Decrease in brain soluble amyloid precursor protein β (sAPPβ) in Alzheimer's disease cortex

Amyloid-β peptide (Aβ) is generated by sequential cleavage of the amyloid precursor protein (APP) by β-site amyloid precursor protein cleaving enzyme 1 (β-secretase, or BACE1) and γ-secretase. Several reports demonstrate increased BACE1 enzymatic activity in brain and cerebrospinal fluid (CSF) from Alzheimer's disease (AD) subjects, suggesting that an increase in BACE1-mediated cleavage of APP drives amyloid pathophysiology in AD. BACE1 cleavage of APP leads to the generation of a secreted N-terminal fragment of APP (sAPPβ). To relate BACE1 activity better to endogenous APP processing in AD and control brains, we have directly measured brain sAPPβ levels using a novel APP β-site specific enzyme-linked immunosorbent assay. We demonstrate a significant reduction in brain cortical sAPPβ levels in AD compared with control subjects. In the same brain samples, BACE1 activity was unchanged, full-length APP and sAPPα levels were significantly reduced, and Aβ peptides were significantly elevated. In conclusion, a reduction in cortical brain sAPPβ together with unchanged BACE1 activity suggests that this is due to reduced full-length APP substrate in late-stage AD subjects. These results highlight the need for multiparameter analysis of the amyloidogenic process to understand better AD pathophysiology in early vs. late-stage AD.     

Characterization of plasma β‐secretase (BACE1) activity and soluble amyloid precursor proteins as potential biomarkers for Alzheimer's disease

Reduction in cerebrospinal fluid (CSF) amyloid β42 (Aβ42) and elevation in total tau and phospho‐thr181 tau consistently differentiate between Alzheimer's disease (AD) and age‐matched control subjects. In contrast, CSF β‐site APP‐cleaving enzyme activity (BACE1) and soluble amyloid precursor proteins α and β (sAPPα and sAPPβ) are without consistent patterns in AD subjects. Plasma sampling is much easier, with fewer side effects, and is readily applied in primary care centers, so we have developed and validated novel plasma BACE activity, sAPPβ, and sAPPα assays and investigated their ability to distinguish AD from age‐matched controls. Plasma BACE activity assay was sensitive and specific, with signal being immunodepleted with a specific BACE1 antibody and inhibited with a BACE1‐specific inhibitor. Plasma sAPPβ and sAPPα assays were specific, with signal diluting linearly, immunodepleted with specific antibodies, and at background levels in APP knockout mice. In rhesus monkeys, BACE1 but not γ‐secretase inhibitor led to significant lowering of plasma sAPPβ with concurrent elevation of plasma sAPPα. AD subjects showed a significant increase in plasma BACE1 activity, sAPPβ, sAPPα, and Aβ42 (P < 0.001) compared with age‐matched controls. In conclusion, plasma BACE activity and sAPP endpoints provide novel investigative biomarkers for AD diagnosis and potential pharmacodynamic biomarkers for secretase inhibitor studies. © 2012 Wiley Periodicals, Inc.     

Low-Density Lipoprotein Receptor-Related Protein-1 (LRP1) C4408R Mutant Promotes Amyloid Precursor Protein (APP) α-Cleavage in Vitro

Previous studies have demonstrated that the low-density lipoprotein receptor-related protein-1 (LRP1) plays conflicting roles in Alzheimer’s disease (AD) pathogenesis, clearing β-amyloid (Aβ) from the brain while also enhancing APP endocytosis and resultant amyloidogenic processing. We have recently discovered that co-expression of mutant LRP1 C-terminal domain (LRP1-CT C4408R) with Swedish mutant amyloid precursor protein (APPswe) in Chinese hamster ovary (CHO) cells decreases Aβ production, while also increasing sAPPα and APP α-C-terminal fragment (α-CTF), compared with CHO cells expressing APPswe alone. Surprisingly, the location of this mutation on LRP1 corresponded with the α-secretase cleavage site of APP. Further experimentation confirmed that in CHO cells expressing APPswe or wild-type APP (APPwt), co-expression of LRP1-CT C4408R decreases Aβ and increases sAPPα and α-CTF compared with co-expression of wild-type LRP1-CT. In addition, LRP1-CT C4408R enhanced the unglycosylated form of LRP1-CT and reduced APP endocytosis as determined by flow cytometry. This finding identifies a point mutation in LRP1 which slows LRP1-CT-mediated APP endocytosis and amyloidogenic processing, while enhancing APP α-secretase cleavage, thus demonstrating a potential novel target for slowing AD pathogenesis.     

Monocular denervation of visual nuclei modulates APP processing and sAPPα production: A possible role on neural plasticity

Amyloid precursor protein (APP) is essential to physiological processes such as synapse formation and neural plasticity. Sequential proteolysis of APP by beta- and gamma-secretases generates amyloid-beta peptide (Aβ), the main component of senile plaques in Alzheimer Disease. Alternative APP cleavage by alpha-secretase occurs within Aβ domain, releasing soluble α-APP (sAPPα), a neurotrophic fragment. Among other functions, sAPPα is important to synaptogenesis, neural survival and axonal growth. APP and sAPPα levels are increased in models of neuroplasticity, which suggests an important role for APP and its metabolites, especially sAPPα, in the rearranging brain. In this work we analyzed the effects of monocular enucleation (ME), a classical model of lesion-induced plasticity, upon APP content, processing and also in secretases levels. Besides, we addressed whether α-secretase activity is crucial for retinotectal remodeling after ME. Our results showed that ME induced a transient reduction in total APP content. We also detected an increase in α-secretase expression and in sAPP production concomitant with a reduction in Aβ and β-secretase contents. These data suggest that ME facilitates APP processing by the non-amyloidogenic pathway, increasing sAPPα levels. Indeed, the pharmacological inhibition of α-secretase activity reduced the axonal sprouting of ipsilateral retinocollicular projections from the intact eye after ME, suggesting that sAPPα is necessary for synaptic structural rearrangement. Understanding how APP processing is regulated under lesion conditions may provide new insights into APP physiological role on neural plasticity.     

Short-term interleukin-1(beta) increases the release of secreted APP(alpha) via MEK1/2-dependent and JNK-dependent alpha-secretase cleavage in neuroglioma U251 cells

Several lines of neuroimmunological evidence correlate the development of the inflammatory responses of the brain with the formation of amyloid plaques associated with the pathogenesis of neurodegenerative disorders such as Alzheimer's disease. Within this context, we tested the ability of interleukin-1beta (IL-1beta) to regulate the processing of beta-amyloid precursor protein (beta-APP) in neuroglioma U251 cells. Our findings have shown that short-term treatment with IL-1beta (2 hr) resulted in a concentration-dependent decrease in the amount of the cell-associated form of beta-APP in U251 cells as compared to untreated cells, whereas a 2-hr treatment with IL-1beta led to increased release of secreted APP(alpha) fragment (sAPP(alpha)) into the conditioned media of the cells. The fact that sAPP(alpha) is an alpha-secretase cleavage metabolite of the cell-associated form of beta-APP, and the observation that IL-1beta-induced sAPP(alpha) release could be blocked by tissue inhibitors of metalloproteinases-1 (alpha-secretase inhibitors), suggested that alpha-secretase might be involved in IL-1beta-induced-sAPP(alpha) release. Moreover, to determine whether an intracellular signaling pathway mediates the IL-1beta-induced increase in sAPP(alpha) secretion, we used various specific signaling inhibitors and found that sAPP(alpha) release is significantly blocked by the mitogen-activated protein kinase (MEK1/2) inhibitor PD98059 and the c-Jun N-terminal kinase inhibitor SP600125. These findings suggested that the mechanism of IL-1beta-induced-sAPP(alpha) release is dependent on MEK1/2- and JNK-activated alpha-secretase cleavage in neuroglioma U251 cells.     

Accumulation of caspase cleaved amyloid precursor protein represents an early neurodegenerative event in aging and in Alzheimer's disease

The activation of caspase-3 and possibly other caspases during apoptosis may lead to the cleavage of the amyloid precursor protein (APP) and subsequent accumulation of APP cleavage products (cAPP). We examined the association between activated caspase-3 and cAPP in human brain by qualitative and quantitative analysis of in situ immunohistochemistry and Western blots. Frontal cortex and hippocampal tissue from age-matched control and Alzheimer's brains (AD) was used. Both activated caspase-3 and cAPP are increased in AD [Braak and Braak (BB) stage IV-VI] compared to aged control (BB stage 0-1) and transitional (BB stage II-III) cases in the hippocampal and frontal cortex. Caspase-3 activation and the accumulation of APP cleavage fragments appear to either parallel or precede neurofibrillary tangle formation. These findings raise the possibility that the activation of caspase-3 and cleavage of APP may be involved with neuronal degeneration and that pathways characteristic of apoptosis are activated in AD.     

The role of the low-density lipoprotein receptor-related protein (LRP1) in Alzheimer's A beta generation: development of a cell-based model system

The clearance and degradation of extracellular Aβ is critical for regulating β-amyloid deposition, a major hallmark of brains of patients with Aβ in Alzheimer’s Disease. The low-density lipoprotein receptor-related protein, LRP1, is a large endocytic receptor that significantly contributes to the balance between degradation and production of Aβ. An extracellular portion of the LRP, known as the cluster II region can bind to the secreted form of APP (sAPP-KPI). We show here that a GST fusion protein containing the cluster II region of LRP can be used as a ‘mini-receptor’ that specifically binds to sAPP-KPI from conditioned cultured medium. The binding between the GST-LRP-cluster II fusion protein and sAPP-KPI can be inhibited with the strong binding ligand of LRP1, called receptor-associated protein (RAP). Furthermore, a cell-based in vitro assay system has been developed to monitor the production of total Aβ and Aβ_1–42 in the presence and absence of RAP in Chinese hamster ovary (CHO) cell lines both deficient in LRP and expressing LRP. A 3-day treatment of the L2 (CHO cells deficient in LRP and overexpressing APP751) and L3 (CHO cells expressing LRP and overexpressing APP751) cell lines with RAP showed a decrease in total Aβ and, interestingly, also a decrease in the ratio of Aβ₄₂/Aβ_total. This cell-based model system and LRP-cluster II mini-receptor will be very useful for screening novel compounds that can reduce Aβ accumulation by inhibiting binding of APP-KPI to LRP1.     

MicroRNA-195 downregulates Alzheimer's disease amyloid-β production by targeting BACE1

Alzheimer's disease (AD) is a progressive neurodegenerative disease, characterized by amyloid-beta (Aβ) deposition and neurofibrillary tangles. Numerous microRNAs have been found to play crucial roles in regulating Aβ production in the process of AD. Previous investigations have reported lower levels of many microRNAs in AD patients and animal models. Here, we examined the role of miR-195 in the process of Aβ formation. Bioinformatics' algorithms predicted miR-195 binding sites within the beta-site APP cleaving enzyme 1 (BACE1) 3'-untranslated region (3'-UTR), and we found the level of miR-195 to be negatively related to the protein level of BACE1 in SAMP8 mice. We confirmed the target site in HEK293 cells by luciferase assay. Overexpression of miR-195 in N2a/WT cells decreased the BACE1 protein level, and inhibition of miR-195 resulted in increase of BACE1 protein level. Furthermore, overexpression of miR-195 in N2a/APP decreased the level of Aβ, while inhibition of miR-195 resulted in an increase of Aβ. Thus, we demonstrated that miR-195 could downregulate the level of Aβ by inhibiting the translation of BACE1. We conclude that miR-195 might provide a therapeutic strategy for AD.     

Lys(203) and Lys(382) are essential for the proteasomal degradation of BACE1

Amyloid β protein (Aβ) is the primary component of senile plaques in Alzheimer's disease brains and its aggregate form is neurotoxic. Aβ is generated through proteolysis of β-amyloid precursor protein (APP) by two proteases: β-secretase and γ-secretase. BACE1, the β-secretase in vivo and the key rate-limiting enzyme that initiates the formation of Aβ, is an attractive drug target for AD therapy. Our previous study demonstrated that BACE1 is ubiquitinated and its degradation and effect on APP cleaving process are mediated by the ubiquitin-proteasome pathway. However, the specific underlying mechanism is still not well defined. In present study, we determined the specific binding sites responsible for the proteasomal degradation of BACE1. Ten fragments of human BACE1 cDNA with each of them containing 1 to 3 Lys codons were cloned, and HEK293 cells transfected with these recombinant plasmids were treated with specific proteasome inhibitor lactacystin. The protein levels of fragment-3 (Pro(149)-Leu(180)), -4 (IIe(179)-Ser(230)) and -8 (Met(349)-Arg(400)) were significantly increased by lactacystin treatment, and immunocytochemical staining results showed that fragment-3, -4 and -8 proteins were colocalized with ubiquitin. Site-directed mutagenesis at Lys(203) and Lys(382) of BACE1 abolished the proteasomal degradation of BACE1 and affected APP processing at β site and Aβ production. Taken together, our study demonstrated that BACE1 Lys(203) and Lys(382) are essential for its proteasomal degradation, and the results may advance our understanding of regulation of BACE1 and APP processing by the ubiquitin proteasome system in AD pathogenesis and shed new insights on its pharmaceutical potential.     

淀粉样前体蛋白胞内域的功能和机制

Alzheimer's disease (AD),a degenerative neurological disorder,is the most common form of dementia among older people,whose symptoms include gradual memory loss,cognitive impairments and deterioration of language skills.Amyloid precursor protein (APP) is cleaved by serials of secretases and generates Aβ,sAPPα/β and APP intracellular domain (AICD).Aβ forms amyloid plaques,together with neurofibrillary tangles (NFTs) which is comprised with hyperphosphorylated tau,are hallmarks ofAD.Aβ,especially in its oligomeric form,plays important roles in AD,causing cell death,calcium influx,loss of spines and repression of long-term potentiation (LTP)[1].However,recent studies indicate that in addition to Aβ,other fragments of APP after its cleavage,such as AICD,play essential roles in AD as well.In this article,the function of AICD and its underlying mechanisms will be reviewed.     

Arsenic affects expression and processing of amyloid precursor protein (APP) in primary neuronal cells overexpressing the Swedish mutation of human APP

Arsenic poisoning due to contaminated water and soil, mining waste, glass manufacture, select agrochemicals, as well as sea food, affects millions of people world wide. Recently, an involvement of arsenic in Alzheimer's disease (AD) has been hypothesized (Gong and O’Bryant, 2010). The present study stresses the hypothesis whether sodium arsenite, and its main metabolite, dimethylarsinic acid (DMA), may affect expression and processing of the amyloid precursor protein (APP), using the cholinergic cell line SN56.B5.G4 and primary neuronal cells overexpressing the Swedish mutation of APP, as experimental approaches.Exposure of cholinergic SN56.B5.G4 cells with either sodium arsenite or DMA decreased cell viability in a concentration- and exposure-time dependent manner, and affected the activities of the cholinergic enzymes acetylcholinesterase and choline acetyltransferase. Both sodium arsenite and DMA exposure of SN56.B5.G4 cells resulted in enhanced level of APP, and sAPP in the membrane and cytosolic fractions, respectively. To reveal any effect of arsenic on APP processing, the amounts of APP cleavage products, sAPPβ, and β-amyloid (Aβ) peptides, released into the culture medium of primary neuronal cells derived from transgenic Tg2576 mice, were assessed by ELISA. Following exposure of neuronal cells by sodium arsenite for 12 h, the membrane-bound APP level was enhanced, the amount of sAPPβ released into the culture medium was slightly higher, while the levels of Aβ peptides in the culture medium were considerably lower as compared to that assayed in the absence of any drug. The sodium arsenite-induced reduction of Aβ formation suggests an inhibition of the APP γ-cleavage step by arsenite. In contrast, DMA exposure of neuronal cells considerably increased formation of Aβ and sAPPβ, accompanied by enhanced membrane APP level. The DMA-induced changes in APP processing may be the result of the enhanced APP expression. Alternatively, increased Aβ production may also be due to stimulation of caspase activity by arsenic compounds, or failure in Aβ degradation.In summary, the present report clearly demonstrates that sodium arsenite and DMA affect processing of APP in vitro.     

BACE (β-secretase) modulates the processing of APLP2 in vivo

BACE is an aspartyl protease that cleaves the amyloid precursor protein (APP) at the beta-secretase cleavage site and is involved in Alzheimer's disease. The aim of our study was to determine whether BACE affects the processing of the APP homolog APLP2. To this end, we developed BACE knockout mice with a targeted insertion of the gene for beta-galactosidase. BACE appeared to be exclusively expressed in neurons as determined by differential staining. BACE was expressed in specific areas in the cortex, hippocampus, cerebellum, pons, and spinal cord. APP processing was altered in the BACE knockouts with Abeta levels decreasing. The levels of APLP2 proteolytic products were decreased in BACE KO mice, but increased in BACE transgenic mice. Overexpression of BACE in cultured cells led to increased APLP2 processing. Our results strongly suggest that BACE is a neuronal protein that modulates the processing of both APP and APLP2.     

Amyloid precursor protein (APP) regulates synaptic structure and function

The amyloid precursor protein (APP) plays a critical role in Alzheimer's disease (AD) pathogenesis. APP is proteolytically cleaved by β- and γ-secretases to generate the amyloid β-protein (Aβ), the core protein component of senile plaques in AD. It is also cleaved by α-secretase to release the large soluble APP (sAPP) luminal domain that has been shown to exhibit trophic properties. Increasing evidence points to the development of synaptic deficits and dendritic spine loss prior to deposition of amyloid in transgenic mouse models that overexpress APP and Aβ peptides. The consequence of loss of APP, however, is unsettled. In this study, we investigated whether APP itself plays a role in regulating synaptic structure and function using an APP knock-out (APP-/-) mouse model. We examined dendritic spines in primary cultures of hippocampal neurons and CA1 neurons of hippocampus from APP-/- mice. In the cultured neurons, there was a significant decrease (~35%) in spine density in neurons derived from APP-/- mice compared to littermate control neurons that were partially restored with sAPPα-conditioned medium. In APP-/- mice in vivo, spine numbers were also significantly reduced but by a smaller magnitude (~15%). Furthermore, apical dendritic length and dendritic arborization were markedly diminished in hippocampal neurons. These abnormalities in neuronal morphology were accompanied by reduction in long-term potentiation. Strikingly, all these changes in vivo were only seen in mice that were 12-15months in age but not in younger animals. We propose that APP, specifically sAPP, is necessary for the maintenance of dendritic integrity in the hippocampus in an age-associated manner. Finally, these age-related changes may contribute to AD pathology independent of Aβ-mediated synaptic toxicity.     

LRP1 modulates APP trafficking along early compartments of the secretory pathway

The amyloid beta peptide (Aβ) is a central player in Alzheimer's disease (AD) pathology. Aβ liberation depends on APP cleavage by β- and γ-secretases. The low density lipoprotein receptor related protein 1 (LRP1) was shown to mediate APP processing at multiple steps. Newly synthesized LRP1 can interact with APP, implying an interaction between these two proteins early in the secretory pathway. We wanted to investigate whether LRP1 mediates APP trafficking along the secretory pathway, and, if so, whether it affects APP processing. Indeed, the early trafficking of APP within the secretory pathway is strongly influenced by its interaction with the C-terminal domain of LRP1. The LRP1-construct expressing an ER-retention motif, LRP-CT KKAA, had the capacity to retard APP traffic to early secretory compartments. In addition, we provide evidence that APP metabolism occurs in close conjunction with LRP1 trafficking, highlighting a new role of lipoprotein receptors in neurodegenerative diseases.     

Presenilin-1 interacts directly with the beta-site amyloid protein precursor cleaving enzyme (BACE1)

A neuropathological hallmark of Alzheimer's disease is the presence of amyloid plaques. The major constituent of these plaques, occurring largely in brain areas important for memory and cognition, is the 40-42 amyloid residues (Abeta). Abeta is derived from the amyloid protein precursor after cleavage by the recently identified beta-secretase (BACE1) and the putative gamma-secretase complex containing presenilin 1 (PS1). In an attempt to develop a functional secretase enzymatic assay in yeast we demonstrate a direct binding between BACE1 and PS1. This interaction was confirmed in vivo using coimmunoprecipitation and colocalization studies in human cultured cells. Our results show that PS1 preferably binds immature BACE1, thus possibly acting as a functional regulator of BACE1 maturation and/or activity.     

Platelet amyloid precursor protein processing: a bio-marker for Alzheimer's disease

The amyloid precursor protein (APP) in brain is processed either by an amyloidogenic pathway by beta-secretase and gamma-secretase to yield Abeta (beta-amyloid 4 kDa) peptide or by alpha-secretase within the beta-amyloid domain to yield non-amyloidogenic products. We have studied blood platelet levels of a 22-kDa fragment containing the Abeta (beta-amyloid 4 kDa) peptide, beta-secretase (BACE1), alpha-secretase (ADAM10), and APP isoform ratios of the 120-130 kDa to 110 kDa peptides from 31 Alzheimer's disease (AD) patients and 10 age-matched healthy control subjects. We found increased levels of Abeta4, increased activation of beta-secretase (BACE1), decreased activation of alpha-secretase (ADAM10) and decreased APP ratios in AD patients compared to normal control subjects. These observations indicate that the blood platelet APP is processed by the same amyloidogenic and non-amyloidogenic pathways as utilized in brain and that APP processing in AD patients is altered compared to control subjects and may be a useful bio-marker for the diagnosis of AD, the progression of disease and for monitoring drug responses in clinical trials.     

常压高氧对淀粉样蛋白前体/早老素1双重转基因小鼠脑内老年斑及β-淀粉样蛋白的影响

目的 探讨常压高氧(40%O_2,60%空气)处理淀粉样蛋白前体/早老素1(APP/PS1)双重转基因小鼠是否发挥神经保护作用.方法 对APP/PS1双重转基因阿尔茨海默病(AD)模型种鼠交配后产下的子代小鼠进行基因分型,待子代达10周龄时,取双重转基因小鼠40只,随机分成A、B、C、D 4组,每组10只,A、B 2组小鼠喂养于常压高氧中8 h/d,A组持续4周,B组持续8周;C、D组喂养于空气中4或8周,分别作为A、B组的对照.高氧处理后采用免疫组织化学、Thioflavin S染色检测小鼠脑组织形态学的变化,Western blot检测APP代谢过程中相关蛋白的表达变化,ELISA定量检测小鼠脑内β-淀粉样蛋白(Aβ)水平的变化.结果 免疫组织化学和Thioflavin S染色均显示,与对照组相比,高氧处理组小鼠皮质和海马内老年斑数量明显减少,B组比A组减少更显著.高氧处理组小鼠脑内C_(99)、C_(83)水平显著高于对照组,Aβ水平明显低于对照组,但各组小鼠脑内全长APP及β位淀粉样前体蛋白裂解酶1(BACE1)蛋白水平无明显改变.ELISA结果提示,B组小鼠海马和皮质内Aβ_(40)[(783.6±97.2)pg/ml]和Aβ_(42)[(175.3±17.1)ps/ml]含量明显低于对照组Aβ_(40)[(1251.6±42.3)pg/ml,t=9.36,P<0.01]和Aβ_(42)[(286.8±13.0)pg/ml,t=13.7,P<0.01]的含量.结论 常压高氧处理能显著减少AD模型小鼠脑内Aβ的产生、沉积及老年斑的形成;这种改变可能通过减少Aβ产生或加速Aβ清除实现.     

Restoring Soluble Amyloid Precursor Protein α Functions as a Potential Treatment for Alzheimer's Disease

Soluble amyloid precursor protein α (sAPPα), a secreted proteolytic fragment of nonamyloidogenic amyloid precursor protein (APP) processing, is known for numerous neuroprotective functions. These functions include but are not limited to proliferation, neuroprotection, synaptic plasticity, memory formation, neurogenesis, and neuritogenesis in cell culture and animal models. In addition, sAPPα influences amyloid‐β (Aβ) production by direct modulation of APP β‐secretase proteolysis as well as Aβ‐related or unrelated tau pathology, hallmark pathologies of Alzheimer's disease (AD). Thus, the restoration of sAPPα levels and functions in the brain by increasing nonamyloidogenic APP processing and/or manipulation of its signaling could reduce AD pathology and cognitive impairment. It is likely that identification and characterization of sAPPα receptors in the brain, downstream effectors, and signaling pathways will pave the way for an attractive therapeutic target for AD prevention or intervention. © 2016 Wiley Periodicals, Inc.