Non-amyloidogenic Amyloid Precursor Protein Research Articles

Levetiracetam Treatment Normalizes Levels of Presynaptic Endocytosis Machinery and Restores Nonamyloidogenic APP Processing in App Knock-in Mice.

Toxic amyloid-beta (Aβ) peptides, produced by sequential proteolytic cleavage of the amyloid precursor protein (APP), play a key role in the initial stage of Alzheimer’s disease (AD). Increasing evidence indicates that Aβ42 induces neuronal circuit hyperexcitability in the early stages of AD pathology. As a result, researchers have investigated treatments that modulate the excitatory/inhibitory imbalance as potential AD therapies. For example, levetiracetam, an atypical antiepileptic drug used to quell hyperexcitability, has garnered recent interest in the AD field, even though its exact mechanism(s) of action remains elusive. Here, we show that in APP knock-in mouse models of amyloid pathology, chronic levetiracetam administration decreases cortical Aβ42 levels and lowers the amyloid plaque burden. In addition, using multiplexed tandem mass tag-quantitative mass spectrometry-based proteomic analysis, we determined that chronic levetiracetam administration selectively normalizes levels of presynaptic endocytic proteins. Finally, we found that levetiracetam treatment selectively lowers beta carboxyl-terminal fragment levels, while the abundance of full-length APP remains unchanged. In summary, this work reports that chronic treatment with levetiracetam serves as a useful therapeutic in AD by normalizing levels of presynaptic endocytic proteins and altering APP cleavage preference, leading to a decrease in both Aβ42 levels and the amyloid plaque burden. These novel findings provide novel evidence for the previously documented therapeutic value of levetiracetam to mitigate AD pathology.

Acute Interleukin‐6 Injection Influences Key Enzymes Involved in Amyloid Precursor Protein Processing in C57BL/6J Mice

Background Amyloid-beta plaques are a hallmark feature of Alzheimer's disease and are the product of aggregated amyloid-beta peptides. These peptides originate from amyloid precursor protein (APP) which is cleaved by beta secretase (BACE1), a key enzyme in the amyloidogenic cascade. Cleavage of APP by alpha secretase (ADAM10) however, does not result in amyloid-beta peptides and is a key enzyme in the non-amyloidogenic cascade. It is widely accepted that exercise reduces amyloid-beta production and plaque build-up in the brain, although it is unclear how exercise may be driving this observation. Interleukin-6 (IL-6) is an exercise induced cytokine and recent work in this area has demonstrated a role for acute exercise induced IL-6 in tissues such as muscle, adipose, and liver, however the role of acute IL-6 in the brain remains ill defined. The purpose of this study is to investigate if acute IL-6 injection will modulate key enzymes involved in APP processing, namely BACE1 and ADAM10. It is hypothesized that acute IL-6 injection will result in a shift towards the non-amyloidogenic processing of APP. Methods Male 10-week-old C57BL/6J mice were intraperitoneally injected with a weight-adjusted dose of recombinant murine IL-6 (3ng/g body mass) or an equivalent volume of saline. Brain tissue was acquired 15 minutes post-injection and the pre-frontal cortex and hippocampus regions were dissected. Results Acute IL-6 injection resulted in lower BACE1 activity in both the cortex (p=0.011) and the hippocampus (p=0.027) compared to the saline injected group. This was accompanied by lower BACE1 (p=0.007) and sAPPb(p=0.019) protein content in the hippocampus of mice injected with IL-6. No differences for BACE1 or sAPPb were observed in the cortex. ADAM10 activity was higher with IL-6 injection compared to saline in the hippocampus (p=0.018) and the cortex (p=0.055). A change in ADAM10 protein content was also observed with the ratio of pro to mature ADAM10 being higher in the cortex (p=0.033) of the IL-6 injected mice as compared to the saline injected control. Similarly, both pro (p=0.017) and mature (p=0.035) ADAM10 protein content were lower in the hippocampus of the IL-6 injected mice as compared to the saline injected control. Conclusions These results demonstrate that acute IL-6 modulates important enzymes involved in APP processing; pushing the cascade towards the non-amyloidogenic arm. This work further highlights regional brain differences in response to acute IL-6 and extends our knowledge of the function of acute IL-6 as a signalling molecule in the brain. Future work should determine if exercise induced IL-6 is in fact the main mediator of non-amyloidogenic APP processing.

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

A prevalent model of Alzheimer's disease (AD) pathogenesis postulates the generation of neurotoxic fragments derived from the amyloid precursor protein (APP) after its internalization to endocytic compartments. The molecular pathways that regulate APP internalization and intracellular trafficking in neurons are incompletely understood. Here, we report that 5xFAD mice, an animal model of AD, expressing signaling-deficient variants of the p75 neurotrophin receptor (p75NTR ) show greater neuroprotection from AD neuropathology than animals lacking this receptor. p75NTR knock-in mice lacking the death domain or transmembrane Cys259 showed lower levels of Aβ species, amyloid plaque burden, gliosis, mitochondrial stress, and neurite dystrophy than global knock-outs. Strikingly, long-term synaptic plasticity and memory, which are completely disrupted in 5xFAD mice, were fully recovered in the knock-in mice. Mechanistically, we found that p75NTR interacts with APP at the plasma membrane and regulates its internalization and intracellular trafficking in hippocampal neurons. Inactive p75NTR variants internalized considerably slower than wild-type p75NTR and showed increased association with the recycling pathway, thereby reducing APP internalization and co-localization with BACE1, the critical protease for generation of neurotoxic APP fragments, favoring non-amyloidogenic APP cleavage. These results reveal a novel pathway that directly and specifically regulates APP internalization, amyloidogenic processing, and disease progression, and suggest that inhibitors targeting the p75NTR transmembrane domain may be an effective therapeutic strategy in AD.

Centrally Active Multitarget Anti-Alzheimer Agents Derived from the Antioxidant Lead CR-6.

Oxidative stress is a major pathogenic factor in Alzheimer's disease, but it should not be tackled alone rather together with other key targets to derive effective treatments. The combination of the scaffold of the polar antioxidant lead 7-methoxy-2,2-dimethylchroman-6-ol (CR-6) with that of the lipophilic cholinesterase inhibitor 6-chlorotacrine results in compounds with favorable brain permeability and multiple activities in vitro (acetylcholinesterase, butyrylcholinesterase, β-site amyloid precursor protein (APP) cleaving enzyme-1 (BACE-1), and Aβ42 and tau aggregation inhibition). In in vivo studies on wild-type and APP/presenilin 1 (PS1) mice, two selected compounds were well tolerated and led to positive trends, albeit statistically nonsignificant in some cases, on memory performance, amyloid pathology (reduced amyloid burden and potentiated non-amyloidogenic APP processing), and oxidative stress (reduced cortical oxidized proteins and increased antioxidant enzymes superoxide dismutase 2 (SOD2), catalase, glutathione peroxidase 1 (GPX1), and heme oxygenase 1 (Hmox1) and transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2)). These compounds emerge as interesting brain-permeable multitarget compounds, with a potential as anti-Alzheimer agents beyond that of the original lead CR-6.

Phosphatidylinositol-4-phosphate 5-kinase type 1α attenuates Aβ production by promoting non-amyloidogenic processing of amyloid precursor protein.

Alzheimer's disease (AD) is characterized by a chronic decline in cognitive function and is pathologically typified by cerebral deposition of amyloid-β peptide (Aβ). The production of Aβ is mediated by sequential proteolysis of amyloid precursor protein (APP) by β- and γ-secretases, and has been implicated as the essential determinant of AD pathology. Previous studies have demonstrated that the level of phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] in the membrane may potentially modulate Aβ production. Given that PI(4,5)P2 is produced by type 1 phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks), we sought to determine whether the level of PIP5K type Iα (PIP5K1A) can affect production of Aβ by modulating the lipid composition of the membrane. Using a HEK-derived cell line that constitutively expresses yellow fluorescent protein-tagged APP (APP-YFP), we demonstrated that overexpression of PIP5K1A results in significant enhancement of non-amyloidogenic APP processing and a concomitant suppression of the amyloidogenic pathway, leading to a marked decrease in secreted Aβ. Consistently, cells overexpressing PIP5K1A exhibited a significant redistribution of APP-YFP from endosomal compartments to the cell surface. Our findings suggest that PIP5K1A may play a critical role in governing Aβ production by modulating membrane distribution of APP, and as such, the pathway may be a valuable therapeutic target for AD.

Amyloid precursor protein-mediated mitochondrial regulation and Alzheimer's disease.

Despite clear evidence of a neuroprotective physiological role of amyloid precursor protein (APP) and its non-amyloidogenic processing products, APP has been investigated mainly in animal and cellular models of amyloid pathology in the context of Alzheimer's disease. The rare familial mutations in APP and presenilin-1/2, which sometimes drive increased amyloid β (Aβ) production, may have unduly influenced Alzheimer's disease research. APP and its cleavage products play important roles in cellular and mitochondrial metabolism, but many studies focus solely on Aβ. Mitochondrial bioenergetic metabolism is essential for neuronal function, maintenance and survival, and multiple reports indicate mitochondrial abnormalities in patients with Alzheimer's disease. In this review, we focus on mitochondrial abnormalities reported in sporadic Alzheimer's disease patients and the role of full-length APP and its non-amyloidogenic fragments, particularly soluble APPα, on mitochondrial bioenergetic metabolism. We do not review the plethora of animal and in vitro studies using mutant APP/presenilin constructs or experiments using exogenous Aβ. In doing so, we aim to invigorate research and discussion around non-amyloidogenic APP processing products and the mechanisms linking mitochondria and complex neurodegenerative disorders such as sporadic Alzheimer's disease. LINKED ARTICLES: This article is part of a themed section on Therapeutics for Dementia and Alzheimer's Disease: New Directions for Precision Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.18/issuetoc.

Distinct invivo roles of secreted APP ectodomain variants APPsα and APPsβ in regulation of spine density, synaptic plasticity, and cognition.

Increasing evidence suggests that synaptic functions of the amyloid precursor protein (APP), which is key to Alzheimer pathogenesis, may be carried out by its secreted ectodomain (APPs). The specific roles of APPsα and APPsβ fragments, generated by non-amyloidogenic or amyloidogenic APP processing, respectively, remain however unclear. Here, we expressed APPsα or APPsβ in the adult brain of conditional double knockout mice (cDKO) lacking APP and the related APLP2. APPsα efficiently rescued deficits in spine density, synaptic plasticity (LTP and PPF), and spatial reference memory of cDKO mice. In contrast, APPsβ failed to show any detectable effects on synaptic plasticity and spine density. The C-terminal 16 amino acids of APPsα (lacking in APPsβ) proved sufficient to facilitate LTP in a mechanism that depends on functional nicotinic α7-nAChRs. Further, APPsα showed high-affinity, allosteric potentiation of heterologously expressed α7-nAChRs in oocytes. Collectively, we identified α7-nAChRs as a crucial physiological receptor specific for APPsα and show distinct invivo roles for APPsα versus APPsβ. This implies that reduced levels of APPsα that might occur during Alzheimer pathogenesis cannot be compensated by APPsβ.

Catalpol Inhibits Amyloid-β Generation Through Promoting α-Cleavage of APP in Swedish Mutant APP Overexpressed N2a Cells.

Amyloid-β (Aβ) peptides play a crucial role in the pathogenesis of Alzheimer’s disease (AD), due to its neurotoxicity. Thus, blocking Aβ generation and aggregation in the brain has been realized as an efficient way for the prevention of AD. The natural product catalpol, isolated from Rehmannia glutinosa, has shown neuroprotective activities through inhibiting soluble Aβ production, degrading Aβ peptide, and attenuating Aβ toxicity and neuroinflammatory responses. In the present study, we aimed to evaluate whether catalpol reduce Aβ generation associated with regulating amyloid precursor protein (APP) proteolytic processing. By using Swedish mutant APP overexpressed N2a (SweAPP N2a) cells treated with catalpol, we found that catalpol was not able to reduce the expression levels of β-secretase (BACE-1) and γ-secretase (PS1, APH-1, PEN-2 and Nicastrin). By contrast, catalpol had a significant promotion effect on the expression of α-secretase (ADAM10) and its proteolytic products, sAPPα and C83, suggesting that catalpol reduced the production of Aβ might be involved in non-amyloidogenic APP pathway. In addition, we confirmed that the extracellular signal-related kinase/cAMP-response element binding protein (ERK/CREB) signaling pathways were responsible for the up-regulation of ADAM10 in catalpol-treated SweAPP N2a cells. The present data, for the first time, have demonstrated that the effect of catalpol on the inhibiting Aβ generation might be closely related to α-cleavage of APP processing.

M344 promotes nonamyloidogenic amyloid precursor protein processing while normalizing Alzheimer’s disease genes and improving memory

Alzheimer's disease (AD) comprises multifactorial ailments for which current therapeutic strategies remain insufficient to broadly address the underlying pathophysiology. Epigenetic gene regulation relies upon multifactorial processes that regulate multiple gene and protein pathways, including those involved in AD. We therefore took an epigenetic approach where a single drug would simultaneously affect the expression of a number of defined AD-related targets. We show that the small-molecule histone deacetylase inhibitor M344 reduces beta-amyloid (Aβ), reduces tau Ser396 phosphorylation, and decreases both β-secretase (BACE) and APOEε4 gene expression. M344 increases the expression of AD-relevant genes: BDNF, α-secretase (ADAM10), MINT2, FE65, REST, SIRT1, BIN1, and ABCA7, among others. M344 increases sAPPα and CTFα APP metabolite production, both cleavage products of ADAM10, concordant with increased ADAM10 gene expression. M344 also increases levels of immature APP, supporting an effect on APP trafficking, concurrent with the observed increase in MINT2 and FE65, both shown to increase immature APP in the early secretory pathway. Chronic i.p. treatment of the triple transgenic (APPsw/PS1M146V/TauP301L) mice with M344, at doses as low as 3 mg/kg, significantly prevented cognitive decline evaluated by Y-maze spontaneous alternation, novel object recognition, and Barnes maze spatial memory tests. M344 displays short brain exposure, indicating that brief pulses of daily drug treatment may be sufficient for long-term efficacy. Together, these data show that M344 normalizes several disparate pathogenic pathways related to AD. M344 therefore serves as an example of how a multitargeting compound could be used to address the polygenic nature of multifactorial diseases.

Exercise and BDNF reduce Aβ production by enhancing α‐secretase processing of APP

Alzheimer's disease (AD) is an age-related neurodegenerative disorder characterized by aggregation of toxic forms of amyloid β peptide (Aβ). Treatment strategies have largely been focused on inhibiting the enzymes (β- and γ-secretases) that liberate Aβ from the amyloid precursor protein (APP). While evidence suggests that individuals who exercise regularly are at reduced risk for AD and studies of animal models demonstrate that running can ameliorate brain Aβ pathology and associated cognitive deficits, the underlying mechanisms are unknown. However, considerable evidence suggests that brain-derived neurotrophic factor (BDNF) mediates beneficial effects of exercise on neuroplasticity and cellular stress resistance. Here, we tested the hypothesis that BDNF promotes non-amyloidogenic APP processing. Using a transgenic mouse model of Alzheimer's disease and cultured human neural cells, we demonstrate that exercise and BDNF reduce production of toxic Aβ peptides through a mechanism involving enhanced α-secretase processing of APP. This anti-amyloidogenic APP processing involves subcellular redistribution of α-secretase and an increase in intracellular neuroprotective APP peptides capable of binding and inhibiting β-secretase. Moreover, our results suggest that BDNF's ability to promote neurite outgrowth is primarily exerted through pathways other than APP processing. Exercise and other factors that enhance BDNF signaling may therefore have both therapeutic and prophylactic value in the battle against AD. Read the Editorial Highlight for this article on page 191.

Nicotinamide mononucleotide inhibits JNK activation to reverse Alzheimer disease

Amyloid-β (Aβ) oligomers have been accepted as major neurotoxic agents in the therapy of Alzheimer’s disease (AD). It has been shown that the activity of nicotinamide adenine dinucleotide (NAD+) is related with the decline of Aβ toxicity in AD. Nicotinamide mononucleotide (NMN), the important precursor of NAD+, is produced during the reaction of nicotinamide phosphoribosyl transferase (Nampt). This study aimed to figure out the potential therapeutic effects of NMN and its underlying mechanisms in APPswe/PS1dE9 (AD-Tg) mice. We found that NMN gave rise to a substantial improvement in behavioral measures of cognitive impairments compared to control AD-Tg mice. In addition, NMN treatment significantly decreased β-amyloid production, amyloid plaque burden, synaptic loss, and inflammatory responses in transgenic animals. Mechanistically, NMN effectively controlled JNK activation. Furthermore, NMN potently progressed nonamyloidogenic amyloid precursor protein (APP) and suppressed amyloidogenic APP by mediating the expression of APP cleavage secretase in AD-Tg mice. Based on our findings, it was suggested that NMN substantially decreases multiple AD-associated pathological characteristically at least partially by the inhibition of JNK activation.

Icariside II Effectively Reduces Spatial Learning and Memory Impairments in Alzheimer's Disease Model Mice Targeting Beta-Amyloid Production.

Icariside II (ICS II) is a broad-spectrum anti-cancer natural compound extracted from Herba Epimedii Maxim. Recently, the role of ICS II has been investigated in central nervous system, especially have a neuroprotective effect in Alzheimer’s disease (AD). In this study, we attempted to investigate the effects of ICS II, on cognitive deficits and beta-amyloid (Aβ) production in APPswe/PS1dE9 (APP/PS1) double transgenic mice. It was found that chronic ICS II administrated not only effectively ameliorated cognitive function deficits, but also inhibited neuronal degeneration and reduced the formation of plaque burden. ICS II significantly suppressed Aβ production via promoting non-amyloidogenic APP cleavage process by up-regulating a disintegrin and metalloproteinase domain 10 (ADAM10) expression, inhibited amyloidogenic APP processing pathway by down-regulating amyloid precursor protein (APP) and β-site amyloid precursor protein cleavage enzyme 1 (BACE1) expression in APP/PS1 transgenic mice. Meanwhile, ICS II attenuated peroxisome proliferator-activated receptor-γ (PPARγ) degradation as well as inhibition of eukaryotic initiation factor α phosphorylation (p-eIF2α) and PKR endoplasmic reticulum regulating kinase phosphorylation (p-PERK). Moreover, phosphodiesterase type 5 inhibitors (PDE5-Is) have recently emerged as a possible therapeutic target for cognitive enhancement via inhibiting Aβ levels, and we also found that ICS II markedly decreased phosphodiesterase-5A (PDE5A) expression. In conclusion, the present study demonstrates that ICS II could attenuate spatial learning and memory impairments in APP/PS1 transgenic mice. This protection appears to be due to the increased ADAM10 expression and decreased expression of both APP and BACE1, resulting in inhibition of Aβ production in the hippocampus and cortex. Inhibition of PPARγ degradation and PERK/eIF2α phosphorylation are involved in the course, therefore suggesting that ICS II might be a promising potential compound for the treatment of AD.

Novel Insights into the Physiological Function of the APP (Gene) Family and Its Proteolytic Fragments in Synaptic Plasticity.

The amyloid precursor protein (APP) is well known to be involved in the pathophysiology of Alzheimer's disease (AD) via its cleavage product amyloid ß (Aß). However, the physiological role of APP, its various proteolytic products and the amyloid precursor-like proteins 1 and 2 (APLP1/2) are still not fully clarified. Interestingly, it has been shown that learning and memory processes represented by functional and structural changes at synapses are altered in different APP and APLP1/2 mouse mutants. In addition, APP and its fragments are implicated in regulating synaptic strength further reinforcing their modulatory role at the synapse. While APLP2 and APP are functionally redundant, the exclusively CNS expressed APLP1, might have individual roles within the synaptic network. The proteolytic product of non-amyloidogenic APP processing, APPsα, emerged as a neurotrophic peptide that facilitates long-term potentiation (LTP) and restores impairments occurring with age. Interestingly, the newly discovered η-secretase cleavage product, An-α acts in the opposite direction, namely decreasing LTP. In this review we summarize recent findings with emphasis on the physiological role of the APP gene family and its proteolytic products on synaptic function and plasticity, especially during processes of hippocampal LTP. Therefore, we focus on literature that provide electrophysiological data by using different mutant mouse strains either lacking full-length or parts of the APP proteins or that utilized secretase inhibitors as well as secreted APP fragments.

Rescue of Hypovitaminosis A Induces Non-Amyloidogenic Amyloid Precursor Protein (APP) Processing.

Retinoic acid, the bioactive metabolite of beta-carotene or vitamin A, plays a pleiotropic, multifunctional role in vertebrate development. Studies in rodents revealed that a diet deficient in vitamin A results in a complex neonatal syndrome (the VAD syndrome), manifested in many organs. In humans, the function of retinoic acid (RA) extends into adulthood, where it has important roles in fertility, vision, and suppression of neoplastic growth. In recent years, it has also been suggested that retinoic acid might potentially act as a therapeutically relevant drug in attenuating or even preventing neurodegenerative diseases such as Alzheimer's disease (AD). Here, we report that VAD leads to an increase in A-beta peptide levels while only minor effects were observed on expression levels of the amyloid precursor protein (APP) processing proteinases in wild type mice. In line with these findings, rescue of hypovitaminosis reduced A-beta amount to baseline and induced sApp-alpha secretion in combination with an increase of alpha-secretase Adam10. By comparing retinoic acid treatment starting from a full nutrition status and a "VAD" situation in human neuroblastoma cells, we show that while intensities of differential gene expression were higher in replenished cells, a large overlap in AD-related, regulated genes was observed. Our data suggest that hypovitaminosis A can contribute to onset or progression of AD by increasing synthesis of A-beta peptides and that several AD-related genes such as ADAM10 or BDNF are regulated by retinoic acid. We suggest that dietary supplementation with retinoic acid derivatives is likely to have a beneficial effect on AD-pathology in individuals with hypovitaminosis and patients with normal vitamin A status.

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.

Substance P and Alzheimer's Disease: Emerging Novel Roles.

Alzheimer`s disease (AD) is an irreversible neurodegenerative disease, clinically characterized by progressive impairments of memory and cognition. The hallmarks of AD are neurofibrillary tangles, mainly constituted by altered phosphorylated and truncated portions of tau protein, and the abnormal extracellular deposition of neurotoxic beta amyloid (Aβ) peptides, derived from the proteolytic processing of amyloid precursor protein (APP). According to the amyloid hypothesis, Aβ is considered to be linked to the selective neurodegeneration seen in AD. Recent evidence points to an increase in voltage-gated potassium (Kv) channel currents in the etiology of Aβ-induced neuronal apoptosis. Substance P (SP) is an 11-aa neuropeptide, member of the tachykinin family, broadly distributed in the Central Nervous System where it acts as a neurotransmitter, neuromodulator, and neurotrophic factor. This peptide may play an important role in neurodegenerative disorders, since reduced levels of SP were found in brain areas and spinal fluid of AD patients. In addition to its neuroprotective properties, it was recently demonstrated that SP is able to stimulate non-amyloidogenic APP processing, thereby reducing the possibility of generation of toxic Aβ peptides in the brain. Recent studies, using in vitro and in vivo models, have also shown that the neuroprotective role of SP against Aβ could be related to its ability of modulate Kv channel currents. In this review, we briefly summarized the current findings on the neurotrophic and neuroprotective effects of SP, providing information about its anti-amyloidogenic and anti-Aβ toxicity role.

The polarity protein Par3 regulates APP trafficking and processing through the endocytic adaptor protein Numb

The processing of amyloid precursor protein (APP) into β-amyloid peptide (Aβ) is a key step in the pathogenesis of Alzheimer’s disease (AD), and trafficking dysregulations of APP and its secretases contribute significantly to altered APP processing. Here we show that the cell polarity protein Par3 plays an important role in APP processing and trafficking. We found that the expression of full length Par3 is significantly decreased in AD patients. Overexpression of Par3 promotes non-amyloidogenic APP processing, while depletion of Par3 induces intracellular accumulation of Aβ. We further show that Par3 functions by regulating APP trafficking. Loss of Par3 decreases surface expression of APP by targeting APP to the late endosome/lysosome pathway. Finally, we show that the effects of Par3 are mediated through the endocytic adaptor protein Numb, and Par3 functions by interfering with the interaction between Numb and APP. Together, our studies show a novel role for Par3 in regulating APP processing and trafficking.

387. Gene Delivery of APOE2 Reduces Amyloid Pathology in Transgenic Mouse Models of Alzheimer's Disease

The deposition of amyloid β-peptides (Aβ), cleavage products of the amyloid precursor protein (APP) by β- and γ-secretases, in brain represents a pathological hallmark of Alzheimer's disease (AD). Apolipoprotein E (APOE) ε4 allele carriers have an increased risk to develop AD and an earlier age of onset, whereas carriers of the ε2 allele have reduced risk and a delayed age of onset. APOE is also a major determinant of brain Aβ and amyloid burden in humans and in several transgenic mouse models of AD (E4>E3>E2). We have previously reported that lentivirus-mediated intraparenchymal gene delivery of APOE2 significantly reduces brain Aβ levels and amyloid plaque burden in PDAPP mice. To extend these findings, we administered an rh.10 serotype adeno-associated viral vector expressing the gene (AAVrh.10-APOE2, 1.0X1010 viral genomes (vg)) directly into the hippocampus of 9-month-old PDAPP mice, a mouse model of AD-related amyloidosis. Eight weeks post-injection, AAVrh.10-APOE2 administration resulted in 5-6 times higher levels of APOE2 expression than targeted replacement (wild-type) mice and a marked decrease in both soluble (~33% reduction. P<0.05) and insoluble Aβ42 levels (~70% reduction. P<0.001) compared to control mice. Given the important role of APOE4 in AD risk and amyloid burden, we next assessed how gene delivery of APOE2 affects amyloid pathology in APP.PS1/TRE4 mice where brain Aβ/amyloid deposition is dependent on APOE4 expression. AAVrh.10-APOE2 (0.25X1010, 0.5X1010, or 1×1010 vg) was bilaterally administered into the hippocampus of 2.5-month-old APP.PS1/TRE4 mice. Eight weeks post-injection, there was a dose-dependent increase in APOE expression and a corresponding dose-dependent decrease in insoluble and soluble Aβ levels in the hippocampus of mice treated with AAVrh.10-APOE2, suggesting that overexpression of APOE2 effectively counteracts the detrimental effects of APOE4 on amyloid pathology. We also investigated the effects of AAVrh.10-APOE2 treatment on various proteins associated with Aβ production and clearance by Western analysis. No significant differences were observed in the relative hippocampal levels of APP, β-secretase, C99 (APP cleavage product of β-secretase), or C83 (a non-amyloidogenic APP cleavage product by α-secretase) between mice treated with AAVrh.10-APOE2 or a control vector, suggesting no effect on Aβ production. By contrast, the levels of insulin-degrading enzyme (IDE, an Aβ-degrading enzyme) and ATG5/LC3 (the signaling pathway responsible for autophagy) were significantly (P<0.001 and P<0.05 respectively) elevated in the hippocampus of AAVrh.10-APOE2-treated mice. Taken together, the data demonstrates that AAVrh.10-mediated delivery of APOE2 effectively reduces Aβ pathology in the hippocampus of APP mutant mice expressing either murine Apoe or APOE4. The latter may be due to an enhancement of Aβ metabolism or clearance. Gene delivery of APOE2 may represent a potential therapeutic strategy for treating or preventing AD.

Inhibition of c-Jun N-terminal kinase activation reverses Alzheimer disease phenotypes in APPswe/PS1dE9 mice.

Growing evidence indicates that the activation of c-Jun N-terminal kinase (JNK) is implicated in the multiple major pathological features of Alzheimer disease (AD). However, whether specific inhibition of JNK activation could prevent disease progression in adult transgenic AD models at moderate stage remains unknown. Here we first investigated the potential disease-modifying therapeutic effect of systemic administration of SP600125, a small-molecule JNK-specific inhibitor, in middle-aged APPswe/PS1dE9 mice. Using behavioral, histological, and biochemical methods, outcomes of SP600125 treatment on neuropathology and cognitive deficits were studied in APPswe/PS1dE9 mice. Compared with vehicle-treated APPswe/PS1dE9 mice, chronic treatment of SP600125 for 12 weeks potently inhibited JNK activation, which resulted in a marked improvement of behavioral measures of cognitive deficits and a dramatic reduction in amyloid plaque burden, β-amyloid production, tau hyperphosphorylation, inflammatory responses, and synaptic loss in these transgenic animals. In particular, we found that SP600125 treatment strongly promoted nonamyloidogenic amyloid precursor protein (APP) processing and inhibited amyloidogenic APP processing via regulating APP-cleavage secretase expression (ie, ADAM10, BACE1, and PS1) in APPswe/PS1dE9 mice. Our findings demonstrate that chronic SP600125 treatment is powerfully effective in slowing down disease progression by markedly reducing multiple pathological features and ameliorating cognitive deficits associated with AD. This study highlights the concept that active JNK actually contributes to the development of the disease, and provides critical preclinical evidence that specific inhibition of JNK activation by SP600125 treatment may be a novel promising disease-modifying therapeutic strategy for the treatment of AD.

Synthesis and biological evaluation of isoxazoline derivatives as potent M₁ muscarinic acetylcholine receptor agonists.

A series of azacyclic compounds substituted with isoxazole and 5-substituted isoxazolines were synthesized as acyclic modifications of the oxime class M1 mACh receptor agonist. Among them, 3-(tetrahydropyrin-3-yl)-5-(2-pyrrolodin-1-yl)isoxazoline compound 4f displayed potent and selective M1 mACh receptor agonist activity in the functional calcium mobilization assay (EC50=31 nM). Introduction of 2-pyrrolidinone and 3-tetrahydropyridine groups are pivotal to the high potency. Moreover, 4f was found to facilitate non-amyloidogenic amyloid precursor protein (APP) processing by significantly increasing ERK1/2 phosphorylation and sAPPα secretion, known disease-modifying effects related to M1 mAChR agonists in Alzheimer's disease (AD).