyloidogenesis.

beta-site APP cleaving enzyme-1 (BACE1), the rate-limiting enzyme for beta-amyloid (Abeta) production, is elevated in Alzheimer's disease (AD). Here, we show that energy deprivation induces phosphorylation of the translation initiation factor eIF2alpha (eIF2alpha-P), which increases the translation of BACE1. Salubrinal, an inhibitor of eIF2alpha-P phosphatase PP1c, directly increases BACE1 and elevates Abeta production in primary neurons. Preventing eIF2alpha phosphorylation by transfection with constitutively active PP1c regulatory subunit, dominant-negative eIF2alpha kinase PERK, or PERK inhibitor P58(IPK) blocks the energy-deprivation-induced BACE1 increase. Furthermore, chronic treatment of aged Tg2576 mice with energy inhibitors increases levels of eIF2alpha-P, BACE1, Abeta, and amyloid plaques. Importantly, eIF2alpha-P and BACE1 are elevated in aggressive plaque-forming 5XFAD transgenic mice, and BACE1, eIF2alpha-P, and amyloid load are correlated in humans with AD. These results strongly suggest that eIF2alpha phosphorylation increases BACE1 levels and causes Abeta overproduction, which could be an early, initiating molecular mechanism in sporadic AD.