Phenserine regulates translation of β-amyloid precursor protein mRNA by a putative interleukin-1 responsive element, a target for drug development

The reduction in levels of the potentially toxic amyloid-β peptide (Aβ) has emerged as one of the most important therapeutic goals in Alzheimer's disease. Key targets for this goal are factors that affect the expression and processing of the Aβ precursor protein (βAPP). Earlier reports from our laboratory have shown that a novel cholinesterase inhibitor, phenserine, reduces βAPP levels in vivo. Herein, we studied the mechanism of phenserine's actions to define the regulatory elements in βAPP processing. Phenserine treatment resulted in decreased secretion of soluble βAPP and Aβ into the conditioned media of human neuroblastoma cells without cellular toxicity. The regulation of βAPP protein expression by phenserine was posttranscriptional as it suppressed βAPP protein expression without altering βAPP mRNA levels. However, phenserine's action was neither mediated through classical receptor signaling pathways, involving extracellular signal-regulated kinase or phosphatidylinositol 3-kinase activation, nor was it associated with the anticholinesterase activity of the drug. Furthermore, phenserine reduced expression of a chloramphenicol acetyltransferase reporter fused to the 5′-mRNA leader sequence of βAPP without altering expression of a control chloramphenicol acetyltransferase reporter. These studies suggest that phenserine reduces Aβ levels by regulating βAPP translation via the recently described iron regulatory element in the 5′-untranslated region of βAPP mRNA, which has been shown previously to be up-regulated in the presence of interleukin-1. This study identifies an approach for the regulation of βAPP expression that can result in a substantial reduction in the level of Aβ.

Cholinergic agonists stimulate secretion of soluble full-length amyloid precursor protein in neuroendocrine cells.

The Abeta peptide of Alzheimer disease is derived from the proteolytic processing of the amyloid precursor proteins (APP), which are considered type I transmembrane glycoproteins. Recently, however, soluble forms of full-length APP were also detected in several systems including chromaffin granules. In this report we used antisera specific for the cytoplasmic sequence of APP to show that primary bovine chromaffin cells secrete a soluble APP, termed solAPPcyt, of an apparent molecular mass of 130 kDa. This APP was oversecreted from Chinese hamster ovary cells transfected with a full-length APP cDNA indicating that solAPPcyt contained both the transmembrane and Abeta sequence. Deglycosylation of solAPPcyt showed that it contained both N- and O-linked sugars, suggesting that this APP was transported through the endoplasmic reticulum-Golgi pathway. Secretion of solAPPcyt from primary chromatin cells was temperature-, time-, and energy-dependent and was stimulated by cell depolarization in a Ca2+-dependent manner. Cholinergic receptor agonists, including acetylcholine, nicotine, or carbachol, stimulated the rapid secretion of solAPPcyt, a process that was inhibited by cholinergic antagonists. Stimulation of solAPPcyt secretion was paralleled by a stimulation of secretion in catecholamines and chromogranin A, indicating that secretion of solAPPcyt was mediated by chromaffin granule vesicles. Taken together, our results show that release of the potentially amyloidogenic solAPPcyt is an active cellular process mediated by both the constitutive and regulated pathways. solAPPcyt was also detected in human cerebrospinal fluid. Combined with the neuronal physiology of chromaffin cells, our data suggest that cholinergic agonists may stimulate the release of this APP in neuronal synapses where it may exert its biological functions. Moreover, vesicular or secreted solAPPcyt may serve as a soluble precursor of Abeta.

Amyloid beta-peptide disrupts carbachol-induced muscarinic cholinergic signal transduction in cortical neurons.

Cholinergic pathways serve important functions in learning and memory processes, and deficits in cholinergic transmission occur in Alzheimer disease (AD). A subset of muscarinic cholinergic receptors are linked to G-proteins that activate phospholipase C, resulting in the liberation of inositol trisphosphate and Ca2+ release from intracellular stores. We now report that amyloid beta-peptide (Abeta), which forms plaques in the brain in AD, impairs muscarinic receptor activation of G proteins in cultured rat cortical neurons. Exposure of rodent fetal cortical neurons to Abeta25-35 and Abeta1-40 resulted in a concentration and time-dependent attenuation of carbachol-induced GTPase activity without affecting muscarinic receptor ligand binding parameters. Downstream events in the signal transduction cascade were similarly attenuated by Abeta. Carbachol-induced accumulation of inositol phosphates (IP, IP2, IP3, and IP4) was decreased and calcium imaging studies revealed that carbachol-induced release of calcium was severely impaired in neurons pretreated with Abeta. Muscarinic cholinergic signal transduction was disrupted with subtoxic levels of exposure to AP. The effects of Abeta on carbachol-induced GTPase activity and calcium release were attenuated by antioxidants, implicating free radicals in the mechanism whereby Abeta induced uncoupling of muscarinic receptors. These data demonstrate that Abeta disrupts muscarinic receptor coupling to G proteins that mediate induction of phosphoinositide accumulation and calcium release, findings that implicate Abeta in the impairment of cholinergic transmission that occurs in AD.

Generation of mice with a 200-kb amyloid precursor protein gene deletion by Cre recombinase-mediated site-specific recombination in embryonic stem cells.

Gene disruptions and deletions of up to 20kb have been generated by homologous recombination with appropriate targeting vectors in murine embryonic stem (ES) cells. Because we could not obtain a deletion of about 200 kb in the mouse amyloid precursor protein gene by the classical technique, we employed strategies involving the insertion of loxP sites upstream and downstream of the region to be deleted by homologous recombination and elicited excision of the loxP-flanked region by introduction of a Cre expression vector into the ES cells. In the first approach, the loxP sequences were inserted in two successive steps and after each step, ES cell clones were isolated and characterized. Deletion of the loxP-flanked sequence was accomplished by introducing the cre gene in a third step. In the second approach, ES cells containing the upstream loxP cassette were electroporated simultaneously with the downstream loxP targeting vector and the Cre expression plasmid. ES cells were obtained that gave rise to chimeric mice capable of germ-line transmission of the deleted amyloid precursor protein allele.

Expression of the C terminus of the amyloid precursor protein alters growth factor responsiveness in stably transfected PC12 cells.

The amyloid precursor protein (APP) is a molecule centrally involved in Alzheimer disease pathology, but whose normal function is still poorly understood. To investigate the consequences of increased intracellular production of various regions of APP on cellular physiology, we stably transfected PC12 cells with the C-terminal 100 amino acids of the human APP. In eight transfected clones that express the APP(C100) protein, exposure to nerve growth factor (NGF) did not promote differentiation. Transfectants continued to divide and failed to elaborate extensive neurites, whereas control PC12 cells, mock-transfected PC12 cells, and a nonexpressing transfected cell line did develop neurites and stopped dividing after NGF stimulation. Unlike NGF treatment, treatment with basic fibroblast growth factor profoundly accelerated neurite outgrowth in transfected cells. Also, a dramatic increase in a tyrosine phosphatase activity was noted. Expression and accumulation of APP C100 protein in PC12 cells results in an abnormal response to growth factor stimulation.

Tumor necrosis factors alpha and beta protect neurons against amyloid beta-peptide toxicity: evidence for involvement of a kappa B-binding factor and attenuation of peroxide and Ca2+ accumulation.

In Alzheimer disease (AD) the amyloid beta-peptide (A beta) accumulates in plaques in the brain. A beta can be neurotoxic by a mechanism involving induction of reactive oxygen species (ROS) and elevation of intracellular free calcium levels ([Ca2+]i). In light of evidence for an inflammatory response in the brain in AD and reports of increased levels of tumor necrosis factor (TNF) in AD brain we tested the hypothesis that TNFs affect neuronal vulnerability to A beta. A beta-(25-35) and A beta-(1-40) induced neuronal degeneration in a concentration- and time-dependent manner. Pretreatment of cultures for 24 hr with TNF-beta or TNF-alpha resulted in significant attenuation of A beta-induced neuronal degeneration. Accumulation of peroxides induced in neurons by A beta was significantly attenuated in TNF-pretreated cultures, and TNFs protected neurons against iron toxicity, suggesting that TNFs induce antioxidant pathways. The [Ca2+]i response to glutamate (quantified by fura-2 imaging) was markedly potentiated in neurons exposed to A beta, and this action of A beta was suppressed in cultures pretreated with TNFs. Electrophoretic mobility-shift assays demonstrated an induction of a kappa beta-binding activity in hippocampal cells exposed to TNFs. Exposure of cultures to I kappa B (MAD3) antisense oligonucleotides, a manipulation designed to induce NF-kappa B, mimicked the protection by TNFs. These data suggest that TNFs protect hippocampal neurons against A beta toxicity by suppressing accumulation of ROS and Ca2+ and that kappa B-dependent transcription is sufficient to mediate these effects. A modulatory role for TNF in the neurodegenerative process in AD is proposed.

Amyloid precursor protein processing is stimulated by metabotropic glutamate receptors.

Stimulation of muscarinic m1 or m3 receptors can, by generating diacylglycerol and activating protein kinase C, accelerate the breakdown of the amyloid precursor protein (APP) to form soluble, nonamyloidogenic derivatives (APPs), as previously shown. This relationship has been demonstrated in human glioma and neuroblastoma cells, as well as in transfected human embryonic kidney 293 cells and PC-12 cells. We now provide evidence that stimulation of metabotropic glutamate receptors (mGluRs), which also are coupled to phosphatidylinositol 4,5-bisphosphate hydrolysis, similarly accelerates processing of APP into nonamyloidogenic APPs. This process is demonstrated both in hippocampal neurons derived from fetal rats and in human embryonic kidney 293 cells transfected with cDNA expression constructs encoding the mGluR 1 alpha subtype. In hippocampal neurons, both an mGluR antagonist, L-(+)-2-amino-3-phosphonopropionic acid, and an inhibitor of protein kinase C, GF 109203X, blocked the APPs release evoked by glutamate receptor stimulation. Ionotropic glutamate agonists, N-methyl-D-aspartate or S(-)-5-fluorowillardiine, failed to affect APPs release. These data show that selective mGluR agonists that initiate signal-transduction events can regulate APP processing in bona fide primary neurons and transfected cells. As glutamatergic neurons in the cortex and hippocampus are damaged in Alzheimer disease, amyloid production in these regions may be enhanced by deficits in glutamatergic neurotransmission.

Serum amyloid P component prevents proteolysis of the amyloid fibrils of Alzheimer disease and systemic amyloidosis.

Extracellular deposition of amyloid fibrils is responsible for the pathology in the systemic amyloidoses and probably also in Alzheimer disease [Haass, C. & Selkoe, D. J. (1993) Cell 75, 1039-1042] and type II diabetes mellitus [Lorenzo, A., Razzaboni, B., Weir, G. C. & Yankner, B. A. (1994) Nature (London) 368, 756-760]. The fibrils themselves are relatively resistant to proteolysis in vitro but amyloid deposits do regress in vivo, usually with clinical benefit, if new amyloid fibril formation can be halted. Serum amyloid P component (SAP) binds to all types of amyloid fibrils and is a universal constituent of amyloid deposits, including the plaques, amorphous amyloid beta protein deposits and neurofibrillary tangles of Alzheimer disease [Coria, F., Castano, E., Prelli, F., Larrondo-Lillo, M., van Duinen, S., Shelanski, M. L. & Frangione, B. (1988) Lab. Invest. 58, 454-458; Duong, T., Pommier, E. C. & Scheibel, A. B. (1989) Acta Neuropathol. 78, 429-437]. Here we show that SAP prevents proteolysis of the amyloid fibrils of Alzheimer disease, of systemic amyloid A amyloidosis and of systemic monoclonal light chain amyloidosis and may thereby contribute to their persistence in vivo. SAP is not an enzyme inhibitor and is protective only when bound to the fibrils. Interference with binding of SAP to amyloid fibrils in vivo is thus an attractive therapeutic objective, achievement of which should promote regression of the deposits.

Spatial distribution of diffuse, primitive, and classic amyloid-beta deposits and blood vessels in the upper laminae of the frontal cortex in Alzheimer disease

The spatial distribution of the diffuse, primitive, and classic amyloid-beta deposits was studied in the upper laminae of the superior frontal gyrus in cases of sporadic Alzheimer disease (AD). Amyloid-beta-stained tissue was counterstained with collagen IV to determine whether the spatial distribution of the amyloid-beta deposits along the cortex was related to blood vessels. In all patients, amyloid-beta deposits and blood vessels were aggregated into distinct clusters and in many patients, the clusters were distributed with a regular periodicity along the cortex. The clusters of diffuse and primitive deposits did not coincide with the clusters of blood vessels in most patients. However, the clusters of classic amyloid-beta deposits coincided with those of the large diameter (>10 microm) blood vessels in all patients and with clusters of small-diameter (< 10 microm) blood vessels in four patients. The data suggest that, of the amyloid-beta subtypes, the clusters of classic amyloid-beta deposits appear to be the most closely related to blood vessels and especially to the larger-diameter, vertically penetrating arterioles in the upper cortical laminae.

Laminar distribution of amyloid-beta (Abeta) deposits in dementia with Lewy bodies: comparison with Alzheimer's disease

Significant amyloid-beta (Abeta) deposition in cases of dementia with Lewy bodies (DLB) may represent concurrent Alzheimer's disease (AD). To test this hypothesis, the laminar distribution of the diffuse, primitive, and classic Abeta deposits was studied in the frontal and temporal cortex in cases of DLB and were compared with AD. In DLB, the diffuse and primitive deposits exhibited two common patterns of distribution; either maximum density occurred in the upper cortical laminae or a bimodal distribution was present with density peaks in the upper and lower laminae. In addition, a bimodal distribution of the classic deposits was observed in approximately half of the cortical areas analysed. A number of differences in the laminar distributions of Abeta deposits were observed in DLB and AD. First, the proportion of the primitive relative to the diffuse and classic deposits present was lower in DLB compared with AD. Second, the primitive deposits were more frequently bimodally distributed in DLB. Third, the density of the diffuse deposits reached a maximum lower in the cortical profile in AD. These data suggest differences in the pattern of cortical degeneration in the two disorders and therefore, DLB cases with significant Abeta pathology may not represent the coexistence of DLB and AD.

Beta-amyloid (beta/A4) deposition in the medial temporal lobe in Down's syndrome: effects of brain region and patient age

The density of diffuse, primitive, classic and compact beta-amyloid (beta/A4) deposits was studied in the medial temporal lobe in 12 cases of Down's syndrome (DS) from 38 to 67 years of age. Total beta/A4 deposit density was greater in the adjacent cortex compared with regions of the hippocampus, and these differences were similar within each age group of patients. The ratio of the primitive to diffuse deposits was greater in the hippocampus than in the adjacent cortex. Total beta/A4 density did not vary significantly with patient age. However, the density of the diffuse deposits exhibited a parabolic, and the primitive, classic and compact deposits an inverted parabolic, response with age. Hence, in DS, (1) beta/A4 density remains relatively constant with age, (2) differences in beta/A4 density between the hippocampus and adjacent cortex are established at an early age, and (3) mature beta/A4 subtype formation depends on brain region and patient age.

The amyloid precursor protein controls PIKfyve function

While the Amyloid Precursor Protein (APP) plays a central role in Alzheimer's disease, its cellular function still remains largely unclear. It was our goal to establish APP function which will provide insights into APP's implication in Alzheimer's disease. Using our recently developed proteo-liposome assay we established the interactome of APP's intracellular domain (known as AICD), thereby identifying novel APP interactors that provide mechanistic insights into APP function. By combining biochemical, cell biological and genetic approaches we validated the functional significance of one of these novel interactors. Here we show that APP binds the PIKfyve complex, an essential kinase for the synthesis of the endosomal phosphoinositide phosphatidylinositol-3,5-bisphosphate. This signalling lipid plays a crucial role in endosomal homeostasis and receptor sorting. Loss of PIKfyve function by mutation causes profound neurodegeneration in mammals. Using C. elegans genetics we demonstrate that APP functionally cooperates with PIKfyve in vivo. This regulation is required for maintaining endosomal and neuronal function. Our findings establish an unexpected role for APP in the regulation of endosomal phosphoinositide metabolism with dramatic consequences for endosomal biology and important implications for our understanding of Alzheimer's disease.

The role of amyloid precursor protein in neuronal and non-neuronal cell lines

Models of Alzheimer’s disease (AD) have provided useful insights into the pathogenesis and mechanistic pathways that lead to its development. One emerging idea about AD is that it may be described as a hypometabolic disorder due to the reduction of glucose uptake in AD brains. Inappropriate processing of Amyloid Precursor Protein (APP) is considered central to the initiation and progression of the disease. Although the exact role of APP misprocessing is unclear, it may play a role in neuronal metabolism before the onset of neurodegeneration. To investigate the potential role of APP in neuronal metabolism, the SHSY5Y neuroblastoma cell line was used to generate cell lines that stably overexpress wild type APP695 or express Swedish mutated-APP observed in familial AD (FAD), both under the control of the neuronal promoter, Synapsin I. The effects of APP on glucose uptake, cellular stress and energy homeostasis were studied extensively. It was found that APP-overexpressing cells exhibited decreased glucose uptake with changes in basal oxygen consumption in comparison to control cell lines. Similar studies were also performed in fibroblasts taken from FAD patients compared with control fibroblasts. Previous studies found FAD-derived fibroblasts displayed altered metabolic profiles, calcium homeostasis and oxidative stress when compared to controls. As such, in this study fibroblasts were studied in terms of their ability to metabolise glucose and their mitochondrial function. Results show that FAD-derived fibroblasts demonstrate no differences in mitochondrial function, or response to oxidative stress compared to control fibroblasts. However, control fibroblasts treated with Aβ1-42 demonstrated changes in glucose uptake. This study highlights the importance of APP expression within non-neuronal cell lines, suggesting that whilst AD is considered a brain-associated disorder, peripheral effects in non-neuronal cell types should also be considered when studying the effects of Aβ on metabolism.

The amyloid precursor protein (APP) binds the PIKfyve complex and modulates its function

Phosphoinositides are important components of eukaryotic membranes that are required for multiple forms of membrane dynamics. Phosphoinositides are involved in defining membrane identity, mediate cell signalling and control membrane trafficking events. Due to their pivotal role in membrane dynamics, phosphoinositide de-regulation contributes to various human diseases. In this review, we will focus on the newly emerging regulation of the PIKfyve complex, a phosphoinositide kinase that converts the endosomal phosphatidylinositol-3-phosphate [PI(3)P] to phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2)], a low abundance phosphoinositide of outstanding importance for neuronal integrity and function. Loss of PIKfyve function is well known to result in neurodegeneration in both mousemodels and human patients. Our recent work has surprisingly identified the amyloid precursor protein (APP), the central molecule in Alzheimer s disease aetiology, as a novel interaction partner of a subunit of the PIKfyve complex, Vac14. Furthermore, it has been shown that APP modulates PIKfyve function and PI(3,5)P2 dynamics, suggesting that the APP gene family functions as regulator of PI(3,5)P2 metabolism. The recent advances discussed in this review suggest a novel, unexpected, â-amyloid-independent mechanism for neurodegeneration in Alzheimer s disease.

Lipids in Amyloid-β processing, aggregation, and toxicity

Aggregation of amyloid-beta (Aβ) peptide is the major event underlying neuronal damage in Alzheimer's disease (AD). Specific lipids and their homeostasis play important roles in this and other neurodegenerative disorders. The complex interplay between the lipids and the generation, clearance or deposition of Aβ has been intensively investigated and is reviewed in this chapter. Membrane lipids can have an important influence on the biogenesis of Aβ from its precursor protein. In particular, increased cholesterol in the plasma membrane augments Aβ generation and shows a strong positive correlation with AD progression. Furthermore, apolipoprotein E, which transports cholesterol in the cerebrospinal fluid and is known to interact with Aβ or compete with it for the lipoprotein receptor binding, significantly influences Aβ clearance in an isoform-specific manner and is the major genetic risk factor for AD. Aβ is an amphiphilic peptide that interacts with various lipids, proteins and their assemblies, which can lead to variation in Aβ aggregation in vitro and in vivo. Upon interaction with the lipid raft components, such as cholesterol, gangliosides and phospholipids, Aβ can aggregate on the cell membrane and thereby disrupt it, perhaps by forming channel-like pores. This leads to perturbed cellular calcium homeostasis, suggesting that Aβ-lipid interactions at the cell membrane probably trigger the neurotoxic cascade in AD. Here, we overview the roles of specific lipids, lipid assemblies and apolipoprotein E in Aβ processing, clearance and aggregation, and discuss the contribution of these factors to the neurotoxicity in AD.