α‐secretase mediated conversion of the amyloid precursor protein derived membrane stub C99 to C83 limits Aβ generation

The Swedish mutation within the amyloid precursor protein (APP) causes early‐onset Alzheimer’s disease due to increased cleavage of APP by BACE1. While β‐secretase shedding of Swedish APP (APPswe) largely results from an activity localized in the late secretory pathway, cleavage of wild‐type APP occurs mainly in endocytic compartments. However, we show that liberation of Aβ from APPswe is still dependent on functional internalization from the cell surface. Inspite the unchanged overall β‐secretase cleaved soluble APP released from APP_swe secretion, mutations of the APPswe internalization motif strongly reduced C99 levels and substantially decreased Aβ secretion. We point out that α‐secretase activity‐mediated conversion of C99 to C83 is the main cause of this Aβ reduction. Furthermore, we demonstrate that α‐secretase cleavage of C99 even contributes to the reduction of Aβ secretion of internalization deficient wild‐type APP. Therefore, inhibition of α‐secretase cleavage increased Aβ secretion through diminished conversion of C99 to C83 in APP695, APP695swe or C99 expressing cells.     

RTN4B‐mediated suppression of Sirtuin 2 activity ameliorates β‐amyloid pathology and cognitive impairment in Alzheimer's disease mouse model

Sirtuin 2 (SIRT2) is an NAD+ dependent deacetylase that is the most abundant sirtuin protein in the brain. Accumulating evidence revealed the role of SIRT2 in a wide range of biological processes and age‐related diseases. However, the pivotal mechanism of SIRT2 played in Alzheimer's disease (AD) remains unknown. Here, we report that pharmacological inactivation of SIRT2 has a beneficial effect in AD. The deacetylase inhibitor of SIRT2 rescued the cognitive impairment in amyloid precursor protein/presenilin 1 transgenic mouse (APP/PS1 mouse), and the BACE1 cleavage was weakened to reduce the β‐amyloid (Aβ) production in the hippocampus. Moreover, we firstly identified that Reticulon 4B (RTN4B) played a crucial role between SIRT2/BACE1 regulation in AD. RTN4B, as a deacetylation substrate for SIRT2, the deacetylation by SIRT2 drived the ubiquitination and degradation of RTN4B and then the disturbed RTN4B interacted with and influenced the expression of BACE1. When we overexpressed RTN4B in neurons of the hippocampus in the AD mouse model, the abnormal Aβ accumulation and cognitive impairment were ameliorated, consistent with the results of SIRT2 inhibition in vivo. Moreover, we showed that the regulatory effect of SIRT2 on BACE1 is dependent on RTN4B. When RTN4B was knocked down, the effects of SIRT2 inhibition on the BACE1 level, Aβ pathology, and AD‐liked behaviors were also blocked. Collectively, we provide evidence that SIRT2 may be a potential target for AD; the new found SIRT2/RTN4B/BACE1 pathological pathway is one of the critical mechanisms for the improvement of SIRT2 on AD.     

Intracellular Itinerary of Internalised β‐Secretase,BACE1, and Its Potential Impact on β‐Amyloid Peptide Biogenesis

β‐Secretase (BACE1) cleavage of the amyloid precursor protein (APP) represents the initial step in the formation of the Alzheimer's disease associated amyloidogenic Aβ peptide. Substantive evidence indicates that APP processing by BACE1 is dependent on intracellular sorting of this enzyme. Nonetheless, knowledge of the intracellular trafficking pathway of internalised BACE1 remains in doubt. Here we show that cell surface BACE1 is rapidly internalised by the AP2/clathrin dependent pathway in transfected cells and traffics to early endosomes and Rab11‐positive, juxtanuclear recycling endosomes, with very little transported to the TGN as has been previously suggested. Moreover, BACE1 is predominantly localised to the early and recycling endosome compartments in different cell types, including neuronal cells. In contrast, the majority of internalised wild‐type APP traffics to late endosomes/lysosomes. To explore the relevance of the itinerary of BACE1 on APP processing, we generated a BACE1 chimera containing the cytoplasmic tail of TGN38 (BACE/TGN38), which cycles between the cell surface and TGN in an AP2‐dependent manner. Wild‐type BACE1 is less efficient in Aβ production than the BACE/TGN38 chimera, highlighting the relevance of the itinerary of BACE1 on APP processing. Overall the data suggests that internalised BACE1 and APP diverge at early endosomes and that Aβ biogenesis is regulated in part by the recycling itinerary of BACE1.     

SCFFbx2‐E3‐ligase‐mediated degradation of BACE1 attenuates Alzheimer’s disease amyloidosis and improves synaptic function

BACE1 (β‐secretase) plays a central role in the β‐amyloidogenesis of Alzheimer’s disease (AD). The ubiquitin–proteasome system, a major intracellular protein quality control system, has been implicated recently in BACE1 metabolism. We report that the SCF^Fbx2‐E3 ligase is involved in the binding and ubiquitination of BACE1 via its Trp 280 residue of F‐box‐associated domain. Physiologically, we found that Fbx2 was expressed in various intracellular organelles in brain neurons and that BACE1 is colocalized with Fbx2 and the amyloid precursor protein (APP), mainly at the early endosome and endoplasmic reticulum. The former are believed to be the major intracellular compartments where the APP is cleaved by BACE1 and β‐amyloid is produced. Importantly, we found that overexpression of Fbx2 in the primary cortical and hippocampal neurons derived from Tg2576 transgenic mice significantly promoted BACE1 degradation and reduced β‐amyloid production. In the search for specific endogenous modulators of Fbx2 expression, we found that PPARγ coactivator‐1α (PGC‐1α) was capable of promoting the degradation of BACE1 through a mechanism involving Fbx2 gene expression. Interestingly, we found that the expression of both Fbx2 and PGC‐1α was significantly decreased in the brains of aging Tg2576 mice. Our in vivo studies using a mouse model of AD revealed that exogenous adenoviral Fbx2 expression in the brain significantly decreased BACE1 protein levels and activity, coincidentally reducing β‐amyloid levels and rescuing synaptic deficits. Our study is the first to suggest that promoting Fbx2 in the brain may represent a novel strategy for the treatment of AD.     

Rho‐associated coiled‐coil kinase 1 activation mediates amyloid precursor protein site‐specific Ser655 phosphorylation and triggers amyloid pathology

Rho‐associated coiled‐coil kinase 1 (ROCK1) is proposed to be implicated in Aβ suppression; however, the role for ROCK1 in amyloidogenic metabolism of amyloid precursor protein (APP) to produce Aβ was unknown. In the present study, we showed that ROCK1 kinase activity and its APP binding were enhanced in AD brain, resulting in increased β‐secretase cleavage of APP. Furthermore, we firstly confirmed that APP served as a substrate for ROCK1 and its major phosphorylation site was located at Ser655. The increased level of APP Ser655 phosphorylation was observed in the brain of APP/PS1 mice and AD patients compared to controls. Moreover, blockade of APP Ser655 phosphorylation, or inhibition of ROCK1 activity with either shRNA knockdown or Y‐27632, ameliorated amyloid pathology and improved learning and memory in APP/PS1 mice. These findings suggest that activated ROCK1 targets APP Ser655 phosphorylation, which promotes amyloid processing and pathology. Inhibition of ROCK1 could be a potential therapeutic approach for AD.     

Specificity of presenilin‐1‐ and presenilin‐2‐dependent γ‐secretases towards substrate processing

The two presenilin‐1 (PS1) and presenilin‐2 (PS2) homologs are the catalytic core of the γ‐secretase complex, which has a major role in cell fate decision and Alzheimer's disease (AD) progression. Understanding the precise contribution of PS1‐ and PS2‐dependent γ‐secretases to the production of β‐amyloid peptide (Aβ) from amyloid precursor protein (APP) remains an important challenge to design molecules efficiently modulating Aβ release without affecting the processing of other γ‐secretase substrates. To that end, we studied PS1‐ and PS2‐dependent substrate processing in murine cells lacking presenilins (PSs) (PS1KO, PS2KO or PS1‐PS2 double‐KO noted PSdKO) or stably re‐expressing human PS1 or PS2 in an endogenous PS‐null (PSdKO) background. We characterized the processing of APP and Notch on both endogenous and exogenous substrates, and we investigated the effect of pharmacological inhibitors targeting the PSs activity (DAPT and L‐685,458). We found that murine PS1 γ‐secretase plays a predominant role in APP and Notch processing when compared to murine PS2 γ‐secretase. The inhibitors blocked more efficiently murine PS2‐ than murine PS1‐dependent processing. Human PSs, especially human PS1, expression in a PS‐null background efficiently restored APP and Notch processing. Strikingly, and contrary to the results obtained on murine PSs, pharmacological inhibitors appear to preferentially target human PS1‐ than human PS2‐dependent γ‐secretase activity.     

Activation of PKA/SIRT1 signaling pathway by photobiomodulation therapy reduces Aβ levels in Alzheimer's disease models

A hallmark of Alzheimer's disease (AD) is the accumulation of amyloid‐β (Aβ), which correlates significantly with progressive cognitive deficits. Although photobiomodulation therapy (PBMT), as a novel noninvasive physiotherapy strategy, has been proposed to improve neuronal survival, decrease neuron loss, ameliorate dendritic atrophy, and provide overall AD improvement, it remains unknown whether and how this neuroprotective process affects Aβ levels. Here, we report that PBMT reduced Aβ production and plaque formation by shifting amyloid precursor protein (APP) processing toward the nonamyloidogenic pathway, thereby improving memory and cognitive ability in a mouse model of AD. More importantly, a pivotal protein, SIRT1, was involved in this process by specifically up‐regulating ADAM10 and down‐regulating BACE1, which is dependent on the cAMP/PKA pathway in APP/PS1 primary neurons and SH‐SY5Y cells stably expressing human APP Swedish mutation (APPswe). We further found that the activity of the mitochondrial photoacceptor cytochrome c oxidase (CcO) was responsible for PBMT‐induced activation of PKA and SIRT1. Together, our research suggests that PBMT as a viable therapeutic strategy has great potential value in improving cognitive ability and combatting AD.     

The Swedish APP mutation alters the effect of genetically reduced BACE1 expression on the APP processing

Inhibition of β-secretase (BACE1) is a key therapeutic approach in Alzheimer's disease (AD), as BACE1 initiates amyloid-β (Aβ) cleavage from the β-amyloid precursor protein (APP). As Aβ reductions in mice lacking one BACE1 allele diverged considerably between studies we investigated the effect of BACE1 knock-out in more detail. With both BACE1 alleles the Swedish mutation (APP23 mice) increased APP processing and shifted it towards the β-secretase pathway as compared with non-mutated APP expressed at a similar level (APP51/16 mice). This effect was much smaller then observed in cell culture. An about 50% decrease in BACE1 enzyme activity resulted in a sub-proportional Aβ reduction with the Swedish mutation (-20%) and even less for non-mutated APP (-16%). In wild-type mice, the Aβ reduction may be even further diminished. Other metabolites of the β-secretase pathway decreased accordingly while the alternative α-secretase pathway increased. Complete BACE1 deletion strongly enhanced these changes. The remaining Aβ signal also described by others can be explained by assay cross-reactivity with other APP metabolites supporting BACE1 as the major β-secretase. Our data indicate that BACE1 is in excess over APP at the cleavage site(s). Alterations in APP expression or substrate properties, therefore, quantitatively change its cleavage and Aβ generation.     

Mitofusin‐2 knockdown increases ER–mitochondria contact and decreases amyloid β‐peptide production

Mitochondria are physically and biochemically in contact with other organelles including the endoplasmic reticulum (ER). Such contacts are formed between mitochondria‐associated ER membranes (MAM), specialized subregions of ER, and the outer mitochondrial membrane (OMM). We have previously shown increased expression of MAM‐associated proteins and enhanced ER to mitochondria Ca²⁺ transfer from ER to mitochondria in Alzheimer's disease (AD) and amyloid β‐peptide (Aβ)‐related neuronal models. Here, we report that siRNA knockdown of mitofusin‐2 (Mfn2), a protein that is involved in the tethering of ER and mitochondria, leads to increased contact between the two organelles. Cells depleted in Mfn2 showed increased Ca²⁺ transfer from ER to mitchondria and longer stretches of ER forming contacts with OMM. Interestingly, increased contact resulted in decreased concentrations of intra‐ and extracellular Aβ₄₀ and Aβ₄₂. Analysis of γ‐secretase protein expression, maturation and activity revealed that the low Aβ concentrations were a result of impaired γ‐secretase complex function. Amyloid‐β precursor protein (APP), β‐site APP‐cleaving enzyme 1 and neprilysin expression as well as neprilysin activity were not affected by Mfn2 siRNA treatment. In summary, our data shows that modulation of ER–mitochondria contact affects γ‐secretase activity and Aβ generation. Increased ER–mitochondria contact results in lower γ‐secretase activity suggesting a new mechanism by which Aβ generation can be controlled.     

Simvastatin abolishes nitric oxide‐ and reactive oxygen species‐induced cyclooxygenase‐2 expression by blocking the nuclear factor κB pathway in rabbit articular chondrocytes

Nitric oxide (NO) and reactive oxygen species (ROS) have been shown to be linked with numerous diseases, including osteoarthritis (OA). Our study aimed to examine the effect of simvastatin on NO‐ or ROS‐induced cyclooxygenase‐2 (COX‐2) expression in OA. Simvastatin has attracted considerable attention since the discovery of its pharmacological effects on different pathogenic processes, including inflammation. Here, we report that simvastatin treatment blocked sodium nitroprusside (SNP)‐ and interleukin 1 beta (IL‐1β)‐induced COX‐2 production. In addition, simvastatin attenuated SNP‐induced NO production and IL‐1β‐induced ROS generation. Treatment with simvastatin prevented SNP‐ and IL‐1β‐induced nuclear factor kappa B (NF‐κB) activity. Inhibiting NO production and ROS generation using N‐acetylcysteine (NAC) and NG‐monomethyl‐ l ‐arginine ( l ‐NMMA), respectively, accelerated the influence of simvastatin on NF‐κB activity. In addition, NAC blocked SNP and simvastatin‐mediated COX‐2 production and NF‐κB activity but did not alter IL‐1β and simvastatin‐mediated COX‐2 expression. l ‐NMMA treatment also abolished IL‐1β‐mediated COX‐2 expression and NF‐κB activation, whereas SNP and simvastatin‐mediated COX‐2 expression were not altered compared with the levels in the SNP and simvastatin‐treated cells. Our findings suggested that simvastatin blocks COX‐2 expression by inhibiting SNP‐induced NO production and IL‐1β‐induced ROS generation by blocking the NF‐κB pathway.     

The stress response neuropeptide CRF increases amyloid‐β production by regulating γ‐secretase activity

The biological underpinnings linking stress to Alzheimer's disease (AD) risk are poorly understood. We investigated how corticotrophin releasing factor (CRF), a critical stress response mediator, influences amyloid‐β (Aβ) production. In cells, CRF treatment increases Aβ production and triggers CRF receptor 1 (CRFR1) and γ‐secretase internalization. Co‐immunoprecipitation studies establish that γ‐secretase associates with CRFR1; this is mediated by β‐arrestin binding motifs. Additionally, CRFR1 and γ‐secretase co‐localize in lipid raft fractions, with increased γ‐secretase accumulation upon CRF treatment. CRF treatment also increases γ‐secretase activity in vitro, revealing a second, receptor‐independent mechanism of action. CRF is the first endogenous neuropeptide that can be shown to directly modulate γ‐secretase activity. Unexpectedly, CRFR1 antagonists also increased Aβ. These data collectively link CRF to increased Aβ through γ‐secretase and provide mechanistic insight into how stress may increase AD risk. They also suggest that direct targeting of CRF might be necessary to effectively modulate this pathway for therapeutic benefit in AD, as CRFR1 antagonists increase Aβ and in some cases preferentially increase Aβ42 via complex effects on γ‐secretase.     

Prostaglandin I2 upregulates the expression of anterior pharynx‐defective‐1α and anterior pharynx‐defective‐1β in amyloid precursor protein/presenilin 1 transgenic mice

Cyclooxygenase‐2 (COX‐2) has been recently identified to be involved in the pathogenesis of Alzheimer's disease (AD). Yet, the role of an important COX‐2 metabolic product, prostaglandin (PG) I₂, in the pathogenesis of AD remains unknown. Using human‐ and mouse‐derived neuronal cells as well as amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice as model systems, we elucidated the mechanism of anterior pharynx‐defective (APH)‐1α and pharynx‐defective‐1β induction. In particular, we found that PGI₂ production increased during the course of AD development. Then, PGI₂ accumulation in neuronal cells activates PKA/CREB and JNK/c‐Jun signaling pathways by phosphorylation, which results in APH‐1α/1β expression. As PGI₂ is an important metabolic by‐product of COX‐2, its suppression by NS398 treatment decreases the expression of APH‐1α/1β in neuronal cells and APP/PS1 mice. More importantly, β‐amyloid protein (Aβ) oligomers in the cerebrospinal fluid (CSF) of APP/PS1 mice are critical for stimulating the expression of APH‐1α/1β, which was blocked by NS398 incubation. Finally, the induction of APH‐1α/1β was confirmed in the brains of patients with AD. Thus, these findings not only provide novel insights into the mechanism of PGI₂‐induced AD progression but also are instrumental for improving clinical therapies to combat AD.     

Aggravation of Alzheimer's disease due to the COX‐2‐mediated reciprocal regulation of IL‐1β and Aβ between glial and neuron cells

Alzheimer's disease (AD) is the most common form of dementia and displays the characteristics of chronic neurodegenerative disorders; amyloid plaques (AP) that contain amyloid β‐protein (Aβ) accumulate in AD, which is also characterized by tau phosphorylation. Epidemiological evidence has demonstrated that long‐term treatment with nonsteroidal anti‐inflammatory drugs (NSAIDs) markedly reduces the risk of AD by inhibiting the expression of cyclooxygenase 2 (COX‐2). Although the levels of COX‐2 and its metabolic product prostaglandin (PG)E₂ are elevated in the brain of AD patients, the mechanisms for the development of AD remain unknown. Using human‐ or mouse‐derived glioblastoma and neuroblastoma cell lines as model systems, we delineated the signaling pathways by which COX‐2 mediates the reciprocal regulation of interleukin‐1β (IL‐1β) and Aβ between glial and neuron cells. In glioblastoma cells, COX‐2 regulates the synthesis of IL‐1β in a PGE₂‐dependent manner. Moreover, COX‐2‐derived PGE₂ signals the activation of the PI3‐K/AKT and PKA/CREB pathways via cyclic AMP; these pathways transactivate the NF‐κB p65 subunit via phosphorylation at Ser 536 and Ser 276, leading to IL‐1β synthesis. The secretion of IL‐1β from glioblastoma cells in turn stimulates the expression of COX‐2 in human or mouse neuroblastoma cells. Similar regulatory mechanisms were found for the COX‐2 regulation of BACE‐1 expression in neuroblastoma cells. More importantly, Aβ deposition mediated the inflammatory response of glial cells via inducing the expression of COX‐2 in glioblastoma cells. These findings not only provide new insights into the mechanisms of COX‐2‐induced AD but also initially define the therapeutic targets of AD.