Intracellular Amyloid Precursor Protein Research Articles

Inhibition of Autophagy Alters Intracellular Transport ofAPP Resulting in Increased APP Processing.

Alzheimer's disease (AD) pathology is characterized by amyloid beta (Aβ) plaques and dysfunctional autophagy. Aβ is generated by sequential proteolytic cleavage of amyloid precursor protein (APP), and the site of intracellular APP processing is highly debated, which may include autophagosomes. Here, we investigated the involvement of autophagy, including the role of ATG9 in APP intracellular trafficking and processing by applying the RUSH system, which allows studying the transport of fluorescently labeled mCherry-APP-EGFP in a systematic way, starting from the endoplasmic reticulum. HeLa cells, expressing the RUSH mCherry-APP-EGFP system, were investigated by live cell imaging, immunofluorescence, and Western blot. We found that mCherry-APP-EGFP passed through the Golgi faster in ATG9 knockout cells. Furthermore, ATG9 deletion shifted mCherry-APP-EGFP from early endosomes and lysosomes toward the plasma membrane concomitant with reduced endocytosis. Importantly, this alteration in mCherry-APP-EGFP transport resulted in increased secreted mCherry-soluble APP and C-terminal fragment-EGFP. These effects were also phenocopied by pharmacological inhibition of ULK1, indicating that autophagy is regulating the intracellular trafficking and processing of APP. These findings contribute to the understanding of the role of autophagy in APP metabolism and could potentially have implications for new therapeutic approaches for AD.

Early impairment of cortical circuit plasticity and connectivity in the 5XFAD Alzheimer’s disease mouse model

Genetic risk factors for neurodegenerative disorders, such as Alzheimer’s disease (AD), are expressed throughout the life span. How these risk factors affect early brain development and function remain largely unclear. Analysis of animal models with high constructive validity for AD, such as the 5xFAD mouse model, may provide insights on potential early neurodevelopmental effects that impinge on adult brain function and age-dependent degeneration. The 5XFAD mouse model over-expresses human amyloid precursor protein (APP) and presenilin 1 (PS1) harboring five familial AD mutations. It is unclear how the expression of these mutant proteins affects early developing brain circuits. We found that the prefrontal cortex (PFC) layer 5 (L5) neurons in 5XFAD mice exhibit transgenic APP overloading at an early post-weaning age. Impaired synaptic plasticity (long-term potentiation, LTP) was seen at 6–8 weeks age in L5 PFC circuit, which was correlated with increased intracellular APP. APP overloading was also seen in L5 pyramidal neurons in the primary visual cortex (V1) during the critical period of plasticity (4–5 weeks age). Whole-cell patch clamp recording in V1 brain slices revealed reduced intrinsic excitability of L5 neurons in 5XFAD mice, along with decreased spontaneous miniature excitatory and inhibitory inputs. Functional circuit mapping using laser scanning photostimulation (LSPS) combined with glutamate uncaging uncovered reduced excitatory synaptic connectivity onto L5 neurons in V1, and a more pronounced reduction in inhibitory connectivity, indicative of altered excitation and inhibition during VC critical period. Lastly, in vivo single-unit recording in V1 confirmed that monocular visual deprivation-induced ocular dominance plasticity during critical period was impaired in 5XFAD mice. Our study reveals plasticity deficits across multiple cortical regions and indicates altered early cortical circuit developmental trajectory as a result of mutant APP/PS1 over-expression.

Inhibition of 37/67kDa Laminin-1 Receptor Restores APP Maturation and Reduces Amyloid-β in Human Skin Fibroblasts from Familial Alzheimer's Disease.

Alzheimer’s disease (AD) is a fatal neurodegenerative disorder caused by protein misfolding and aggregation, affecting brain function and causing dementia. Amyloid beta (Aβ), a peptide deriving from amyloid precursor protein (APP) cleavage by-and γ-secretases, is considered a pathological hallmark of AD. Our previous study, together with several lines of evidence, identified a strict link between APP, Aβ and 37/67kDa laminin receptor (LR), finding the possibility to regulate intracellular APP localization and maturation through modulation of the receptor. Here, we report that in fibroblasts from familial AD (fAD), APP was prevalently expressed as an immature isoform and accumulated preferentially in the transferrin-positive recycling compartment rather than in the Golgi apparatus. Moreover, besides the altered mitochondrial network exhibited by fAD patient cells, the levels of pAkt and pGSK3 were reduced in respect to healthy control fibroblasts and were accompanied by an increased amount of secreted Aβ in conditioned medium from cell cultures. Interestingly, these features were reversed by inhibition of 37/67kDa LR by NSC47924 a small molecule that was able to rescue the “typical” APP localization in the Golgi apparatus, with consequences on the Aβ level and mitochondrial network. Altogether, these findings suggest that 37/67kDa LR modulation may represent a useful tool to control APP trafficking and Aβ levels with implications in Alzheimer’s disease.

Reduction of AHI1 in the serum of Taiwanese with probable Alzheimer’s disease

ObjectiveThe development of blood-based biomarkers for early diagnosis and treatment of Alzheimer’s disease (AD) is desirable. In AD model mouse brain and neuronal cells, Abelson helper integration site-1 (AHI1) protein is reduced. AHI1 facilitates intracellular amyloid precursor protein (APP) translocation to inhibit amyloidogenic pathology of AD, and thus may be an AD biomarker. MethodsThis study was conducted among 32 AD patients and 54 healthy control (HC) subjects. AHI1-related protein levels from initially collected serum samples in each group were screened using Western blotting. The protein concentrations of AHI1 and amyloid-β (Aβ), peptide(s) derived from APP, from all serum samples were analyzed using ELISA. ResultsIn AD serum, AHI1 and a large truncated C-terminal APP fragment were significantly reduced. The average concentrations of serum AHI1 and Aβ in AD were significantly lower than those in HC. Notably, AHI1 concentration in HC serum was decreased in an age-dependent manner, while it was consistently low in AD serum and had no correlation with Aβ or mini-mental state examination score. The receiver operating characteristic analysis on all subjects demonstrated an area under curve (AUC) value of 0.7 for AHI1 on AD diagnosis, while the AUC increased to 0.82 on the subjects younger than 77 years old, suggesting a good diagnostic performance of serum AHI1 for AD especially at relatively young age. ConclusionAn early event of AHI1 reduction in the body of AD patients was observed. Serum AHI1 may be valuable for early diagnosis of AD.

Azure B affects amyloid precursor protein metabolism in PS70 cells

Alzheimer's disease (AD), the most common form of dementia, is characterized by abundant deposition of amyloid-β (Aβ) peptide that is the result of sequential cleavage of amyloid precursor protein (APP) by β-secretase and γ-secretase. Several studies have documented that inhibition of Aβ peptide synthesis or facilitating its degradation is one of the attractive therapeutic strategies in AD. Methylene blue (MethB), which has recently been investigated in Phase II clinical trials, is a prominent inhibitor in reducing Aβ oligomers. Herein, we wonder whether the mitigating effects of MethB on amyloid metabolism are related to the activity of its major metabolite, azure B. The goal of this study was to investigate the effects of azure B, which is also a cholinesterase inhibitor, on APP processing by using Chinese hamster ovary cells stably expressing human wild-type APP and presenilin 1 (PS70). Azure B significantly decreased the levels of secreted APPα (sAPPα) and Aβ40/42 in culture medium with a dose-dependent manner. A significant decrease was also observed in the levels of intracellular APP without affecting the cell viability. In parallel with the decrease of APP and APP metabolites, the activity of β-secretase 1 (BACE1) was significantly attenuated compared to control. Overall, our results show that azure B has a large contribution for the pharmacological profile of MethB in APP metabolism.

Coconut oil decreases expression of amyloid precursor protein (APP) and secretion of amyloid peptides through inhibition of ADP-ribosylation factor 1 (ARF1)

Alzheimer’s disease (AD), affecting 5.3 million people in the U.S., impairs portions of the brain controlling memories. In humans, mutations in the amyloid precursor protein (APP) gene has been implicated in increased plaque formation, which can block the communication between nerve cells, decrease dendritic formation and increase cell death, and promote neuroinflammation. As coconut oil has been suggested to alleviate the symptoms in AD patients, we examined the impact of coconut oil on APP expression and secretion of amyloid peptides in N2a cells expressing the human APP gene (N2a/APP695). We found that coconut oil treatment decreased APP expression in N2a cells and reduced the secretion of amyloid peptides Aβ40 and Aβ42. Moreover, coconut oil treatment promoted differentiation of N2a cells. Our data suggest that ADP-Ribosylation Factor 1 (ARF1) may contribute to the effects of coconut oil on APP expression and secretion of Aβ. A high ARF1 expression was also detected in the primary neuronal cells from the mice overexpressing the Swedish mutant APP. Immunostaining results revealed that APP is co-localized with ARF1 in the Golgi apparatus and this interaction is impaired after coconut oil treatment. Furthermore, knockdown of ARF-1 using siRNA decreased secretion of amyloid peptides, confirming the impact of ARF1 on the secretion of amyloid peptides. ConclusionThese results suggest that coconut oil decreases intracellular ARF1 expression, thereby resulting in an inhibition of APP and amyloid β secretion. This study reveals a novel mechanism for intracellular APP processing in neuronal cells.

Trafficking in Alzheimer's Disease: Modulation of APP Transport and Processing by the Transmembrane Proteins LRP1, SorLA, SorCS1c, Sortilin, and Calsyntenin.

The amyloid precursor protein (APP), one key player in Alzheimer's disease (AD), is extensively processed by different proteases. This leads to the generation of diverging fragments including the amyloid β (Aβ) peptide, which accumulates in brains of AD patients. Subcellular trafficking of APP is an important aspect for its proteolytic conversion, since the various secretases which cleave APP are located in different cellular compartments. As a consequence, altered subcellular targeting of APP is thought to directly affect the degree to which Aβ is generated. The mechanisms underlying intracellular APP transport are critical to understand AD pathogenesis and can serve as a target for future pharmacological interventions. In the recent years, a number of APP interacting proteins were identified which are implicated in sorting of APP, thereby influencing APP processing at different angles of the secretory or endocytic pathway. This review provides an update on the proteolytic processing of APP and the interplay of the transmembrane proteins low-density lipoprotein receptor-related protein 1, sortilin-receptor with A-type repeats, SorCS1c, sortilin, and calsyntenin. We discuss the specific interactions with APP, the capacity to modulate the intracellular itinerary and the proteolytic conversion of APP, a possible involvement in the clearance of Aβ, and the implications of these transmembrane proteins in AD and other neurodegenerative diseases.

Regulatory effects of simvastatin and apoJ on APP processing and amyloid-β clearance in blood-brain barrier endothelial cells

Amyloid-β peptides (Aβ) accumulate in cerebral capillaries indicating a central role of the blood-brain barrier (BBB) in the pathogenesis of Alzheimer's disease (AD). Although a relationship between apolipoprotein-, cholesterol- and Aβ metabolism is evident, the interconnecting mechanisms operating in brain capillary endothelial cells (BCEC) are poorly understood. ApoJ (clusterin) is present in HDL that regulates cholesterol metabolism which is disturbed in AD. ApoJ levels are increased in AD brains and in plasma of cerebral amyloid angiopathy (CAA) patients. ApoJ may bind, prevent fibrillization, and enhance clearance of Aβ. We here define a connection of apoJ and cellular cholesterol homeostasis in amyloid precursor protein (APP) processing/Aβ metabolism at the BBB. Silencing of apoJ in primary porcine (p)BCEC decreased intracellular APP and Aβ oligomer levels while the addition of purified apoJ to pBCEC increased intracellular APP and enhanced Aβ clearance across the pBCEC monolayer. Treatment of pBCEC with Aβ(1–40) increased expression of apoJ and receptors involved in amyloid transport including lipoprotein receptor-related protein 1 [LRP1]. In accordance, cerebromicrovascular endothelial cells isolated from 3×Tg AD mice showed elevated expression levels of apoJ and LRP1 as compared to Non-Tg animals. Treatment of pBCEC with HMGCoA-reductase inhibitor simvastatin markedly increased intracellular and secreted apoJ levels, in parallel increased secreted Aβ oligomers and reduced Aβ uptake and cell-associated Aβ oligomers. Simvastatin effects on apoJ, APP processing, and LRP1 expression in BCEC were confirmed in the mouse model. We suggest a close and complex interaction of apoJ, cholesterol homeostasis, and APP/Aβ processing and clearance at the BBB.

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.

Effects of phenothiazine-structured compounds on APP processing in Alzheimer's disease cellular model

The excess accumulation of amyloid-β (Aβ) peptides derived from the sequential cleavage of amyloid precursor protein (APP) by secretases, is one of the toxic key events leading to neuronal loss in Alzheimer's disease (AD). Studies have shown that cholinergic activity may also be involved in the regulation of APP metabolism. In the current study, we have investigated the roles of toluidine blue O (TBO) and thionine (TH), newly recognized phenothiazine-derived cholinesterase inhibitors, on the metabolism of APP in Chinese hamster ovary cells stably expressing human APP751 and presenilin 1 (PS70 cells). We assessed the effects of both compounds on the levels of Aβ, soluble APP-α (sAPPα), intracellular APP and β-site APP-cleaving enzyme 1 (BACE1). After treatment of PS70 cells with TBO or TH without any side effect on cell viability, the levels of secreted Aβ40, Aβ42 and sAPPα were assayed by specific sandwich ELISAs while APP and BACE1 in cell lysates were analyzed using Western blot. The secreted Aβ40, Aβ42 and sAPPα in TBO- and TH-treated cells were found to be reduced in a dose-dependent manner compared to vehicle-treated cells. Results suggest that TH mitigated the Aβ pathology by lowering APP levels whereas reduced Aβ caused by TBO treatment seems to be the outcome of both less substrate availability and amyloidogenic APP processing. Taken together, our results represent the first report demonstrating that TBO and TH can affect amyloid metabolism in vitro.

Decreasing the expression of PICALM reduces endocytosis and the activity of β-secretase: implications for Alzheimer's disease.

BackgroundPolymorphisms in the gene for phosphatidylinositol binding clathrin assembly protein (PICALM), an endocytic-related protein, are associated with a small, increased risk of developing Alzheimer’s disease (AD), strongly suggesting that changes in endocytosis are involved in the aetiology of the disease. We have investigated the involvement of PICALM in the processing of amyloid precursor protein (APP) to understand how PICALM could be linked to the development of AD. We used siRNA to deplete levels of PICALM, its isoforms and clathrin heavy chain in the human brain-derived H4 neuroglioma cell line that expresses endogenous levels of APP. We then used Western blotting, ELISA and immunohistochemistry to detect intra- and extracellular protein levels of endocytic-related proteins, APP and APP metabolites including β-amyloid (Aβ). Levels of functional endocytosis were quantified using ALEXA 488-conjugated transferrin and flow cytometry as a marker of clathrin-mediated endocytosis (CME).ResultsFollowing depletion of all the isoforms of PICALM by siRNA in H4 cells, levels of intracellular APP, intracellular β-C-terminal fragment (β-CTF) and secreted sAPPβ (APP fragments produced by β-secretase cleavage) were significantly reduced but Aβ40 was not affected. Functional endocytosis was significantly reduced after both PICALM and clathrin depletion, highlighting the importance of PICALM in this process. However, depletion of clathrin did not affect APP but did reduce β-CTF levels. PICALM depletion altered the intracellular distribution of clathrin while clathrin reduction affected the subcellular pattern of PICALM labelling. Both PICALM and clathrin depletion reduced the expression of BACE1 mRNA and PICALM siRNA reduced protein levels. Individual depletion of PICALM isoforms 1 and 2 did not affect APP levels while clathrin depletion had a differential effect on the isoforms, increasing isoform 1 while decreasing isoform 2 expression.ConclusionsThe depletion of PICALM in brain-derived cells has significant effects on the processing of APP, probably by reducing CME. In particular, it affects the production of β-CTF which is increasingly considered to be an important mediator in AD independent of Aβ. Thus a decrease in PICALM expression in the brain could be beneficial to slow or prevent the development of AD.Electronic supplementary materialThe online version of this article (doi:10.1186/s12868-016-0288-1) contains supplementary material, which is available to authorized users.

The Amyloid Precursor Protein is rapidly transported from the Golgi apparatus to the lysosome and where it is processed into beta-amyloid.

BackgroundAlzheimer’s disease (AD) is characterized by cerebral deposition of β-amyloid peptide (Aβ). Aβ is produced by sequential cleavage of the Amyloid Precursor Protein (APP) by β- and γ-secretases. Many studies have demonstrated that the internalization of APP from the cell surface can regulate Aβ production, although the exact organelle in which Aβ is produced remains contentious. A number of recent studies suggest that intracellular trafficking also plays a role in regulating Aβ production, but these pathways are relatively under-studied. The goal of this study was to elucidate the intracellular trafficking of APP, and to examine the site of intracellular APP processing.ResultsWe have tagged APP on its C-terminal cytoplasmic tail with photoactivatable Green Fluorescent Protein (paGFP). By photoactivating APP-paGFP in the Golgi, using the Golgi marker Galactosyltranferase fused to Cyan Fluorescent Protein (GalT-CFP) as a target, we are able to follow a population of nascent APP molecules from the Golgi to downstream compartments identified with compartment markers tagged with red fluorescent protein (mRFP or mCherry); including rab5 (early endosomes) rab9 (late endosomes) and LAMP1 (lysosomes). Because γ-cleavage of APP releases the cytoplasmic tail of APP including the photoactivated GFP, resulting in loss of fluorescence, we are able to visualize the cleavage of APP in these compartments. Using APP-paGFP, we show that APP is rapidly trafficked from the Golgi apparatus to the lysosome; where it is rapidly cleared. Chloroquine and the highly selective γ-secretase inhibitor, L685, 458, cause the accumulation of APP in lysosomes implying that APP is being cleaved by secretases in the lysosome. The Swedish mutation dramatically increases the rate of lysosomal APP processing, which is also inhibited by chloroquine and L685, 458. By knocking down adaptor protein 3 (AP-3; a heterotetrameric protein complex required for trafficking many proteins to the lysosome) using siRNA, we are able to reduce this lysosomal transport. Blocking lysosomal transport of APP reduces Aβ production by more than a third.ConclusionThese data suggests that AP-3 mediates rapid delivery of APP to lysosomes, and that the lysosome is a likely site of Aβ production.

DECREASING PICALM EXPRESSION ALTERS THE AMYLOIDOGENIC PROCESSING OF AMYLOID PRECURSOR PROTEIN

Changes in endocytosis in Alzheimer's disease (AD) were originally identified more than 15 years ago but it was not known whether they were causative or an effect of the disease. More recent studies on the genetics of AD have identified several genes encoding endocytic proteins which are associated with a small, increased risk of developing AD, strongly suggesting that changes in endocytosis are indeed involved in the aetiology of the disease. One of these genes encodes for the protein phosphatidylinositol binding clathrin assembly protein (PICALM). The ‘Amyloid Hypothesis’ suggests that the build-up of amyloid-beta (Aβ) is a primary causal event in AD. The Aβ peptide is cleaved from amyloid precursor protein (APP) by the β- and γ- secretases, primarily after APP has undergone endocytosis and within the endosomal/lysosomal system. We examined whether decreasing the expression of PICALM affected the processing of APP into Aβ to further our understanding of the importance of the role of this protein in the pathophysiology of AD. Levels of PICALM were depleted by siRNA using oligofectamine for 48 hours in human brain-derived H4 neuroglioma cells that endogenously express APP. Cells were lysed and media collected to detect intra- and extracellular protein levels of endocytic-related proteins, APP and APP metabolites including Aβ. Levels of endocytosis were quantified using ALEXA 488-conjugated transferrin as a marker of clathrin-mediated endocytosis and quantified using flow cytometry. PICALM levels were depleted by more than 85% with siRNA. Levels of intracellular APP, intracellular β-CTF and secreted sAPP β (APP fragments produced by β-secretase cleavage) were significantly reduced suggesting an inhibition of the amyloidogenic pathway. Meanwhile levels of transferrin uptake were significantly reduced in PICALM siRNA-treated cells after 15 and 30 minutes suggesting an inhibition of endocytosis. These results suggest that in cells with endogenous levels of APP, representative of levels of APP in AD in man, PICALM can modulate the amyloidogenic processing of APP to Aβ, primarily via its effect on levels and rates of endocytosis.

Increased expression of the receptor for advanced glycation end products in neurons and astrocytes in a triple transgenic mouse model of Alzheimer’s disease

The receptor for advanced glycation end products (RAGE) has been reported to have a pivotal role in the pathogenesis of Alzheimer's disease (AD). This study investigated RAGE levels in the hippocampus and cortex of a triple transgenic mouse model of AD (3xTg-AD) using western blotting and immunohistochemical double-labeling to assess cellular localization. Analysis of western blots showed that there were no differences in the hippocampal and cortical RAGE levels in 10-month-old adult 3xTg-AD mice, but significant increases in RAGE expression were found in the 22- to 24-month-old aged 3xTg-AD mice compared with those of age-matched controls. RAGE-positive immunoreactivity was observed primarily in neurons of aged 3xTg-AD mice with very little labeling in non-neuronal cells, with the notable exception of RAGE presence in astrocytes in the hippocampal area CA1. In addition, RAGE signals were co-localized with the intracellular amyloid precursor protein (APP)/amyloid beta (Aβ) but not with the extracellular APP/Aβ. In aged 3xTg-AD mice, expression of human tau was observed in the hippocampal area CA1 and co-localized with RAGE signals. The increased presence of RAGE in the 3xTg-AD animal model showing critical aspects of AD neuropathology indicates that RAGE may contribute to cellular dysfunction in the AD brain.

Suppression of amyloid‐β production by 24S‐hydroxycholesterolviainhibition of intracellular amyloid precursor protein trafficking

Cholesterol can be converted to 24S-hydroxycholesterol (24SOHC) by neuronal cholesterol 24-hydroxylase. In mouse models of Alzheimer's disease (AD), increasing 24SOHC levels reduced AD pathology. However, mechanisms underlying the effects of 24SOHC on amyloid-β (Aβ) production have remained unclear. Here we report that 24SOHC treatment reduces Aβ production and increases endoplasmic reticulum (ER)-resident immature amyloid precursor protein (APP) levels in human neuroblastoma SH-SY5Y cells and CHO cells stably expressing human APP. Treatment with 1-10 μM 24SOHC (equivalent to the concentrations detected in human brain homogenates) diminished Aβ production (IC50=4.6 μM for Aβ40) without affecting secretase activities. To evaluate the intracellular APP transport, we established an in vitro vesicle formation assay. We found that APP budding via COPII vesicles was diminished by 70% in 24SOHC-treated cells. The proteomics and immunoblotting analysis revealed that 24SOHC induced the expression of glucose-regulated protein 78 (GRP78), an ER chaperone, through unfolded protein response pathways, and enhanced the formation of the APP/GRP78 complex. Knockdown of GRP78 diminished the inhibitory effects of 24SOHC on Aβ production. These results suggest that 24SOHC down-regulates APP trafficking via enhancement of the complex formation of APP with up-regulated GRP78 in the ER, resulting in suppression of Aβ production.

Intracellular Amyloid Precursor Protein Sorting and Amyloid- Secretion Are Regulated by Src-Mediated Phosphorylation of Mint2

Mint adaptor proteins bind to the membrane-bound amyloid precursor protein (APP) and affect the production of pathogenic amyloid-β (Aβ) peptides related to Alzheimer's disease (AD). Previous studies have shown that loss of each of the three Mint proteins delays the age-dependent production of amyloid plaques in transgenic mouse models of AD. However, the cellular and molecular mechanisms underlying Mints effect on amyloid production are unclear. Because Aβ generation involves the internalization of membrane-bound APP via endosomes and Mints bind directly to the endocytic motif of APP, we proposed that Mints are involved in APP intracellular trafficking, which in turn, affects Aβ generation. Here, we show that APP endocytosis was attenuated in Mint knock-out neurons, revealing a role for Mints in APP trafficking. We also show that the endocytic APP sorting processes are regulated by Src-mediated phosphorylation of Mint2 and that internalized APP is differentially sorted between autophagic and recycling trafficking pathways. A Mint2 phosphomimetic mutant favored endocytosis of APP along the autophagic sorting pathway leading to increased intracellular Aβ accumulation. Conversely, the Mint2 phospho-resistant mutant increased APP localization to the recycling pathway and back to the cell surface thereby enhancing Aβ42 secretion. These results demonstrate that Src-mediated phosphorylation of Mint2 regulates the APP endocytic sorting pathway, providing a mechanism for regulating Aβ secretion.

Role of Phosphatidylinositol Clathrin Assembly Lymphoid-Myeloid Leukemia (PICALM) in Intracellular Amyloid Precursor Protein (APP) Processing and Amyloid Plaque Pathogenesis

One of the pathological hallmarks of Alzheimer disease is the accumulation of amyloid plaques in the extracellular space in the brain. Amyloid plaques are primarily composed of aggregated amyloid β peptide (Aβ), a proteolytic fragment of the transmembrane amyloid precursor protein (APP). For APP to be proteolytically cleaved into Aβ, it must be internalized into the cell and trafficked to endosomes where specific protease complexes can cleave APP. Several recent genome-wide association studies have reported that several single nucleotide polymorphisms (SNPs) in the phosphatidylinositol clathrin assembly lymphoid-myeloid leukemia (PICALM) gene were significantly associated with Alzheimer disease, suggesting a role in APP endocytosis and Aβ generation. Here, we show that PICALM co-localizes with APP in intracellular vesicles of N2a-APP cells after endocytosis is initiated. PICALM knockdown resulted in reduced APP internalization and Aβ generation. Conversely, PICALM overexpression increased APP internalization and Aβ production. In vivo, PICALM was found to be expressed in neurons and co-localized with APP throughout the cortex and hippocampus in APP/PS1 mice. PICALM expression was altered using AAV8 gene transfer of PICALM shRNA or PICALM cDNA into the hippocampus of 6-month-old APP/PS1 mice. PICALM knockdown decreased soluble and insoluble Aβ levels and amyloid plaque load in the hippocampus. Conversely, PICALM overexpression increased Aβ levels and amyloid plaque load. These data indicate that PICALM, an adaptor protein involved in clathrin-mediated endocytosis, regulates APP internalization and subsequent Aβ generation. PICALM contributes to amyloid plaque load in brain likely via its effect on Aβ metabolism.

Somatostatin receptor subtype-4 agonist NNC 26–9100 decreases extracellular and intracellular Aβ1–42 trimers

Soluble amyloid β-protein (Aβ) oligomers are primary mediators of synaptic dysfunction associated with the progression of Alzheimer's disease. Such Aβ oligomers exist dependent on their rates of aggregation and metabolism. Use of selective somatostatin receptor-subtype agonists have been identified as a potential means to mitigate Aβ accumulation in the brain, via regulation of the enzyme neprilysin. Herein, we first evaluated the impact of the somatostatin receptor subtype-4 agonist 1-[3-[N-(5-Bromopyridin-2-yl)-N-(3,4-dichlorobenzyl)amino]propyl]-3-[3-(1H-imidazol-4-yl)propyl]thiourea (NNC 26–9100) on learning and memory in 12-month SAMP8 mice (i.c.v. injection). NNC 26–9100 (0.2μg-dose) was shown to enhance both learning (T-maze) and memory (object recognition) compared to vehicle controls. Cortical and hippocampal tissues were evaluated subsequent to NNC 26–9100 (0.2μg) or vehicle administration for changes in neprilysin activity, along with protein expression of amyloid-precursor protein (APP), neprilysin, and Aβ1–42 oligomers within respective cellular fractions (extracellular, intracellular and membrane). NNC 26–9100 increased neprilysin activity in cortical tissue, with an associated protein expression increase in the extracellular fraction and decreased in the intracellular fraction. A decrease in intracellular APP expression was found with treatment in both cortical and hippocampal tissues. NNC 26–9100 also significantly decreased expression of Aβ1–42 trimers within both the extracellular and intracellular cortical fractions. No expression changes were found in membrane fractions for any protein. These finding suggest the potential use of selective SSTR4 agonists to mitigate toxic oligomeric forms of Aβ1–42 in critical regions of the brain identified with learning and memory decline.

Effects of conjugated linoleic acid on cleavage of amyloid precursor protein via PPARγ

Conjugated linoleic acid (CLA) plays important roles in physiological conditions. The aim of present study was to explore the effects of CLA on the cleavage of amyloid precursor protein (APP) and the potential mechanism involved. The effects of CLA on intracellular APP, BACE1 (β-site APP Cleaving Enzyme1, BACE1), a disintegrin and metalloprotease (ADAM10) and extracellular sAPPα (soluble) were analyzed by RT-PCR, Western blot and ELISA in SH-SY5Y cells. Our study indicated that CLA significantly decreased the expression of BACE1 and increased the extracellular secretion of sAPPα, but not affected the levels of APP and ADAM10. The study also revealed that the nuclear receptor peroxisome proliferators activated receptor γ (PPARγ) played an important role in the CLA-induced intracellular BACE1 decrease, as well as the extracellular sAPPα increase through knockdown of PPARγ transcription using siRNA. We hypothesize that CLA acts as an agonist or ligand, which binds with PPARγ and leads to the increase in APP cleavage via α-secretase-mediated pathway and the decrease in the deposition of Aβ.

Cellular effects of APOE4: Implications for Alzheimer's disease

The dominant risk factor for Alzheimer's disease (AD) is the epsilon-4 allele of apolipoprotein E (ApoE4). This allele confers increased risk for sporadic as well as familial AD disease. Individuals with two copies of the ApoE e4 allele have an approximately eight-fold increased risk of AD and have a significantly lower age of onset compared to AD patients not carrying this allele. Recent data indicate that the greater risk of AD associated with the ApoE4 isoform might relate to ApoE's susceptibility to proteolysis and neurotoxicity. However, the exact molecular mechanisms underlying ApoE and amyloid precursor protein (APP) interactions sub serving the risk-factor effect of ApoE4 remain unclear. The amyloid precursor protein, APP, has been shown to function as a molecular switch: cleavage at the beta, gamma, and caspase sites results in the production of four peptides-sAPPb (from which N-APP is derived), Ab, Jcasp, and C31-that mediate neurite retraction, synaptic reorganization, and ultimately programmedcell death. In contrast, cleavage at the alpha site produces the trophic peptide sAPPa and the inhibitor of APP gamma-site cleavage, aCTF. The decision between these two proteolytic pathways is governed at least in part by ligand binding: interaction with the axon guidance and trophic factor netrin-1 increases a-site cleavage, whereas interaction with the anti-trophin Ab inhibits alpha-site cleavage and increases net production of the four neurite-retractive peptides. Since this system exhibits positive feedback (e.g., Ab begets more Ab via APP processing, whereas aCTF inhibits Ab production), it is by definition prionic. Therefore, it is of interest to determine whether ApoE isoforms impact this trophic-anti-trophic peptide balance differentially, and, if so, by what mechanism. HEK-293T cells, B103-APP (rat neuroblastoma cells stably expressing human wild-type APP), 7W CHO cells (stably transfected with human APP751) and A172 human glioblastoma cells were used to determine whether ApoE, and in particular ApoE4, triggered alternative cellular APP processing that would provide a basis for understanding the cellular toxicity that has been reported previously. Transient transfection of cells was performed with Lipofectamine 2000. Whole-cell extracts were prepared and typically, 100mg of cell extract was loaded for SDS-PAGE and Western blot analysis. A total of 150-200 μg protein was also subjected to immunoprecipitation using specific antibodies to pull down the desired protein. The immunoprecipitated proteins were subjected to SDS-PAGE and Western blotting. The sAPPa, sAPPb, and Ab levels were obtained using the PerkinElmer (PE) AlphaLisa assay, and measurements were performed on an AlphaLisa reader. Immunocytochemistry was performed on cells 24 hrs after transfection with APP, ApoE, or the two in combination. The ApoE expression constructs were a kind gift from Dr. Yadong Huang of the Gladstone Institute. Cells were probed with primary antibodies and stained with an AlexaFluor secondary antibody (either 488 or 555). APP transcriptional activity was performed using the Gal4-APP system with a luciferase readout. Cells were harvested 48 h after transfection and the luciferase and b-galactosidase activities were determined with the Promega luciferase assay kit and the Promega b-galactosidase assay kit, respectively. ApoE3, ApoE4, and its proteolytic product ApoE4delta (ApoEdelta272) co-immunoprecipitated with full-length APP both in cell lysates and conditioned media. Interaction of APP with ApoE4 or ApoE4delta was accompanied by decreased sAPPa secretion and reduced sAPPa/Aß and sAPPa/sAPPß ratios as compared to ApoE3. Both ApoE4 and ApoE4δ reduced interaction between APP and Fe65 and also suppressed Fe65-mediated intracellular APPs.