Transient pharmacologic lowering of Aβ production prior to deposition results in sustained reduction of amyloid plaque pathology

Background

Alzheimer??s disease (AD) is the leading cause of dementia among the elderly. Disease modifying therapies targeting A?? that are in development have been proposed to be more effective if treatment was initiated prior to significant accumulation of A?? in the brain, but optimal timing of treatment initiation has not been clearly established in the clinic. We compared the efficacy of transient pharmacologic reduction of brain A?? with a ??-secretase inhibitor (GSI ) for 1?C3?months (M) treatment windows in APP Tg2576 mice and subsequent aging of the mice to either 15M or 18M.

Results

These data show that reducing A?? production in a 2-3M windows both initiated and discontinued before detectable A?? deposition has the most significant impact on A?? loads up to 11M after treatment discontinuation. In contrast, initiation of treatment for 3M windows from 7-10M or 12-15M shows progressively decreasing efficacy.

Conclusions

These data have major implications for clinical testing of therapeutics aimed at lowering A?? production, indicating that; i) these therapies may have little efficacy unless tested as prophylactics or in the earliest preclinical stage of AD where there is no or minimal A?? accumulation and ii) lowering A?? production transiently during a critical pre-deposition window potentially provides long-lasting efficacy after discontinuation of the treatment.     

Inhibition of γ-secretase worsens memory deficits in a genetically congruous mouse model of Danish dementia

Background

A mutation in the BRI2/ITM2b gene causes familial Danish dementia (FDD). BRI2 is an inhibitor of amyloid-?? precursor protein (APP) processing, which is genetically linked to Alzheimer??s disease (AD) pathogenesis. The FDD mutation leads to a loss of BRI2 protein and to increased APP processing. APP haplodeficiency and inhibition of APP cleavage by ??-secretase rescue synaptic/memory deficits of a genetically congruous mouse model of FDD (FDD_KI). ??-cleavage of APP yields the ??-carboxyl-terminal (??-CTF) and the amino-terminal-soluble APP?? (sAPP??) fragments. ??-secretase processing of ??-CTF generates A??, which is considered the main cause of AD. However, inhibiting A?? production did not rescue the deficits of FDD_KI mice, suggesting that sAPP??/??-CTF, and not A??, are the toxic species causing memory loss.

Results

Here, we have further analyzed the effect of ??-secretase inhibition. We show that treatment with a ??-secretase inhibitor (GSI) results in a worsening of the memory deficits of FDD_KI mice. This deleterious effect on memory correlates with increased levels of the ??/??-CTFs APP fragments in synaptic fractions isolated from hippocampi of FDD_KI mice, which is consistent with inhibition of ??-secretase activity.

Conclusion

This harmful effect of the GSI is in sharp contrast with a pathogenic role for A??, and suggests that the worsening of memory deficits may be due to accumulation of synaptic-toxic ??/??-CTFs caused by GSI treatment. However, ??-secretase cleaves more than 40 proteins; thus, the noxious effect of GSI on memory may be dependent on inhibition of cleavage of one or more of these other ??-secretase substrates. These two possibilities do not need to be mutually exclusive. Our results are consistent with the outcome of a clinical trial with the GSI Semagacestat, which caused a worsening of cognition, and advise against targeting ??-secretase in the therapy of AD. Overall, the data also indicate that FDD_KI is a valuable mouse model to study AD pathogenesis and predict the clinical outcome of therapeutic agents for AD.     

siRNA against presenilin 1 (PS1) down regulates amyloid β42 production in IMR-32 cells

Background  

One of the pathological hallmarks of Alzheimer's disease (AD) is the deposition of the ~4 kDa amyloid β protein (Aβ) within lesions known as senile plaques. Aβ is also deposited in the walls of cerebral blood vessels in many cases of AD. A substantial proportion of the Aβ that accumulates in the AD brain is deposited as Amyloid, which is highly insoluble, proteinaceous material with a β-pleated-sheet conformation and deposited extracellularly in the form of 5-10 nm wide straight fibrils. As γ-secretase catalyzes the final cleavage that releases the Aβ42 or 40 from amyloid β -protein precursor (APP), therefore, it is a potential therapeutic target for the treatment of AD. γ-Secretase cleavage is performed by a high molecular weight protein complex containing presenilins (PSs), nicastrin, Aph-1 and Pen-2. Previous studies have demonstrated that the presenilins (PS1 and PS2) are critical components of a large enzyme complex that performs γ-secretase cleavage.     

Zileuton Improves Memory Deficits,Amyloid and Tau Pathology in a Mouse Model of Alzheimer’s Disease with Plaques and Tangles

The 5-lipoxygenase (5LO) enzyme is widely distributed within the central nervous system. Previous works showed that this protein is up-regulated in Alzheimer’s disease (AD), and plays an active role in the development of brain amyloidosis in the APP transgenic mice. In the present paper, we studied the effect of its pharmacological inhibition on the entire AD-like phenotype of a mouse model with plaques and tangles, the 3×Tg mice. Compared with mice receiving placebo, the group treated with zileuton, a specific 5LO inhibitor, manifested a significant improvement of their memory impairments. The same animals had a significant reduction in Aβ levels and deposition, which was secondary to a down-regulation of the γ-secretase pathway. Additionally, while total tau levels were unchanged for both groups, zileuton-treated mice had a significant reduction in its phosphorylation state and insoluble forms, secondary to a decreased activation of the cdk5 kinase. These data establish a functional role for 5LO in the pathogenesis of the full spectrum of the AD-like phenotype and represent the successful completion of the initial step for the preclinical development of 5LO inhibitors as viable therapeutic agents for AD.     

Synaptic and Endosomal Localization of Active γ-Secretase in Rat Brain

Background

A key player in the development of Alzheimer''s disease (AD) is the γ-secretase complex consisting of at least four components: presenilin, nicastrin, Aph-1 and Pen-2. γ-Secretase is crucial for the generation of the neurotoxic amyloid β-peptide (Aβ) but also takes part in the processing of many other substrates. In cell lines, active γ-secretase has been found to localize primarily to the Golgi apparatus, endosomes and plasma membranes. However, no thorough studies have been performed to show the subcellular localization of the active γ-secretase in the affected organ of AD, namely the brain.

Principal Findings

We show by subcellular fractionation of rat brain that high γ-secretase activity, as assessed by production of Aβ40, is present in an endosome- and plasma membrane-enriched fraction of an iodixanol gradient. We also prepared crude synaptic vesicles as well as synaptic membranes and both fractions showed high Aβ40 production and contained high amounts of the γ-secretase components. Further purification of the synaptic vesicles verified the presence of the γ-secretase components in these compartments. The localization of an active γ-secretase in synapses and endosomes was confirmed in rat brain sections and neuronal cultures by using a biotinylated γ-secretase inhibitor together with confocal microscopy.

Significance

The information about the subcellular localization of γ-secretase in brain is important for the understanding of the molecular mechanisms of AD. Furthermore, the identified fractions can be used as sources for highly active γ-secretase.     

Sortilin, SorCS1b, and SorLA Vps10p sorting receptors, are novel γ-secretase substrates

Background

The mammalian Vps10p sorting receptor family is a group of 5 type I membrane homologs (Sortilin, SorLA, and SorCS1-3). These receptors bind various cargo proteins via their luminal Vps10p domains and have been shown to mediate a variety of intracellular sorting and trafficking functions. These proteins are highly expressed in the brain. SorLA has been shown to be down regulated in Alzheimer's disease brains, interact with ApoE, and modulate Aβ production. Sortilin has been shown to be part of proNGF mediated death signaling that results from a complex of Sortilin, p75^NTR and proNGF. We have investigated and provide evidence for γ-secretase cleavage of this family of proteins.

Results

We provide evidence that these receptors are substrates for presenilin dependent γ-secretase cleavage. γ-Secretase cleavage of these sorting receptors is inhibited by γ-secretase inhibitors and does not occur in PS1/PS2 knockout cells. Like most γ-secretase substrates, we find that ectodomain shedding precedes γ-secretase cleavage. The ectodomain cleavage is inhibited by a metalloprotease inhibitor and activated by PMA suggesting that it is mediated by an α-secretase like cleavage.

Conclusion

These data indicate that the α- and γ-secretase cleavages of the mammalian Vps10p sorting receptors occur in a fashion analogous to other known γ-secretase substrates, and could possibly regulate the biological functions of these proteins.     

Flavonoid-mediated presenilin-1 phosphorylation reduces Alzheimer's disease beta-amyloid production

Glycogen synthase kinase 3 (GSK-3) dysregulation is implicated in the two Alzheimer's disease (AD) pathological hallmarks: β-amyloid plaques and neurofibrillary tangles. GSK-3 inhibitors may abrogate AD pathology by inhibiting amyloidogenic γ-secretase cleavage of amyloid precursor protein (APP). Here, we report that the citrus bioflavonoid luteolin reduces amyloid-β (Aβ) peptide generation in both human 'Swedish' mutant APP transgene-bearing neuron-like cells and primary neurons. We also find that luteolin induces changes consistent with GSK-3 inhibition that ( i ) decrease amyloidogenic γ-secretase APP processing, and ( ii ) promote presenilin-1 (PS1) carboxyl-terminal fragment (CTF) phosphorylation. Importantly, we find GSK-3α activity is essential for both PS1 CTF phosphorylation and PS1-APP interaction. As validation of these findings in vivo , we find that luteolin, when applied to the Tg2576 mouse model of AD, decreases soluble Aβ levels, reduces GSK-3 activity, and disrupts PS1-APP association. In addition, we find that Tg2576 mice treated with diosmin, a glycoside of a flavonoid structurally similar to luteolin, display significantly reduced Aβ pathology. We suggest that GSK-3 inhibition is a viable therapeutic approach for AD by impacting PS1 phosphorylation-dependent regulation of amyloidogenesis.     

Binding of longer Aβ to transmembrane domain 1 of presenilin 1 impacts on Aβ42 generation

Background

Amyloid-β peptide ending at 42nd residue (Aβ42) is believed as a pathogenic peptide for Alzheimer disease. Although γ-secretase is a responsible protease to generate Aβ through a processive cleavage, the proteolytic mechanism of γ-secretase at molecular level is poorly understood.

Results

We found that the transmembrane domain (TMD) 1 of presenilin (PS) 1, a catalytic subunit for the γ-secretase, as a key modulatory domain for Aβ42 production. Aβ42-lowering and -raising γ-secretase modulators (GSMs) directly targeted TMD1 of PS1 and affected its structure. A point mutation in TMD1 caused an aberrant secretion of longer Aβ species including Aβ45 that are the precursor of Aβ42. We further found that the helical surface of TMD1 is involved in the binding of Aβ45/48 and that the binding was altered by GSMs as well as TMD1 mutation.

Conclusions

Binding between PS1 TMD1 and longer Aβ is critical for Aβ42 production.     

Increased expression of PS1 is sufficient to elevate the level and activity of γ-secretase in vivo

Increase in the generation and deposition of amyloid-β (Aβ) plays a central role in the development of Alzheimer's Disease (AD). Elevation of the activity of γ-secretase, a key enzyme required for the generation for Aβ, can thus be a potential risk factor in AD. However, it is not known whether γ-secretase can be upregulated in vivo. While in vitro studies showed that expression of all four components of γ-secretase (Nicastrin, Presenilin, Pen-2 and Aph-1) are required for upregulation of γ-secretase, it remains to be established as to whether this is true in vivo. To investigate whether overexpressing a single component of the γ-secretase complex is sufficient to elevate its level and activity in the brain, we analyzed transgenic mice expressing either wild type or familial AD (fAD) associated mutant PS1. In contrast to cell culture studies, overexpression of either wild type or mutant PS1 is sufficient to increase levels of Nicastrin and Pen-2, and elevate the level of active γ-secretase complex, enzymatic activity of γ-secretase and the deposition of Aβ in brains of mice. Importantly, γ-secretase comprised of mutant PS1 is less active than that of wild type PS1-containing γ-secretase; however, γ-secretase comprised of mutant PS1 cleaves at the Aβ42 site of APP-CTFs more efficiently than at the Aβ40 site, resulting in greater accumulation of Aβ deposits in the brain. Our data suggest that whereas fAD-linked PS1 mutants cause early onset disease, upregulation of PS1/γ-secretase activity may be a risk factor for late onset sporadic AD.     

Tg2576 cortical neurons that express human Ab are susceptible to extracellular Aβ-induced, K+ efflux dependent neurodegeneration

Background

One of the key pathological features of AD is the formation of insoluble amyloid plaques. The major constituent of these extracellular plaques is the beta-amyloid peptide (Aβ), although Aβ is also found to accumulate intraneuronally in AD. Due to the slowly progressive nature of the disease, it is likely that neurons are exposed to sublethal concentrations of both intracellular and extracellular Aβ for extended periods of time.

Results

In this study, we report that daily exposure to a sublethal concentration of Aβ_1-40 (1 µM) for six days induces substantial apoptosis of cortical neurons cultured from Tg2576 mice (which express substantial but sublethal levels of intracellular Aβ). Notably, untreated Tg2576 neurons of similar age did not display any signs of apoptosis, indicating that the level of intracellular Aβ present in these neurons was not the cause of toxicity. Furthermore, wildtype neurons did not become apoptotic under the same chronic Aβ_1-40 treatment. We found that this apoptosis was linked to Tg2576 neurons being unable to maintain K⁺ homeostasis following Aβ treatment. Furthermore, blocking K⁺ efflux protected Tg2576 neurons from Aβ-induced neurotoxicity. Interestingly, chronic exposure to 1 µM Aβ_1-40 caused the generation of axonal swellings in Tg2576 neurons that contained dense concentrations of hyperphosphorylated tau. These were not observed in wildtype neurons under the same treatment conditions.

Conclusions

Our data suggest that when neurons are chronically exposed to sublethal levels of both intra- and extra-cellular Aβ, this causes a K⁺-dependent neurodegeneration that has pathological characteristics similar to AD.     

Expression profiling in APP23 mouse brain: inhibition of Aβ amyloidosis and inflammation in response to LXR agonist treatment

Background

Recent studies demonstrate that in addition to its modulatory effect on APP processing, in vivo application of Liver X Receptor agonist T0901317 (T0) to APP transgenic and non-transgenic mice decreases the level of Aβ_42. Moreover, in young Tg2576 mice T0 completely reversed contextual memory deficits. Compared to other tissues, the regulatory functions of LXRs in brain remain largely unexplored and our knowledge so far is limited to the cholesterol transporters and apoE. In this study we applied T0 to APP23 mice for various times and examined gene and protein expression. We also performed a series of experiments with primary brain cells derived from wild type and LXR knockout mice subjected to various LXR agonist treatments and inflammatory stimuli.

Results

We demonstrate an upregulation of genes related to lipid metabolism/transport, metabolism of xenobiotics and detoxification. Downregulated genes are involved in immune response and inflammation, cell death and apoptosis. Additional treatment experiments demonstrated an increase of soluble apolipoproteins E and A-I and a decrease of insoluble Aβ. In primary LXR^wt but not in LXRα^-/-β^-/- microglia and astrocytes LXR agonists suppressed the inflammatory response induced by LPS or fibrillar Aβ.

Conclusion

The results show that LXR agonists could alleviate AD pathology by acting on amyloid deposition and brain inflammation. An increased understanding of the LXR controlled regulation of Aβ aggregation and clearance systems will lead to the development of more specific and powerful agonists targeting LXR for the treatment of AD.     

Cerebrospinal fluid Presenilin-1 increases at asymptomatic stage in genetically determined Alzheimer’s disease

Background

Presenilin-1 (PS1), the active component of the intramembrane γ-secretase complex, can be detected as soluble heteromeric aggregates in cerebrospinal fluid (CSF). The aim of this study was to examine the different soluble PS1 complexes in the lumbar CSF (CSF-PS1) of individuals with Alzheimer’s disease (AD), particularly in both symptomatic and asymptomatic genetically determined AD, in order to evaluate their potential as early biomarkers.

Methods

Western blotting, differential centrifugation and co-immunoprecipitation served to determine and characterize CSF-PS1 complexes. We also monitored the assembly of soluble PS1 into complexes in a cell model, and the participation of Aβ in the dynamics and robustness of the stable PS1 complexes.

Results

There was an age-dependent increase in CSF-PS1 levels in cognitively normal controls, the different complexes represented in similar proportions. The total levels of CSF-PS1, and in particular the proportion of the stable 100–150 kDa complexes, increased in subjects with autosomal dominant AD that carried PSEN1 mutations (eight symptomatic and six asymptomatic ADAD) and in Down syndrome individuals (ten demented and ten non-demented DS), compared with age-matched controls (n?=?23), even prior to the appearance of symptoms of dementia. The proportion of stable CSF-PS1 complexes also increased in sporadic AD (n?=?13) and mild-cognitive impaired subjects (n?=?12), relative to age-matched controls (n?=?17). Co-immunoprecipitation demonstrated the association of Aβ oligomers with soluble PS1 complexes, particularly the stable complexes.

Conclusions

Our data suggest that CSF-PS1 complexes may be useful as an early biomarker for AD, reflecting the pathology at asymptomatic state.

     

7-Deoxy-trans-dihydronarciclasine Reduces β-Amyloid and Ameliorates Memory Impairment in a Transgenic Model of Alzheimer’s Disease

The critical pathological feature of Alzheimer’s disease (AD) is the accumulation of β-amyloid (Aβ), the main constituent of amyloid plaques. β-amyloid precursor protein (APP) undergoes amyloidogenic cleavage by β- and γ-secretase generating Aβ at endosomes or non-amyloidogenic processing by α-secretase precluding the production of Aβ at the plasma membrane. Recently, several natural products have been widely researched on the prevention of Aβ accumulation for AD treatment. We previously reported that Lycoris chejuensis K. Tae et S. Ko (CJ), which originated from Jeju Island in Korea, improved the disrupted memory functions and reduced Aβ production in vivo. Here, we further explored the effect of its active component, 7-deoxy-trans-dihydronarciclasine (coded as E144), on Aβ generation and the underlying mechanism. Our results showed that E144 reduced the level of APP, especially its mature form, in HeLa cells overexpressing human APP with the Swedish mutation. Concomitantly, E144 decreased the levels of Aβ, sAPPβ, sAPPα, and C-terminal fragment. In addition, administration of E144 normalized the behavioral deficits in Tg2576 mice, an APP transgenic mouse model of AD. E144 also decreased the Aβ and APP levels in the cerebral cortex of Tg2576 mice. Thus, we propose that E144 could be a potential drug candidate for an anti-amyloid disease-modifying AD therapy.     

Neuropeptide-inducible upregulation of proteasome activity precedes nuclear factor kappa B activation in androgen-independent prostate cancer cells

Background

Upregulation of nuclear factor kappa B (NF??B) activity and neuroendocrine differentiation are two mechanisms known to be involved in prostate cancer (PC) progression to castration resistance. We have observed that major components of these pathways, including NF??B, proteasome, neutral endopeptidase (NEP) and endothelin 1 (ET-1), exhibit an inverse and mirror image pattern in androgen-dependent (AD) and -independent (AI) states in vitro.

Methods

We have now investigated for evidence of a direct mechanistic connection between these pathways with the use of immunocytochemistry (ICC), western blot analysis, electrophoretic mobility shift assay (EMSA) and proteasome activity assessment.

Results

Neuropeptide (NP) stimulation induced nuclear translocation of NF??B in a dose-dependent manner in AI cells, also evident as reduced total inhibitor ??B (I??B) levels and increased DNA binding in EMSA. These effects were preceded by increased 20?S proteasome activity at lower doses and at earlier times and were at least partially reversed under conditions of NP deprivation induced by specific NP receptor inhibitors, as well as NF??B, I??B kinase (IKK) and proteasome inhibitors. AD cells showed no appreciable nuclear translocation upon NP stimulation, with less intense DNA binding signal on EMSA.

Conclusions

Our results support evidence for a direct mechanistic connection between the NPs and NF??B/proteasome signaling pathways, with a distinct NP-induced profile in the more aggressive AI cancer state.     

Intracellular Trafficking of Presenilin 1 Is Regulated by ��-Amyloid Precursor Protein and Phospholipase D1

Excessive accumulation of β-amyloid peptides in the brain is a major cause for the pathogenesis of Alzheimer disease. β-Amyloid is derived from β-amyloid precursor protein (APP) through sequential cleavages by β- and γ-secretases, whose enzymatic activities are tightly controlled by subcellular localization. Delineation of how intracellular trafficking of these secretases and APP is regulated is important for understanding Alzheimer disease pathogenesis. Although APP trafficking is regulated by multiple factors including presenilin 1 (PS1), a major component of the γ-secretase complex, and phospholipase D1 (PLD1), a phospholipid-modifying enzyme, regulation of intracellular trafficking of PS1/γ-secretase and β-secretase is less clear. Here we demonstrate that APP can reciprocally regulate PS1 trafficking; APP deficiency results in faster transport of PS1 from the trans-Golgi network to the cell surface and increased steady state levels of PS1 at the cell surface, which can be reversed by restoring APP levels. Restoration of APP in APP-deficient cells also reduces steady state levels of other γ-secretase components (nicastrin, APH-1, and PEN-2) and the cleavage of Notch by PS1/γ-secretase that is more highly correlated with cell surface levels of PS1 than with APP overexpression levels, supporting the notion that Notch is mainly cleaved at the cell surface. In contrast, intracellular trafficking of β-secretase (BACE1) is not regulated by APP. Moreover, we find that PLD1 also regulates PS1 trafficking and that PLD1 overexpression promotes cell surface accumulation of PS1 in an APP-independent manner. Our results clearly elucidate a physiological function of APP in regulating protein trafficking and suggest that intracellular trafficking of PS1/γ-secretase is regulated by multiple factors, including APP and PLD1.An important pathological hallmark of Alzheimer disease (AD)⁴ is the formation of senile plaques in the brains of patients. The major components of those plaques are β-amyloid peptides (Aβ), whose accumulation triggers a cascade of neurodegenerative steps ending in formation of senile plaques and intraneuronal fibrillary tangles with subsequent neuronal loss in susceptible brain regions (1, 2). Aβ is proteolytically derived from the β-amyloid precursor protein (APP) through sequential cleavages by β-secretase (BACE1), a novel membrane-bound aspartyl protease (3, 4), and by γ-secretase, a high molecular weight complex consisting of at least four components: presenilin (PS), nicastrin (NCT), anterior pharynx-defective-1 (APH-1), and presenilin enhancer-2 (PEN-2) (5, 6). APP is a type I transmembrane protein belonging to a protein family that includes APP-like protein 1 (APLP1) and 2 (APLP2) in mammals (7, 8). Full-length APP is synthesized in the endoplasmic reticulum (ER) and transported through the Golgi apparatus. Most secreted Aβ peptides are generated within the trans-Golgi network (TGN), also the major site of steady state APP in neurons (9–11). APP can be transported to the cell surface in TGN-derived secretory vesicles if not proteolyzed to Aβ or an intermediate metabolite. At the cell surface APP is either cleaved by α-secretase to produce soluble sAPPα (12) or reinternalized for endosomal/lysosomal degradation (13, 14). Aβ may also be generated in endosomal/lysosomal compartments (15, 16). In contrast to neurotoxic Aβ peptides, sAPPα possesses neuroprotective potential (17, 18). Thus, the subcellular distribution of APP and proteases that process it directly affect the ratio of sAPPα to Aβ, making delineation of the mechanisms responsible for regulating trafficking of all of these proteins relevant to AD pathogenesis.Presenilin (PS) is a critical component of the γ-secretase. Of the two mammalian PS gene homologues, PS1 and PS2, PS1 encodes the major form (PS1) in active γ-secretase (19, 20). Nascent PSs undergo endoproteolytic cleavage to generate an amino-terminal fragment (NTF) and a carboxyl-terminal fragment (CTF) to form a functional PS heterodimer (21). Based on observations that PSs possess two highly conserved aspartate residues indispensable for γ-secretase activity and that specific transition state analogue γ-secretase inhibitors bind to PS1 NTF/CTF heterodimers (5, 22), PSs are believed to be the catalytic component of the γ-secretase complex. PS assembles with three other components, NCT, APH-1, and PEN-2, to form the functional γ-secretase (5, 6). Strong evidence suggests that PS1/γ-secretase resides principally in the ER, early Golgi, TGN, endocytic and intermediate compartments, most of which (except the TGN) are not major subcellular sites for APP (23, 24). In addition to generating Aβ and cleaving APP to release the APP intracellular domain, PS1/γ-secretase cleaves other substrates such as Notch (25), cadherin (26), ErbB4 (27), and CD44 (28), releasing their respective intracellular domains. Interestingly, PS1/γ-secretase cleavage of different substrates seems to occur at different subcellular compartments; APP is mainly cleaved at the TGN and early endosome domains, whereas Notch is predominantly cleaved at the cell surface (9, 11, 29). Thus, perturbing intracellular trafficking of PS1/γ-secretase may alter interactions between PS1/γ-secretase and APP, contributing to either abnormal Aβ generation and AD pathogenesis or decreased access of PS1/γ-secretase to APP such that Aβ production is reduced. However, mechanisms regulating PS1/γ-secretase trafficking warrant further investigation.In addition to participating in γ-secretase activity, PS1 regulates intracellular trafficking of several membrane proteins, including other γ-secretase components (nicastrin, APH-1, and PEN-2) and the substrate APP (reviewed in Ref. 30). Intracellular APP trafficking is highly regulated and requires other factors such as mint family members and SorLA (2). Moreover, we recently found that phospholipase D1 (PLD1), a phospholipid-modifying enzyme that regulates membrane trafficking events, can interact with PS1, and can regulate budding of APP-containing vesicles from the TGN and delivery of APP to the cell surface (31, 32). Interestingly, Kamal et al. (33) identified an axonal membrane compartment that contains APP, BACE1, and PS1 and showed that fast anterograde axonal transport of this compartment is mediated by APP and kinesin-I, implying a traffic-regulating role for APP. Increased APP expression is also shown to decrease retrograde axonal transport of nerve growth factor (34). However, whether APP indeed regulates intracellular trafficking of proteins including BACE1 and PS1/γ-secretase requires further validation. In the present study we demonstrate that intracellular trafficking of PS1, as well as that of other γ-secretase components, but not BACE1, is regulated by APP. APP deficiency promotes cell surface delivery of PS1/γ-secretase complex and facilitates PS1/γ-secretase-mediated Notch cleavage. In addition, we find that PLD1 also regulates intracellular trafficking of PS1 through a different mechanism and more potently than APP.     

Effects of heparin and enoxaparin on APP processing and Aβ production in primary cortical neurons from Tg2576 mice

Background

Alzheimer''s disease (AD) is caused by accumulation of Aβ, which is produced through sequential cleavage of β-amyloid precursor protein (APP) by the β-site APP cleaving enzyme (BACE1) and γ-secretase. Enoxaparin, a low molecular weight form of the glycosaminoglycan (GAG) heparin, has been reported to lower Aβ plaque deposition and improve cognitive function in AD transgenic mice.

Methodology/Principal Findings

We examined whether heparin and enoxaparin influence APP processing and inhibit Aβ production in primary cortical cell cultures. Heparin and enoxaparin were incubated with primary cortical cells derived from Tg2576 mice, and the level of APP and proteolytic products of APP (sAPPα, C99, C83 and Aβ) was measured by western blotting. Treatment of the cells with heparin or enoxaparin had no significant effect on the level of total APP. However, both GAGs decreased the level of C99 and C83, and inhibited sAPPα and Aβ secretion. Heparin also decreased the level of β-secretase (BACE1) and α-secretase (ADAM10). In contrast, heparin had no effect on the level of ADAM17.

Conclusions/Significance

The data indicate that heparin and enoxaparin decrease APP processing via both α- and β-secretase pathways. The possibility that GAGs may be beneficial for the treatment of AD needs further study.     

Ceramide sphingolipid signaling mediates Tumor Necrosis Factor (TNF)-dependent toxicity via caspase signaling in dopaminergic neurons

Background

The A?? peptide that accumulates in Alzheimer??s disease (AD) is derived from amyloid precursor protein (APP) following proteolysis by ??- and ??-secretases. Substantial evidence indicates that alterations in APP trafficking within the secretory and endocytic pathways directly impact the interaction of APP with these secretases and subsequent A?? production. Various members of the low-density lipoprotein receptor (LDLR) family have been reported to play a role in APP trafficking and processing and are important risk factors in AD. We recently characterized a distinct member of the LDLR family called LDLR-related protein 10 (LRP10) that shuttles between the trans-Golgi Network (TGN), plasma membrane (PM), and endosomes. Here we investigated whether LRP10 participates in APP intracellular trafficking and A?? production.

Results

In this report, we provide evidence that LRP10 is a functional APP receptor involved in APP trafficking and processing. LRP10 interacts directly with the ectodomain of APP and colocalizes with APP at the TGN. Increased expression of LRP10 in human neuroblastoma SH-SY5Y cells induces the accumulation of mature APP in the Golgi and reduces its presence at the cell surface and its processing into A??, while knockdown of LRP10 expression increases A?? production. Mutations of key motifs responsible for the recycling of LRP10 to the TGN results in the aberrant redistribution of APP with LRP10 to early endosomes and a concomitant increase in APP ??-cleavage into A??. Furthermore, expression of LRP10 is significantly lower in the post-mortem brain tissues of AD patients, supporting a possible role for LRP10 in AD.

Conclusions

The present study identified LRP10 as a novel APP sorting receptor that protects APP from amyloidogenic processing, suggesting that a decrease in LRP10 function may contribute to the pathogenesis of Alzheimer??s disease.     

Calpastatin levels affect calpain activation and calpain proteolytic activity in APP transgenic mouse model of Alzheimer's disease

The intracellular Ca(2+)-dependent protease calpain and the specific calpain endogenous inhibitor calpastatin are widely distributed, with the calpastatin/calpain ratio varying among tissues and species. Increased Ca(2+) and calpain activation have been implicated in Alzheimer's disease (AD), with scant data available on calpastatin/calpain ratio in AD. Information is lacking on calpain activation and calpastatin levels in transgenic mice that exhibit AD-like pathology. We studied calpain and calpastatin in Tg2576 mice and in their wild type littermates (control mice). We found that in control mice calpastatin level varies among brain regions; it is significantly higher in the cerebellum than in the hippocampus, frontal and temporal cortex, whereas calpain levels are similar in all these regions. In the Tg2576 mice, calpain is activated, calpastatin is diminished, and calpain-dependent proteolysis is observed in brain regions affected in AD and in transgenic mice (especially hippocampus). In contrast, no differences are observed between the Tg2576 and the control mice in the cerebellum, which does not exhibit AD-like pathology. The results are consistent with the notion that a high level of calpastatin in the cerebellum renders the calpain in this brain region less liable to be activated; in the other brain parts, in which calpastatin is low, calpain is more easily activated in the presence of increased Ca(2+), and in turn the activated calpain leads to further diminution in calpastatin (a known calpain substrate). The results indicate that calpastatin is an important factor in the regulation of calpain-induced protein degradation in the brains of the affected mice, and imply a role for calpastatin in attenuating AD pathology. Promoting calpastatin expression may be used to ameliorate some manifestations of AD.     

Reciprocal relationship between APP positioning relative to the membrane and PS1 conformation

Background

Several familial Alzheimer disease (FAD) mutations within the transmembrane region of the amyloid precursor protein (APP) increase the Aβ_42/40 ratio without increasing total Aβ production. In the present study, we analyzed the impact of FAD mutations and γ-secretase modulators (GSMs) that alter the Aβ_42/40 ratio on APP C-terminus (CT) positioning relative to the membrane, reasoning that changes in the alignment of the APP intramembranous domain and presenilin 1 (PS1) may impact the PS1/γ-secretase cleavage site on APP.

Results

By using a Förster resonance energy transfer (FRET)-based technique, fluorescent lifetime imaging microscopy (FLIM), we show that Aβ_42/40 ratio-modulating factors which target either APP substrate or PS1/γ-secretase affect proximity of the APP-CT to the membrane and change PS1 conformation.

Conclusions

Thus, we propose that there is a reciprocal relationship between APP-CT positioning relative to the membrane and PS1 conformation, suggesting that factors that modulate either APP positioning in the membrane or PS1 conformation could be exploited therapeutically.     

Arjunolic acid protects against DNCB-induced atopic dermatitis-like symptoms in mice by restoring a normal cytokine balance

Purpose

Atopic dermatitis (AD) is a chronically relapsing, pruritic, eczematous skin disorder accompanying allergic inflammation. AD is triggered by oxidative stress and immune imbalance. The effect of oral arjunolic acid (AA) on 2,4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis in mice was investigated.

Methods

Repeated epicutaneous application of DNCB to the ear and shaved dorsal skin of mice was performed to induce AD-like symptoms and skin lesions: 250mg/kg AA was given orally for three weeks to assess its anti-pruritic effects. Serum levels of tumor necrosis factor (TNF)-α, interleukin (IL)-4, IL-6, IL-10, immunoglobulin (Ig)E and caspase-3 were assessed by ELISA.

Results

We found that AA alleviated DNCB-induced AD-like symptoms as quantified by skin lesions, dermatitis score, ear thickness and scratching behavior. Levels of reactive oxygen species in the AA group were significantly inhibited compared with those in the DNCB group. In parallel, AA blocked a DNCBinduced reduction in serum levels of IL-4 and IL-10 associated with an attenuation of DNCB-induced increases in serum TNF-α, IL-6, IgE and caspase-3.

Conclusions

The results indicate that AA suppresses DNCB-induced AD in mice via redox balance and immune modulation, and could be a safe clinical treatment for AD.