Mechanisms of Aβ Clearance and Degradation by Glial Cells

Amyloid beta-protein (Abeta) is the major component of neuritic (amyloid) plaques in Alzheimer's disease, and its deposition is an early and constant event in the complex pathogenetic cascade of the disease. Although many studies have focused on the biosynthetic processing of the beta-amyloid precursor protein and on the production and polymerization of Abeta, understanding the degradation and clearance of Abeta has received very little attention. By incubating the conditioned medium of metabolically labeled Abeta-secreting cells with media of various cultured cell lines, we observed a time-dependent decrease in the amount of Abeta in the mixed media. The factor principally responsible for this decrease was a secreted metalloprotease released by both neural and non-neural cells. Among the cells examined, the microglial cell line, BV-2, produced the most Abeta-degrading activity. The protease was completely blocked by the metalloprotease inhibitor, 1,10-phenanthroline, and partially inhibited by EDTA, whereas inhibitors of other protease classes produced little or no inhibition. Substrate analysis suggests that the enzyme was a non-matrix metalloprotease. The protease cleaved both Abeta1-40 and Abeta1-42 peptides secreted by beta-amyloid precursor protein-transfected cells but failed to degrade low molecular weight oligomers of Abeta that form in the culture medium. Lipopolysaccharide, a stimulator of macrophages/microglia, activated BV-2 cells to increase their Abeta-degrading metalloprotease activity. We conclude that secreted Abeta1-40 and Abeta1-42 peptides are constitutively degraded by a metalloprotease released by microglia and other neural cells, providing a potential mechanism for the clearance of Abeta in brain tissue.

Amyloid-β proteins (Aβ) of 42 (Aβ42) and 40 aa (Aβ40) accumulate as senile plaques (SP) and cerebrovascular amyloid protein deposits that are defining diagnostic features of Alzheimer's disease (AD). A number of rare mutations linked to familial AD (FAD) on the Aβ precursor protein (APP), Presenilin-1 (PS1), Presenilin- 2 (PS2), Adamalysin10, and other genetic risk factors for sporadic AD such as the ε4 allele of Apolipoprotein E (ApoE-ε4) foster the accumulation of Aβ and also induce the entire spectrum of pathology associated with the disease. Aβ accumulation is therefore a key pathological event and a prime target for the prevention and treatment of AD. APP is sequentially processed by β-site APP cleaving enzyme (BACE1) and γ-secretase, a multisubunit PS1/PS2-containing integral membrane protease, to generate Aβ. Although Aβ accumulates in all forms of AD, the only pathways known to be affected in FAD increase Aβ production by APP gene duplication or via base substitutions on APP and γ-secretase subunits PS1 and PS2 that either specifically increase the yield of the longer Aβ42 or both Aβ40 and Aβ42. However, the vast majority of AD patients accumulate Aβ without these known mutations. This led to proposals that impairment of Aβ degradation or clearance may play a key role in AD pathogenesis. Several candidate enzymes, including Insulin-degrading enzyme (IDE), Neprilysin (NEP), Endothelin-converting enzyme (ECE), Angiotensin converting enzyme (ACE), Plasmin, and Matrix metalloproteinases (MMPs) have been identified and some have even been successfully evaluated in animal models. Several studies also have demonstrated the capacity of γ-secretase inhibitors to paradoxically increase the yield of Aβ and we have recently established that the mechanism is by skirting Aβ degradation. This review outlines major cellular pathways of Aβ degradation to provide a basis for future efforts to fully characterize the panel of pathways responsible for Aβ turnover.

The Contribution of the Amyloid Hypothesis to the Understanding of Alzheimer's Disease: A Critical Overview

Accumulation of the amyloid-beta (Abeta) peptide in the brain is a crucial factor in the development of Alzheimer disease. Expression of transforming growth factor-beta1 (TGF-beta1), an immunosuppressive cytokine, has been associated in vivo with Abeta accumulation in transgenic mice and recently with Abeta clearance by activated microglia, suggesting its deleterious and beneficial effects in neuronal cells. In this study, we demonstrated that TGF-beta1 stimulated the production of matrix metalloproteinase-2 (MMP-2) in a time- and dose-dependent manner in a human monocytic THP-1 cell line. Notably, we found that Abeta1-42 consistently inhibited the TGF-beta1-induced production of MMP-2, the endogenous gene containing Smad response elements, whereas the reverse peptide, Abeta42-1, evidenced little effect. Additionally, Abeta1-42 reduced TGF-beta1-induced increase in plasminogen activator inhibitor-1 (PAI-1). This inhibitory effect of Abeta1-42 was also seen in human astroglial T98G cell line. Furthermore, Abeta1-42 significantly induced the expression of Smad7, which appears in turn to mediate the Abeta suppression of the TGF-beta1-induced MMP-2 production. Indeed, Smad7 overexpression mimicked the inhibitory effect of Abeta1-42 on TGF-beta1-induced MMP-2 production. Importantly, Abeta1-42 markedly suppressed the transactivation of the transfected reporter construct, p3TP-Lux, which contains TGF-beta1-inducible Smad response elements. This was concomitant with a decreased MMP-2 production in TGF-beta1-treated cells. Inhibition of cellular Smad7 levels via the small interference RNA method significantly ameliorated the Abeta1-42-mediated suppression of TGF-beta1-inducible transcription reporter activity, thereby restoring MMP-2 induction, whereas Smad7 transfection down-regulated TGF-beta1-inducible transcription reporter activity. Collectively, these data suggest that Abeta1-42 may play an important role in the negative regulation of TGF-beta1-induced MMP-2 production via Smad7 expression.

Amyloid β-protein (Aβ) containing amyloid plaques and abnormal phosphorylated τ-protein containing neurofibrillary tangles (NFTs) are hallmark lesions of Alzheimer's disease. Both Aβ plaques and NFTs show hierarchical patterns in which the areas of the brain are subsequently affected by Aβ plaques and NFTs, respectively (Braak and Braak, 1991; Thal et al., 2002). Aβ plaques start to develop in the neocortex (phase 1) and spread from there into allocortical regions (phase 2), diencephalon, basal forebrain and striatum (phase 3), midbrain and medulla oblongata (phase 4), and finally into the pons and the cerebellum (phase 5) (Thal et al., 2002). The first NFTs in the brain hemispheres are found in the transentorhinal cortex (stage I), then in the entorhinal cortex (stage II), the hippocampus (stage III), the temporal cortex (stage IV), further neocortical areas except the primary fields (stage V), and, finally, also in primary cortical areas, such as the primary visual cortex (stage VI) (Braak and Braak, 1991). Axonal connections between subsequently affected brain regions suggest that AD pathology spreads along neuronal pathways (Thal et al., 2002; Braak and Del Tredici, 2011). Insufficient clearance of Aβ has been considered to play an essential role in the pathogenesis of AD. Clearance mechanisms that contribute to Aβ elimination from brain are cellular enzymatic proteolysis in glial cells, neurons or in the extracellular space (Qiu et al., 1998; Yamaguchi et al., 1998; Iwata et al., 2000; Thal et al., 2000; Farris et al., 2003), transport through the blood-brain barrier (Shibata et al., 2000; Ito et al., 2007), and perivascular drainage (Weller et al., 2008) (Figure ​(Figure1A1A). Figure 1 Schematic representation of Aβ clearance and propagation. (A) Aβ clearance mechanisms: enzymatic clearance within neurons (N) and glial cells [here shown in the example is an astrocyte (AG)] or in the extracellular space (ECS) (Qiu et ... Here, I will discuss the potential impact of impaired Aβ clearance on propagation mechanisms for Aβ and τ.

Lipoprotein lipase (LPL) is a member of a lipase family known to hydrolyze triglyceride molecules in plasma lipoprotein particles. LPL also plays a role in the binding of lipoprotein particles to cell-surface molecules, including sulfated glycosaminoglycans (GAGs). LPL is predominantly expressed in adipose and muscle but is also highly expressed in the brain where its specific roles are unknown. It has been shown that LPL is colocalized with senile plaques in Alzheimer disease (AD) brains, and its mutations are associated with the severity of AD pathophysiological features. In this study, we identified a novel function of LPL; that is, LPL binds to amyloid β protein (Aβ) and promotes cell-surface association and uptake of Aβ in mouse primary astrocytes. The internalized Aβ was degraded within 12 h, mainly in a lysosomal pathway. We also found that sulfated GAGs were involved in the LPL-mediated cellular uptake of Aβ. Apolipoprotein E was dispensable in the LPL-mediated uptake of Aβ. Our findings indicate that LPL is a novel Aβ-binding protein promoting cellular uptake and subsequent degradation of Aβ.

Cerebral clearance of amyloid β-protein (Aβ) is decreased in early-onset and late-onset Alzheimer's disease (AD). Aβ is cleared from the brain by enzymatic degradation and by transport out of the brain. More than 20 Aβ-degrading enzymes have been described. Increasing the degradation of Aβ offers an opportunity to decrease brain Aβ levels in AD patients. This review discusses the direct and indirect approaches which have been used in experimental systems to alter the expression and/or activity of Aβ-degrading enzymes. Also discussed are the enzymes' regulatory mechanisms, the conformations of Aβ they degrade, where in the scheme of Aβ production, extracellular release, cellular uptake, and intracellular degradation they exert their activities, and changes in their expression and/or activity in AD and its animal models. Most of the experimental approaches require further confirmation. Based upon each enzyme's effects on Aβ (some of the enzymes also possess β-secretase activity and may therefore promote Aβ production), its direction of change in AD and/or its animal models, and the Aβ conformation(s) it degrades, investigating the effects of increasing the expression of neprilysin in AD patients would be of particular interest. Increasing the expression of insulin-degrading enzyme, endothelin-converting enzyme-1, endothelin-converting enzyme-2, tissue plasminogen activator, angiotensin-converting enzyme, and presequence peptidase would also be of interest. Increasing matrix metalloproteinase-2, matrix metalloproteinase-9, cathepsin-B, and cathepsin-D expression would be problematic because of possible damage by the metalloproteinases to the blood brain barrier and the cathepsins' β-secretase activity. Many interventions which increase the enzymatic degradation of Aβ have been shown to decrease AD-type pathology in experimental models. If a safe approach can be found to increase the expression or activity of selected Aβ-degrading enzymes in human subjects, then the possibility that this approach could slow the AD progression should be examined in clinical trials.

Sarsasapogenin has been reported to improve dementia symptoms somehow, probably through modulating the function of cholinergic system, suppressing neurofibrillary tangles, and inhibiting inflammation. However, the role of sarsasapogenin in response to beta-amyloid (Aβ) remains to be delineated. This study aimed to determine the therapeutic effect of sarsasapogenin-13 (AA13, a sarsasapogenin derivative) on learning and memory impairments in Aβ-injected mice, as well as the role of AA13 in neuroglia-mediated antiinflammation and Aβ clearance. Focusing on the role of AA13 in regulating glial responses to Aβ, we conducted behavioral, morphological, and protein expression studies to explore the effects of AA13 on Aβ clearance and inflammatory regulation. The results indicated that oral administration of AA13 attenuated the memory deficits of intracerebroventricular (i.c.v.) Aβ-injected mice; also, AA13 protected neuroglial cells against Aβ-induced cytotoxicity. The further mechanical studies demonstrated that AA13 reversed the upregulation of proinflammatory M1 markers and increased the expression of antiinflammatory M2 markers in Aβ-treated cells. Furthermore, AA13 facilitated Aβ clearance through promoting Aβ phagocytosis and degradation. AA13 modulated the expression of fatty acid translocase (CD36), insulin-degrading enzyme (IDE), neprilysin (NEP), and endothelin-converting enzyme (ECE) in neuroglia. The present study indicated that the neuroprotective effect of AA13 might relate to its modulatory effects on microglia activation state, phagocytic ability, and expression of Aβ-degrading enzymes, which makes it a promising therapeutic agent in the early stage of Alzheimer's disease (AD).

There is increasing evidence that deficient clearance of β-amyloid (Aβ) contributes to its accumulation in late-onset Alzheimer disease (AD). Several Aβ-degrading enzymes, including neprilysin (NEP), endothelin-converting enzyme (ECE), and angiotensin-converting enzyme (ACE) reduce Aβ levels and protect against cognitive impairment in mouse models of AD. In post-mortem human brain tissue we have found that the activity of these Aβ-degrading enzymes rise with age and increases still further in AD, perhaps as a physiological response that helps to minimize the build-up of Aβ. ECE-1/-2 and ACE are also rate-limiting enzymes in the production of endothelin-1 (ET-1) and angiotensin II (Ang II), two potent vasoconstrictors, increases in the levels of which are likely to contribute to reduced blood flow in AD. This review considers the possible interdependence between Aβ-degrading enzymes, ischemia and Aβ in AD: ischemia has been shown to increase Aβ production both in vitro and in vivo, whereas increased Aβ probably enhances ischemia by vasoconstriction, mediated at least in part by increased ECE and ACE activity. In contrast, NEP activity may help to maintain cerebral perfusion, by reducing the accumulation of Aβ in cerebral blood vessels and lessening its toxicity to vascular smooth muscle cells. In assessing the role of Aβ-degrading proteases in the pathogenesis of AD and, particularly, their potential as therapeutic agents, it is important to bear in mind the multifunctional nature of these enzymes and to consider their effects on other substrates and pathways.

The pathological hallmark of Alzheimer disease is the senile plaque principally composed of tightly aggregated amyloid-beta fibrils (fAbeta), which are thought to be resistant to degradation and clearance. In this study, we explored whether proteases capable of degrading soluble Abeta (sAbeta) could degrade fAbeta as well. We demonstrate that matrix metalloproteinase-9 (MMP-9) can degrade fAbeta and that this ability is not shared by other sAbeta-degrading enzymes examined, including endothelin-converting enzyme, insulin-degrading enzyme, and neprilysin. fAbeta was decreased in samples incubated with MMP-9 compared with other proteases, assessed using thioflavin-T. Furthermore, fAbeta breakdown with MMP-9 but not with other proteases was demonstrated by transmission electron microscopy. Proteolytic digests of purified fAbeta were analyzed with matrix-assisted laser desorption ionization time-of-flight mass spectrometry to identify sites of Abeta that are cleaved during its degradation. Only MMP-9 digests contained fragments (Abeta(1-20) and Abeta(1-30)) from fAbeta(1-42) substrate; the corresponding cleavage sites are thought to be important for beta-pleated sheet formation. To determine whether MMP-9 can degrade plaques formed in vivo, fresh brain slices from aged APP/PS1 mice were incubated with proteases. MMP-9 digestion resulted in a decrease in thioflavin-S (ThS) staining. Consistent with a role for endogenous MMP-9 in this process in vivo, MMP-9 immunoreactivity was detected in astrocytes surrounding amyloid plaques in the brains of aged APP/PS1 and APPsw mice, and increased MMP activity was selectively observed in compact ThS-positive plaques. These findings suggest that MMP-9 can degrade fAbeta and may contribute to ongoing clearance of plaques from amyloid-laden brains.

Vitamin E: Curse or Benefit in Alzheimer's Disease? A Systematic Investigation of the Impact of α-, γ- and δ-Tocopherol on Aß Generation and Degradation in Neuroblastoma Cells.

Extracellular amyloid plaques, intracellular neurofibrillary tangles, and loss of basal forebrain cholinergic neurons in the brains of Alzheimer's disease (AD) patients may be the end result of abnormalities in lipid metabolism and peroxidation that may be caused, or exacerbated, by beta-amyloid peptide (Abeta). Apolipoprotein E (apoE) is a major apolipoprotein in the brain, mediating the transport and clearance of lipids and Abeta. ApoE-dependent dendritic and synaptic regeneration may be less efficient with apoE4, and this may result in, or unmask, age-related neurodegenerative changes. The increased risk of AD associated with apoE4 may be modulated by diet, vascular risk factors, and genetic polymorphisms that affect the function of other transporter proteins and enzymes involved in brain lipid homeostasis. Diet and apoE lipoproteins influence membrane lipid raft composition and the properties of enzymes, transporter proteins, and receptors mediating Abeta production and degradation, tau phosphorylation, glutamate and glucose uptake, and neuronal signal transduction. The level and isoform of apoE may influence whether Abeta is likely to be metabolized or deposited. This review examines the current evidence for diet, lipid homeostasis, and apoE in the pathogenesis of AD. Effects on the cholinergic system and response to cholinesterase inhibitors by APOE allele carrier status are discussed briefly.

Alzheimer’s disease (AD) is a degenerative neurological disease that primarily affects the elderly. Drug therapy is the main strategy for AD treatment, but current treatments suffer from poor efficacy and a number of side effects. Non-drug therapy is attracting more attention and may be a better strategy for treatment of AD. Hypoxia is one of the important factors that contribute to the pathogenesis of AD. Multiple cellular processes synergistically promote hypoxia, including aging, hypertension, diabetes, hypoxia/obstructive sleep apnea, obesity, and traumatic brain injury. Increasing evidence has shown that hypoxia may affect multiple pathological aspects of AD, such as amyloid-beta metabolism, tau phosphorylation, autophagy, neuroinflammation, oxidative stress, endoplasmic reticulum stress, and mitochondrial and synaptic dysfunction. Treatments targeting hypoxia may delay or mitigate the progression of AD. Numerous studies have shown that oxygen therapy could improve the risk factors and clinical symptoms of AD. Increasing evidence also suggests that oxygen therapy may improve many pathological aspects of AD including amyloid-beta metabolism, tau phosphorylation, neuroinflammation, neuronal apoptosis, oxidative stress, neurotrophic factors, mitochondrial function, cerebral blood volume, and protein synthesis. In this review, we summarized the effects of oxygen therapy on AD pathogenesis and the mechanisms underlying these alterations. We expect that this review can benefit future clinical applications and therapy strategies on oxygen therapy for AD.

Cerebral amyloid angiopathy (CAA) is a cerebrovascular disorder marked by the deposition of amyloid-beta (Aβ) peptides within the walls of small- and medium-sized cerebral vessels, including arteries and capillaries but rarely veins. This vascular amyloid burden compromises vessel integrity, causes hemorrhages, and contributes to cognitive decline. Efficient Aβ clearance is critical for preventing its pathological accumulation. Thus, understanding the molecular players within the vascular microenvironment is essential. Laminin, a key glycoprotein of the vascular basal lamina (BL), is fundamental to maintaining structural stability of the vessels and regulating interactions among endothelial cells, pericytes, and the extracellular matrix. However, controversial findings exist on how laminin regulates Aβ aggregation and clearance, with both inhibitory and facilitative effects reported. Genetic variations in laminin subunits, their cell-specific expression pattern, and BL remodeling during CAA further complicate this relationship. This review synthesizes current knowledge on vascular Aβ deposition and elimination in CAA, with a particular focus on the critical roles of the BL/laminin and ApoE in shaping the perivascular microenvironment. First, we introduce Aβ processing relevant to CAA and the mechanisms of Aβ clearance in the CNS. Next, laminin-Aβ interactions and their functions in Aβ clearance are summarized. Thirdly, laminin changes and BL remodeling in CAA are discussed. Finally, we discuss the knowledge gap in the field and fundamental questions that need to be answered in future research. Defining the functions of the BL and ApoE within the pathological context of Aβ-rich vasculature may yield new insights into CAA pathogenesis and reveal therapeutic targets to limit vascular amyloid accumulation. Our goal is to provide a concise review on this matter in order to facilitate new hypotheses in the field.

To observe the clinical effect of moxibustion with deqi on Alzheimer's disease (AD) rats, and evaluate its effect on β-amyloid (Aβ) transport and enzymatic degradation proteins, to explore its molecular mechanism for improving cognitive function. Sixty SPF-grade male SD rats were randomly divided into a blank group (8 rats), a sham-operation group (8 rats) and a model establishment group (44 rats). The rats in the model establishment group were injected with Aβ1-42 at bilateral ventricles to establish AD model. Among the 38 rats with successful model establishment, 8 rats were randomly selected as the model group, and the remaining rats were treated with mild moxibustion at "Dazhui" (GV 14), once a day, 40 min each time, for 28 days. According to whether deqi appeared and the occurrence time of deqi, the rats were divided into a deqi group (12 rats), a delayed deqi group (10 rats) and a non-deqi group (8 rats). After the intervention, the Morris water maze test was applied to evaluate the cognitive function; the HE staining was applied to observe the brain morphology; the Western blot method was applied to measure the protein expression of Aβ and its receptor mediated transport [low-density lipoprotein receptor-related protein (LRP) 1, receptor for advanced glycation end products (RAGE), apolipoprotein E (ApoE)] and enzymatic degradation [neprilysin (NEP), insulin degrading enzyme (IDE), endothelin converting enzyme (ECE)-1 and angiotensin converting enzyme (ACE) 2]. Compared with the sham-operation group, in the model group, the escape latency was prolonged (P<0.01), and the times of platform crossing and the ratio of platform quadrant to total time were reduced (P<0.01); the brain tissue was seriously damaged; the expression of hippocampal Aβ and RAGE was increased (P<0.01), and the expression of hippocampal LRP1, ApoE, NEP, IDE, ECE-1 and ACE2 was decreased (P<0.01). Compared with the model group, the escape latency was shortened in the deqi group (P<0.05, P<0.01), and the escape latency in the delayed deqi group and the non-deqi group was shortened from Day 2 to Day 5 (P<0.05, P<0.01), and the times of platform crossing and the ratio of platform quadrant to total time were increased in the deqi group and the delayed deqi group (P<0.01, P<0.05); the brain damage in each moxibustion group was reduced, which was smallest in the deqi group, followed by the delayed deqi group and the non-deqi group; the expression of Aβ and RAGE was decreased (P<0.01, P<0.05) and the expression of LRP1 and IDE was increased in each moxibustion group (P<0.01, P<0.05); the expression of ApoE was increased in the deqi group and the delayed deqi group (P<0.01, P<0.05); the expression of NEP was increased in deqi group (P<0.05), and the expression of ECE-1 and ACE2 was increased in the deqi group and the delayed deqi group (P<0.05). Compared with the delayed deqi group and the non-deqi group, the escape latency in the deqi group was shortened from Day 3 to Day 5 (P<0.05), and the times of platform crossing and the ratio of platform quadrant to total time were increased (P<0.05, P<0.01). Compared with the non-deqi group, the expression of Aβ was reduced (P<0.05), the expression of LRP1 and ApoE was increased in the deqi group (P<0.05). The expression of NEP in the deqi group was higher than that in the delayed deqi group and the non-deqi group (P<0.05). Compared with non-deqi, moxibustion with deqi could promote Aβ transport and degradation, thereby reducing Aβ level in the brain and improving cognitive function for AD rats.