Enhancement of BACE1 Activity by p25/Cdk5-Mediated Phosphorylation in Alzheimer’s Disease

The activity of beta-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is elevated during aging and in sporadic Alzheimer’s disease (AD), but the underlying mechanisms of this change are not well understood. p25/Cyclin-dependent kinase 5 (Cdk5) has been implicated in the pathogenesis of several neurodegenerative diseases, including AD. Here, we describe a potential mechanism by which BACE activity is increased in AD brains. First, we show that BACE1 is phosphorylated by the p25/Cdk5 complex at Thr252 and that this phosphorylation increases BACE1 activity. Then, we demonstrate that the level of phospho-BACE1 is increased in the brains of AD patients and in mammalian cells and transgenic mice that overexpress p25. Furthermore, the fraction of p25 prepared from iodixanol gradient centrifugation was unexpectedly protected by protease digestion, suggesting that p25/Cdk5-mediated BACE1 phosphorylation may occur in the lumen. These results reveal a link between p25 and BACE1 in AD brains and suggest that upregulated Cdk5 activation by p25 accelerates AD pathogenesis by enhancing BACE1 activity via phosphorylation.     

Hypoxia-inducible factor 1alpha (HIF-1alpha)-mediated hypoxia increases BACE1 expression and beta-amyloid generation

The incidence of Alzheimer disease (AD) and vascular dementia is greatly increased following cerebral ischemia and stroke in which hypoxic conditions occur in affected brain areas. beta-Amyloid peptide (Abeta), which is derived from the beta-amyloid precursor protein (APP) by sequential proteolytic cleavages from beta-secretase (BACE1) and presenilin-1 (PS1)/gamma-secretase, is widely believed to trigger a cascade of pathological events culminating in AD and vascular dementia. However, a direct molecular link between hypoxic insults and APP processing has yet to be established. Here, we demonstrate that acute hypoxia increases the expression and the enzymatic activity of BACE1 by up-regulating the level of BACE1 mRNA, resulting in increases in the APP C-terminal fragment-beta (betaCTF) and Abeta. Hypoxia has no effect on the level of PS1, APP, and tumor necrosis factor-alpha-converting enzyme (TACE, an enzyme known to cleave APP at the alpha-secretase cleavage site). Sequence analysis, mutagenesis, and gel shift studies revealed binding of HIF-1 to the BACE1 promoter. Overexpression of HIF-1alpha increases BACE1 mRNA and protein level, whereas down-regulation of HIF-1alpha reduced the level of BACE1. Hypoxic treatment fails to further potentiate the stimulatory effect of HIF-1alpha overexpression on BACE1 expression, suggesting that hypoxic induction of BACE1 expression is primarily mediated by HIF-1alpha. Finally, we observed significant reduction in BACE1 protein levels in the hippocampus and the cortex of HIF-1alpha conditional knock-out mice. Our results demonstrate an important role for hypoxia/HIF-1alpha in modulating the amyloidogenic processing of APP and provide a molecular mechanism for increased incidence of AD following cerebral ischemic and stroke injuries.     

Chronic copper exposure exacerbates both amyloid and tau pathology and selectively dysregulates cdk5 in a mouse model of AD

Excess copper exposure is thought to be linked to the development of Alzheimer's disease (AD) neuropathology. However, the mechanism by which copper affects the CNS remains unclear. To investigate the effect of chronic copper exposure on both beta-amyloid and tau pathologies, we treated young triple transgenic (3×Tg-AD) mice with 250 ppm copper-containing water for a period of 3 or 9 months. Copper exposure resulted in altered amyloid precursor protein processing; increased accumulation of the amyloid precursor protein and its proteolytic product, C99 fragment, along with increased generation of amyloid-beta peptides and oligomers. These changes were found to be mediated via up-regulation of BACE1 as significant increases in BACE1 levels and deposits were detected around plaques in mice following copper exposure. Furthermore, tau pathology within hippocampal neurons was exacerbated in copper-exposed 3×Tg-AD group. Increased tau phosphorylation was closely correlated with aberrant cdk5/p25 activation, suggesting a role for this kinase in the development of copper-induced tau pathology. Taken together, our data suggest that chronic copper exposure accelerates not only amyloid pathology but also tau pathology in a mouse model of AD.     

Casein Kinase 2 dependent phosphorylation of eIF4B regulates BACE1 expression in Alzheimer’s disease

Alzheimer’s disease (AD) is the most common age-related neurodegenerative disorder. Increased Aβ production plays a fundamental role in the pathogenesis of the disease and BACE1, the protease that triggers the amyloidogenic processing of APP, is a key protein and a pharmacological target in AD. Changes in neuronal activity have been linked to BACE1 expression and Aβ generation, but the underlying mechanisms are still unclear. We provide clear evidence for the role of Casein Kinase 2 in the control of activity-driven BACE1 expression in cultured primary neurons, organotypic brain slices, and murine AD models. More specifically, we demonstrate that neuronal activity promotes Casein Kinase 2 dependent phosphorylation of the translation initiation factor eIF4B and this, in turn, controls BACE1 expression and APP processing. Finally, we show that eIF4B expression and phosphorylation are increased in the brain of APPPS1 and APP-KI mice, as well as in AD patients. Overall, we provide a definition of a mechanism linking brain activity with amyloid production and deposition, opening new perspectives from the therapeutic standpoint.Subject terms: Kinases, Alzheimer''s disease, Neuronal physiology, Pathogenesis     

Altered amyloid-beta metabolism and deposition in genomic-based beta-secretase transgenic mice

Amyloid-beta (Abeta) the primary component of the senile plaques found in Alzheimer's disease (AD) is generated by the rate-limiting cleavage of amyloid precursor protein (APP) by beta-secretase followed by gamma-secretase cleavage. Identification of the primary beta-secretase gene, BACE1, provides a unique opportunity to examine the role this unique aspartyl protease plays in altering Abeta metabolism and deposition that occurs in AD. The current experiments seek to examine how modulating beta-secretase expression and activity alters APP processing and Abeta metabolism in vivo. Genomic-based BACE1 transgenic mice were generated that overexpress human BACE1 mRNA and protein. The highest expressing BACE1 transgenic line was mated to transgenic mice containing human APP transgenes. Our biochemical and histochemical studies demonstrate that mice overexpressing both BACE1 and APP show specific alterations in APP processing and age-dependent Abeta deposition. We observed elevated levels of Abeta isoforms as well as significant increases of Abeta deposits in these double transgenic animals. In particular, the double transgenics exhibited a unique cortical deposition profile, which is consistent with a significant increase of BACE1 expression in the cortex relative to other brain regions. Elevated BACE1 expression coupled with increased deposition provides functional evidence for beta-secretase as a primary effector in regional amyloid deposition in the AD brain. Our studies demonstrate, for the first time, that modulation of BACE1 activity may play a significant role in AD pathogenesis in vivo.     

Mild Oxidative Stress Induces Redistribution of BACE1 in Non-Apoptotic Conditions and Promotes the Amyloidogenic Processing of Alzheimer’s Disease Amyloid Precursor Protein

BACE1 is responsible for β-secretase cleavage of the amyloid precursor protein (APP), which represents the first step in the production of amyloid β (Aβ) peptides. Previous reports, by us and others, have indicated that the levels of BACE1 protein and activity are increased in the brain cortex of patients with Alzheimer’s disease (AD). The association between oxidative stress (OS) and AD has prompted investigations that support the potentiation of BACE1 expression and enzymatic activity by OS. Here, we have established conditions to analyse the effects of mild, non-lethal OS on BACE1 in primary neuronal cultures, independently from apoptotic mechanisms that were shown to impair BACE1 turnover. Six-hour treatment of mouse primary cortical cells with 10–40 µM hydrogen peroxide did not significantly compromise cell viability but it did produce mild oxidative stress (mOS), as shown by the increased levels of reactive radical species and activation of p38 stress kinase. The endogenous levels of BACE1 mRNA and protein were not significantly altered in these conditions, whereas a toxic H₂O₂ concentration (100 µM) caused an increase in BACE1 protein levels. Notably, mOS conditions resulted in increased levels of the BACE1 C-terminal cleavage product of APP, β-CTF. Subcellular fractionation techniques showed that mOS caused a major rearrangement of BACE1 localization from light to denser fractions, resulting in an increased distribution of BACE1 in fractions containing APP and markers for trans-Golgi network and early endosomes. Collectively, these data demonstrate that mOS does not modify BACE1 expression but alters BACE1 subcellular compartmentalization to favour the amyloidogenic processing of APP, and thus offer new insight in the early molecular events of AD pathogenesis.     

Rheb GTPase Regulates β-Secretase Levels and Amyloid β Generation

The β-site amyloid precursor protein (APP)-cleaving enzyme 1 (β-secretase, BACE1) initiates amyloidogenic processing of APP to generate amyloid β (Aβ), which is a hallmark of Alzheimer disease (AD) pathology. Cerebral levels of BACE1 are elevated in individuals with AD, but the molecular mechanisms are not completely understood. We demonstrate that Rheb GTPase (Ras homolog enriched in brain), which induces mammalian target of rapamycin (mTOR) activity, is a physiological regulator of BACE1 stability and activity. Rheb overexpression depletes BACE1 protein levels and reduces Aβ generation, whereas the RNAi knockdown of endogenous Rheb promotes BACE1 accumulation, and this effect by Rheb is independent of its mTOR signaling. Moreover, GTP-bound Rheb interacts with BACE1 and degrades it through proteasomal and lysosomal pathways. Finally, we demonstrate that Rheb levels are down-regulated in the AD brain, which is consistent with an increased BACE1 expression. Altogether, our study defines Rheb as a novel physiological regulator of BACE1 levels and Aβ generation, and the Rheb-BACE1 circuitry may have a role in brain biology and disease.     

Cholesterol-depletion corrects APP and BACE1 misstrafficking in NPC1-deficient cells

Cholesterol accumulation in Niemann-Pick type C disease (NPC) causes increased levels of the amyloid-precursor-protein C-terminal fragments (APP-CTFs) and intracellular amyloid-β peptide (Aβ), the two central molecules in Alzheimer's disease (AD) pathogenesis. We previously reported that cholesterol accumulation in NPC-cells leads to cholesterol-dependent increased APP processing by β-secretase (BACE1) and decreased APP expression at the cell surface (Malnar et al. Biochim Biophys Acta. 1802 (2010) 682-691.). We hypothesized that increased formation of APP-CTFs and Aβ in NPC disease is due to cholesterol-mediated altered endocytic trafficking of APP and/or BACE1. Here, we show that APP endocytosis is prerequisite for enhanced Aβ levels in NPC-cells. Moreover, we observed that NPC cells show cholesterol dependent sequestration and colocalization of APP and BACE1 within enlarged early/recycling endosomes which can lead to increased β-secretase processing of APP. We demonstrated that increased endocytic localization of APP in NPC-cells is likely due to both its increased internalization and its decreased recycling to the cell surface. Our findings suggest that increased cholesterol levels, such as in NPC disease and sporadic AD, may be the upstream effector that drives amyloidogenic APP processing characteristic for Alzheimer's disease by altering endocytic trafficking of APP and BACE1.     

Inactive variants of death receptor p75NTR reduce Alzheimer’s neuropathology by interfering with APP internalization

A prevalent model of Alzheimer’s disease (AD) pathogenesis postulates the generation of neurotoxic fragments derived from the amyloid precursor protein (APP) after its internalization to endocytic compartments. The molecular pathways that regulate APP internalization and intracellular trafficking in neurons are incompletely understood. Here, we report that 5xFAD mice, an animal model of AD, expressing signaling‐deficient variants of the p75 neurotrophin receptor (p75^NTR) show greater neuroprotection from AD neuropathology than animals lacking this receptor. p75^NTR knock‐in mice lacking the death domain or transmembrane Cys²⁵⁹ showed lower levels of Aβ species, amyloid plaque burden, gliosis, mitochondrial stress, and neurite dystrophy than global knock‐outs. Strikingly, long‐term synaptic plasticity and memory, which are completely disrupted in 5xFAD mice, were fully recovered in the knock‐in mice. Mechanistically, we found that p75^NTR interacts with APP at the plasma membrane and regulates its internalization and intracellular trafficking in hippocampal neurons. Inactive p75^NTR variants internalized considerably slower than wild‐type p75^NTR and showed increased association with the recycling pathway, thereby reducing APP internalization and co‐localization with BACE1, the critical protease for generation of neurotoxic APP fragments, favoring non‐amyloidogenic APP cleavage. These results reveal a novel pathway that directly and specifically regulates APP internalization, amyloidogenic processing, and disease progression, and suggest that inhibitors targeting the p75^NTR transmembrane domain may be an effective therapeutic strategy in AD.     

Partial reduction of BACE1 has dramatic effects on Alzheimer plaque and synaptic pathology in APP Transgenic Mice

The aspartyl protease beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) initiates processing of amyloid precursor protein (APP) into amyloid beta (Abeta) peptide, the major component of Alzheimer disease (AD) plaques. To determine the role that BACE1 plays in the development of Abeta-driven AD-like pathology, we have crossed PDAPP mice, a transgenic mouse model of AD overexpressing human mutated APP, onto mice with either a homozygous or heterozygous BACE1 gene knockout. Analysis of PDAPP/BACE(-/-) mice demonstrated that BACE1 is absolutely required for both Abeta generation and the development of age-associated plaque pathology. Furthermore, synaptic deficits, a neurodegenerative pathology characteristic of AD, were also reversed in the bigenic mice. To determine the extent of BACE1 reduction required to significantly inhibit pathology, PDAPP mice having a heterozygous BACE1 gene knock-out were evaluated for Abeta generation and for the development of pathology. Although the 50% reduction in BACE1 enzyme levels caused only a 12% decrease in Abeta levels in young mice, it nonetheless resulted in a dramatic reduction in Abeta plaques, neuritic burden, and synaptic deficits in older mice. Quantitative analyses indicate that brain Abeta levels in young APP transgenic mice are not the sole determinant for the changes in plaque pathology mediated by reduced BACE1. These observations demonstrate that partial reductions of BACE1 enzyme activity and concomitant Abeta levels lead to dramatic inhibition of Abeta-driven AD-like pathology, making BACE1 an excellent target for therapeutic intervention in AD.     

The role of amyloid precursor protein processing by BACE1, the beta-secretase, in Alzheimer disease pathophysiology

Amyloid plaques, composed of the amyloid beta-protein (Abeta), are hallmark neuropathological lesions in Alzheimer disease (AD) brain. Abeta fulfills a central role in AD pathogenesis, and reduction of Abeta levels should prove beneficial for AD treatment. Abeta generation is initiated by proteolysis of amyloid precursor protein (APP) by the beta-secretase enzyme BACE1. Bace1 knockout (Bace1(-/-)) mice have validated BACE1 as the authentic beta-secretase in vivo. BACE1 is essential for Abeta generation and represents a suitable drug target for AD therapy, especially because this enzyme is up-regulated in AD. However, although initial data indicated that Bace1(-/-) mice lack an overt phenotype, the BACE1-mediated processing of APP and other substrates may be important for specific biological processes. In this minireview, topics range from the initial identification of BACE1 to the fundamental knowledge gaps that remain in our understanding of this protease. We address pertinent questions such as putative causes of BACE1 elevation in AD and discuss why, nine years since the identification of BACE1, treatments that address the underlying pathological mechanisms of AD are still lacking.     

Constitutive JAK2/STAT1 activation regulates endogenous BACE1 expression in neurons

The protease BACE1 (β-site APP-cleaving enzyme 1) is essential for the generation of amyloid beta (Aβ) from amyloid precursor protein (APP). Although BACE1 is expressed primarily in neurons, which are a principal source of Aβ in the brain, the mechanism that underlies basal expression of BACE1 in neurons has not been studied thoroughly. In the present study, we found that endogenous BACE1 expression was mediated by constitutive JAK2/STAT1 activation in neurons. Inhibition of the JAK2/STAT1 signaling pathway, using AG490 (a JAK2 inhibitor), a dominant-negative form of STAT1, and SOCS1 and SOCS3 overexpression, reduced levels of BACE1 promoter activity, expression of endogenous BACE1, and generation of Aβ. These results were recapitulated in the SH-SY5Y neuronal cell line, primary cultured neurons, and mouse brains. Therefore, we propose that constitutive JAK2/STAT1 activation mediates endogenous BACE1 expression in neurons and that inhibition of JAK2/STAT1 signaling abrogates basal levels of BACE1 expression and Aβ generation.     

Failure of the interaction between presenilin 1 and the substrate of gamma-secretase to produce Abeta in insect cells

Aggregates of beta-amyloid peptide (Abeta) are the major component of the amyloid core of the senile plaques observed in Alzheimer's disease (AD). Abeta results from the amyloidogenic processing of its precursor, the amyloid precursor protein (APP), by beta- and gamma-secretase activities. If beta-secretase has recently been identified and termed BACE, the identity of gamma-secretase is still obscure. Studies with knock-out mice showed that presenilin 1 (PS1), of which mutations are known to be the first cause of inherited AD, is mandatory for the gamma-secretase activity. However, the proteolytic activity of PS1 remains a matter of debate. Here we used transfected Sf9 insect cells, a cellular model lacking endogenous beta- and/or gamma-secretase activities, to characterize the role of BACE and PS1 in the amyloidogenic processing of human APP. We show that, in Sf9 cells, BACE performs the expected beta-secretase cleavage of APP, generating C99. We also show that C99, which is a substrate of gamma-secretase, tightly binds to the human PS1. Despite this interaction, Sf9 cells still do not produce Abeta. This strongly argues against a direct proteolytic activity of PS1 in APP processing, and points toward an implication of PS1 in trafficking/presenting its substrate to the gamma-secretase.