Baicalein reduces β‐amyloid and promotes nonamyloidogenic amyloid precursor protein processing in an Alzheimer's disease transgenic mouse model

Baicalein, a flavonoid isolated from the roots of Scutellaria baicalensis, is known to modulate γ‐aminobutyric acid (GABA) type A receptors. Given prior reports demonstrating benefits of GABA_A modulation for Alzheimer's disease (AD) treatment, we wished to determine whether this agent might be beneficial for AD. CHO cells engineered to overexpress wild‐type amyloid precursor protein (APP), primary culture neuronal cells from AD mice (Tg2576) and AD mice were treated with baicalein. In the cell cultures, baicalein significantly reduced the production of β‐amyloid (Aβ) by increasing APP α‐processing. These effects were blocked by the GABA_A antagonist bicuculline. Likewise, AD mice treated daily with i.p. baicalein for 8 weeks showed enhanced APP α‐secretase processing, reduced Aβ production, and reduced AD‐like pathology together with improved cognitive performance. Our findings suggest that baicalein promotes nonamyloidogenic processing of APP, thereby reducing Aβ production and improving cognitive performance, by activating GABA_A receptors. © 2013 Wiley Periodicals, Inc.     

Functional role of the low-density lipoprotein receptor-related protein in Alzheimer's disease

Alzheimer's disease (AD) is the most common age-related neurodegenerative disorder, characterized by neuronal loss, neurofibrillary tangle formation and the extracellular deposition of amyloid-beta (Abeta) plaques. The amyloid precursor protein (APP) and the enzymes responsible for Abeta generation seem to be the base elements triggering the destructive processes. Initially, the low-density lipoprotein receptor-related protein (LRP) was genetically linked to AD and later it emerged to impact on many fundamental events related to this disease. LRP is not only involved in Abeta clearance but is also the major receptor of several AD-associated ligands, e.g. apolipoprotein E and alpha2-macroglobulin. APP processing is mediated by LRP on many levels. Enhanced APP internalization through LRP decreases cell surface APP levels and thereby reduces APP shedding. As a consequence of increased APP internalization LRP enhances Abeta secretion. These effects could be attributed to the cytoplasmic tails of LRP and APP. The receptors bind via their NPXY motifs to the two PID domains of FE65 and form a tripartite complex. However, it appears that the second NPVY motif of LRP is the one responsible for the observed influence over APP metabolism. A more in-depth knowledge of the mechanisms regulating APP cleavage may offer additional targets for therapeutic intervention.     

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.     

From Acetylcholine to Amyloid: Neurotransmitters and the Pathology of Alzheimer's Disease

Brain amyloid deposits play a central role in the histopathology of Alzheimer's disease (AD), as evidenced by increased formation of amyloid β peptides (Aβ) in genetic forms of AD that are caused by mutations in the presenilin genes, or the amyloid β protein precursor (APP) gene. Neuronal deafferentation in AD brain may also be associated with accelerated Aβ formation, because APP processing is regulated by neuronal activity, presumably via several G protein-coupled neurotransmitter receptors. Subtype-selective agonists including muscarinic m1 receptor ligands may be useful for the pharmacological reduction of Aβ formation.     

Functional role of lipoprotein receptors in Alzheimer's disease

The LDL receptor gene family constitutes a class of structurally closely related cell surface receptors fulfilling diverse functions in different organs, tissues, and cell types. The LDL receptor is the prototype of this family, which also includes the VLDLR, ApoER2/LRP8, LRP1 and LRP1B, as well as Megalin/GP330, SorLA/LR11, LRP5, LRP6 and MEGF7. Recently several lines of evidence have positioned the LDL receptor gene family as one of the key players in Alzheimer's disease (AD) research. Initially this receptor family was of high interest due to its key function in cholesterol/apolipoprotein E (ApoE) uptake, with the epsilon4 allele of ApoE as the strongest genetic risk factor for late-onset AD. It has been established that the cholesterol metabolism of the cell has a strong impact on the production of Abeta, the major component of the plaques found in the brain of AD-patients. The original report that soluble amyloid precursor protein (APP) containing the kunitz proteinase inhibitor (KPI) domain might act as a ligand for LRP1 led to a complex investigation of the interaction of both proteins and their potential function in AD development. Meanwhile, it has been demonstrated that LRP1 might bind to APP independent of the KPI domain in APP. This APP - LRP1 interaction is facilitated through a trimeric complex of APP-FE65-LRP1, which has a functional role in APP processing. Along with LRP1, APP is transported from the early secretory compartments to the cell surface and subsequently internalised into the endosomal / lysosomal compartments. Recent investigations indicate that ApoER2 and SorLA fulfil a similar role in shifting APP localisation in the cell, which affects APP processing and the production of the APP derived amyloid beta-peptide (Abeta). In addition to the effect of lipoprotein receptors on APP processing and Abeta production, LRP1 has been shown to bind Abeta directly or indirectly through Abeta-lactoferrin, Abeta-alpha2M and Abeta-ApoE complexes in vitro and in vivo. Based on these observations two LRP1 mediated clearance mechanisms of Abeta are proposed to play a crucial role in the prevention of AD: either Abeta-uptake into a cell with its subsequent degradation or its transport out of the brain over the blood brain barrier into the periphery. Following this export Abeta is degraded in the liver, where LRP1 potentially conducts the removal of Abeta from the blood stream. Although the involvement of LDLR family members in AD is not yet fully understood it becomes clear that they can directly affect APP production, Abeta-clearance and Abeta-transport over the blood brain barrier.     

Cholesterol and Alzheimer's disease: is there a link?

The Abeta-amyloid peptide (Abeta), the main component of amyloid plaques, is derived by proteolytic cleavage from the amyloid precursor protein (APP). Epidemiologic and biochemical data suggest a link between cholesterol, APP processing, Abeta, and Alzheimer's disease. Two recent epidemiologic studies indicate that there is a decreased prevalence of AD associated with the use of cholesterol-lowering drugs that inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase inhibitors or statins). Experiments in cell culture and in vivo demonstrate that treatment with statins reduces production of Abeta. The authors discuss how cholesterol might modulate Abeta deposit formation. As neurons receive only small amounts of exogenous cholesterol, statins that efficiently cross the blood-brain barrier may reduce the amount of neuronal cholesterol below a critical level. Decreased neuronal cholesterol levels inhibit the Abeta-forming amyloidogenic pathway possibly by removing APP from cholesterol- and sphingolipid-enriched membrane microdomains. In addition, depletion of cellular cholesterol levels reduces the ability of Abeta to act as a seed for further fibril formation. These intriguing relationships raise the hopes that cholesterol-lowering strategies may influence the progression of AD.     

Betaine suppressed Aβ generation by altering amyloid precursor protein processing

Betaine was an endogenous catabolite of choline, which could be isolated from vegetables and marine products. Betaine could promote the metabolism of homocysteine in healthy subjects and was used for hyperlipidemia, coronary atherosclerosis, and fatty liver in clinic. Recent findings shown that Betaine rescued neuronal damage due to homocysteine induced Alzheimer’s disease (AD) like pathological cascade, including tau hyperphosphorylation and amyloid-β (Aβ) deposition. Aβ was derived from amyloid precursor protein (APP) processing, and was a triggering factor for AD pathological onset. Here, we demonstrated that Betaine reduced Aβ levels by altering APP processing in N2a cells stably expressing Swedish mutant of APP. Betaine increased α-secretase activity, but decreased β-secretase activity. Our data indicate that Betaine might play a protective role in Aβ production.     

Alterations in glutamate receptor 2/3 subunits and amyloid precursor protein expression during the course of Alzheimer’s disease and Lewy body variant

Alterations in the processing and patterns of trophic and/or toxic factors might lead to the increased neuronal vulnerability in the entorhinal cortex in Alzheimer’s disease (AD) and Lewy body variant (LBV). Therefore, patterns and levels of amyloid precursor protein (APP) and glutamate receptor (gluR) expression in the entorhinal cortex and hippocampus in relation to disease severity were investigated. Sections from the hippocampus and entorhinal cortex were single and double immunolabeled for APP, gluR2/3, and n-methyl-d-aspartate receptor (NMDA-R). Within the hippocampus and entorhinal cortex, image analysis revealed progressively decreased APP and gluR2/3 levels during the course of AD and LBV, whereas levels of NMDA-R were unaltered. Furthermore, the present study showed a positive correlation and close co-localization of APP and gluR2/3 immunoreactivity in neurons, suggesting a possible interaction between these two factors. In conclusion, these data imply that alterations in neuronal APP and gluR2/3 expression in the entorhinal cortex lead to increased susceptibility to neurodegeneration and might be markers of vulnerability. Received: 17 March 1997 / Revised, accepted: 9 June 1997     

Signal transduction therapeutics

It is now widely accepted that abnormal processing of the Alzheimer's amyloid precursor protein (APP) can contribute significantly to Alzheimer's disease (AD). APP can be processed proteolytically to give rise to several fragments, including toxic beta-amyloid (Abeta) fragments that are subsequently deposited as amyloid plaques in brains of AD patients. Data from several groups have revealed that APP processing can be regulated by phosphorylation and phosphorylation-dependent events. Consequently, the key players controlling such signal transduction cascades, the protein kinases and phosphatases, as well as their corresponding regulatory proteins, take on added importance. By characterizing how altered cell signaling might contribute to APP processing, one can identify potential targets for signal transduction therapeutics. Here, we review APP phosphorylation and phosphorylation-dependent events in APP processing, with particular focus on phosphatases that impact on APP processing, and their binding and regulatory proteins. Particular attention is given to protein phosphatase 1 (PP1), as it seems to have a central role not only in the regulation of APP cleavage events but also in the molecular control of neurotransmission and in age-related memory deterioration. The development of specific drugs targeting protein phosphatase binding proteins would constitute potential therapeutic agents with a high degree of specificity. The identification of such targets provides novel therapeutic avenues for normal aging and for neurodegenerative conditions such as AD.     

In vitro effects of metrifonate on neuronal amyloid precursor protein processing and protein kinase C level

Alteration in the processing of the amyloid precursor protein (APP) is a central event in the formation of amyloid deposits in the brains of individuals with Alzheimer's disease (AD). It has been suggested that acetylcholinesterase (AChE) inhibitors, which promote the cholinergic function and consequently improve the cognitive deficits, may also exert a neuroprotective effect by activating normal APP processing. We now report that an irreversible AChE inhibitor (metrifonate) increase the cell-associated APP level in a basal forebrain neuronal culture and also elevate the amount of APP secreted into the medium. The alterations in APP processing were accompanied by increased protein kinase C (PKC) levels. The results suggest that AChE inhibitors modulate the metabolism of APP, possibly via their stimulatory effects on PKC. Since changes in the activity and level of PKC may be involved in the pathogenesis of AD, it is concluded that the beneficial effect of metrifonate in AD therapy may be due not only to the stimulatory cholinergic function, but also to its activating effect on PKC.     

Amyloid beta peptide induces cytochrome C release from isolated mitochondria

Amyloid beta peptide (Abeta) is a neurotoxic metabolic product of the amyloid precursor protein (APP). Abeta is strongly implicated in the pathology of Alzheimer's disease (AD) and can be formed intracellularly. In this study, we show that the addition of Abeta to isolated mouse brain mitochondria can directly induce cytochrome c (Cyt c) release and mitochondrial swelling, which were partially inhibited by cyclosporin A (CsA). These results suggest that the Abetaaccumulated intracellularly by APP processing might exert neurotoxicity by interacting with mitochondria and inducing mitochondrial swelling and release of Cyt c, which activates caspase-3 and finally can lead to apoptosis in neuronal cells and to neurodegeneration in AD.     

Reversible in vivo phosphorylation of tau induced by okadaic acid and by unspecific brain lesion in rat

Alzheimer's disease (AD) is histopathologically characterised by the formation of neurofibrillary tangles (NFTs) that are largely composed of hyperphosphorylated tau protein (PHF-tau), and senile plaques which contain aggregates of the Abeta peptide. Formation of PHF-tau and amyloidogenic processing of the amyloid precursor protein (APP) might be related to a disturbance in the balance between protein phosphorylation and dephosphorylation. In the present study, the effects of injections into the cerebral cortex of either okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A, or saline were investigated. Both kinds of injections induced a reversible phosphorylation of tau, albeit to a different extent. The secretion of soluble APP was reduced after OA but not affected after injection of saline. It is concluded that phosphorylation of tau, similar though not identical to those seen in AD can be induced in vivo by inhibition of protein dephosphorylation as well as by unspecific lesion of cortical neurones. It might, therefore, be suggested that phosphorylation processes involved in PHF-formation in AD similarly reflect a neuronal response to injury.     

Is understanding the biological function of APP important in understanding Alzheimer's disease?

The presence of mutations around the A beta sequence in APP provides strong argument for the involvement of APP, and A beta in particular, in pathogenesis of Alzheimer's disease (AD). In vitro studies demonstrated that A beta may cause neuronal death, supporting the hypothetical involvement of A beta in neurodegeneration in AD. However, concentrations of A beta required for neuronal death are nonphysiologically high. Nevertheless, the predominant idea in the field is that it is sufficient to postulate A beta as a major culprit in AD development. The question we pose is whether the potentially important involvement of A beta precludes the etiological (primary) involvement (not pathological, i.e., secondary) of APP functions. We do not have an adequate answer to this question. Current knowledge about APP functions indicates that APP is critically required for the maintenance of neuronal and synaptic structure and function. Because AD is a disease of neuronal and synaptic deterioration, APP may be involved during the course of AD pathogenesis, perhaps secondarily. To ponder the question whether APP may be etiologically involved in AD, much needs to be learned about APP functions. This article is intended to provide a foundation for this challenging task.     

Cellular localization of messenger RNA encoding amyloid-beta-protein in normal tissue and in Alzheimer disease

Amyloid precursor protein (APP) gene encodes the short peptide fragment amyloid-beta-protein present in senile plaque cores, cerebrovascular amyloid, and intracellular neurofibrillary tangles in Alzheimer disease (AD). Using in situ hybridization with biotin-labeled RNA probes, we found distinctive patterns of APP gene expression in different regions of postmortem human brain. Strong hybridization signal for APP messenger RNA (mRNA) was detected in specific classes of neurons, fascicular oligodendroglia, satellite glia, and presumptive microglia. Weaker signal was seen in other neuronal classes, fascicular astrocytes, and vascular endothelial cells, but no signal was seen in protoplasmic astrocytes. Human thymus also shows a restricted pattern of hybridization with high signal in reticular epithelial cells, and much lower signal in lymphocytes. In AD patients, neuronal hybridization for APP mRNA was specifically increased in hippocampus, but not cerebellar and visual cortex when compared to hybridization for neuron-specific enolase mRNA. Most neurons with neurofibrillary tangles had strong APP mRNA signal. These results suggest that APP gene expression is highly regulated in normal tissue, that many different cell classes in brain express the APP gene, and that neuronal expression may increase specifically in brain regions where widespread injury occurs in AD. Amyloid deposits in brains of AD patients might be explained by local production of precursor protein in endothelial cells, neurons or glia.     

APP processing and the APP-KPI domain involvement in the amyloid cascade

Alternative APP mRNA splicing can generate isoforms of APP containing a Kunitz protease inhibitor (KPI) domain. KPI is one of the main serine protease inhibitors. Protein and mRNA KPI(+)APP levels are elevated in Alzheimer's disease (AD) brain and are associated with increased amyloid beta deposition. In the last years increasing evidence on multiple points in the amyloid cascade where KPI(+)APP is involved has been accumulated, admitting an outstanding position in the pathogenesis of AD to the KPI domain. This review focuses on the APP processing, the molecular activity of KPI and its physiological and pathological roles and the KPI involvement in the amyloid cascade through the nerve growth factor, the lipoprotein receptor-related protein, the tumor necrosis factor-alpha converting enzyme and the Notch1 protein.     

Retromer disruption promotes amyloidogenic APP processing

Retromer deficiency has been implicated in sporadic AD and animals deficient in retromer components exhibit pronounced neurodegeneration. Because retromer performs retrograde transport from the endosome to the Golgi apparatus and neuronal Aβ is found in late endosomal compartments, we speculated that retromer malfunction might enhance amyloidogenic APP processing by promoting interactions between APP and secretase enzymes in late endosomes. We have evaluated changes in amyloid precursor protein (APP) processing and trafficking as a result of disrupted retromer activity by knockdown of Vps35, a vacuolar sorting protein that is an essential component of the retromer complex. Knocking down retromer activity produced no change in the quantity or cellular distribution of total cellular APP and had no affect on internalization of cell-surface APP. Retromer deficiency did, however, increase the ratio of secreted Aβ42:Aβ40 in HEK-293 cells over-expressing APP695, due primarily to a decrease in Aβ40 secretion. Recent studies suggest that the retromer-trafficked protein, Wntless, is secreted at the synapse in exosome vesicles and that these same vesicles contain Aβ. We therefore hypothesized that retromer deficiency may be associated with altered exosomal secretion of APP and/or secretase fragments. Holo-APP, Presenilin and APP C-terminal fragments were detected in exosomal vesicles secreted from HEK-293 cells. Levels of total APP C-terminal fragments were significantly increased in exosomes secreted by retromer deficient cells. These data suggest that reduced retromer activity can mimic the effects of familial AD Presenilin mutations on APP processing and promote export of amyloidogenic APP derivatives.     

Cholinesterase inhibitor affects the amyloid precursor protein isoforms in patients with Alzheimer's disease

An altered platelet ratio of amyloid precursor protein (APP) isoforms might be a diagnostic, predictive, or therapeutic marker for Alzheimer's disease (AD). Our purpose was to test the hypothesis that this ratio might serve as a therapeutic marker for AD patients treated with the cholinesterase inhibitor, galantamine. Thirty-nine patients (mean age 76.6 +/- 9.4 years) with AD were treated with galantamine for 12 weeks. Patients were evaluated at baseline, 4 and 12 weeks by cognitive testing along with a determination of their platelet APP isoform ratio. Western blotting was performed to calculate the APP isoform ratio. At the end of the treatment, cognitive scores significantly improved, and the ratio of the high-molecular-weight (130 kDa) isoform to the low-molecular-weight (110-106 kDa) isoforms increased. These results suggest that cholinesterase inhibition might be involved in APP processing.     

Antibody-bound beta-amyloid precursor protein stimulates the production of tumor necrosis factor-alpha and monocyte chemoattractant protein-1 by cortical neurons

Alzheimer's disease (AD) is an age-related neurodegenerative disorder characterized by the accumulation of extracellular depositions of fibrillar beta-amyloid (A beta), which is derived from the alternative processing of beta-amyloid precursor protein (APP). Although APP is thought to function as a cell surface receptor, its mode of action still remains elusive. In this study, we found that the culture medium derived from cortical neurons treated with an anti-APP antibody triggers the death of naive neurons. Biochemical and immunocytochemical analyses revealed the presence, both in the conditioned medium and in neurons, of increased levels of tumor necrosis factor-alpha and monocyte chemoattractant protein-1. Furthermore, the expression of these proinflammatory mediators occurred through a c-Jun N-terminal protein kinase/c-Jun-dependent mechanism. Taken together, our findings provide evidence for a novel mechanism whereby neuronal APP in its full-length configuration induces neuronal death. Such a mechanism might be relevant to neuroinflammatory processes as those observed in AD.     

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.