Aβ Processing Research Articles

TMP21 Transmembrane Domain Regulates γ-Secretase Cleavage

TMP21 has been shown to be associated with the γ-secretase complex and can specifically regulate γ-cleavage without affecting ϵ-mediated proteolysis. To explore the basis of this activity, TMP21 modulation of γ-secretase activity was investigated independent of ϵ-cleavage using an amyloid-β precursor proteinϵ (APPϵ) construct which lacks the amyloid intracellular domain domain. The APPϵ construct behaves similarly to the full-length precursor protein with respect to α- and β-cleavages and is able to undergo normal γ-processing. Co-expression of APPϵ and TMP21 resulted in the accumulation of membrane-embedded higher molecular weight Aβ-positive fragments, consistent with an inhibition of γ-secretase cleavage. The APPϵ system was used to examine the functional domains of TMP21 through the investigation of a series of TMP21-p24a chimera proteins. It was found that chimeras containing the transmembrane domain bound to the γ-secretase complex and could decrease γ-secretase proteolytic processing. This was confirmed though investigation of a synthetic peptide corresponding to the TMP21 transmembrane helix. The isolated TMP21 TM peptide but not the homologous p24a domain was able to reduce Aβ production in a dose-dependent fashion. These observations suggest that the TMP21 transmembrane domain promotes its association with the presenilin complex that results in decreased γ-cleavage activity.

Matrix metalloproteinases-2 and -3 are reduced in cerebrospinal fluid with low beta-amyloid 1–42 levels

Matrix metalloproteinases (MMPs), a family of extracellular soluble or membrane bound endopeptidases, are implicated in many physiological and pathophysiological functions—based on their capability to cleave all protein components of the extracellular matrix. Recent studies have implicated several forms of MMPs in chronic neurodegenerative diseases like Alzheimer's disease (AD), vascular dementia (VD), and Parkinson's disease (PD). The aim of the present study was to analyse eight MMPs (MMP-1, -2, -3, -7, -8, -9, -10, -13) in the human cerebrospinal fluid (CSF) and to correlate with the well established biomarkers beta-amyloid 1–42 (Aβ), total-tau and phospho-tau-181. Our data show a significant decrease of MMP-2 and MMP-3 levels in the CSF in samples with significantly reduced Aβ levels. It is concluded that MMP-2 and MMP-3 are directly linked to Aβ in the brain and a dysfunction may influence the processing of Aβ.

Mitochondrial superoxide: a key player in Alzheimer's disease

Our recent study characterized a role for mitochondrial superoxide in the pathology of Alzheimer’s disease [1]. Using the Tg2576 Alzheimer's disease (AD) mouse model in combination with a mouse that overexpresses the mitochondrial antioxidant enzyme superoxide dismutase (SOD-2), we showed that severe deficits in the spatial and associative memory of AD mice could be prevented by scavenging of superoxide. SOD-2 overexpression also resulted in a reduction in amyloid-β (Aβ) plaque deposition without affecting the levels of soluble and fibrillar Aβ. It did however lead to a reduction in the Aβ 42/40 ratio resulting in an Aβ pool composition less favorable for aggregation. These findings point towards the involvement of mitochondrial superoxide in AD pathology perhaps through its effects on Aβ processing. Here we discuss these findings and comment on the future directions that this research may lead to. Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by the neuropathological deposition of extracellular amyloid-β (Aβ) plaques and intracellular tau neurofibrillary tangles [2]. The disease also is characterized by the devastating loss of memory and cognitive functions, which is thought to arise from the increased production of Aβ [3]. The neurotoxic role of Aβ has long been established [3], however, it is not clear how it contributes to the cognitive deficits characteristic of AD. In addition to Aβ, several studies have implicated oxidative stress in the etiology of AD. Oxidative stress occurs mainly as a

The anti-inflammatory drug CNI-1493 regulates the intracellular processing of Aβ in different cell types of the brain

The anti-inflammatory drug CNI-1493 regulates the intracellular processing of Aβ in different cell types of the brain

Focal cerebral ischemia in rats alters APP processing and expression of Aβ peptide degrading enzymes in the thalamus

We have previously demonstrated aggregation of amyloid precursor protein (APP) and β-amyloid (Aβ) to dense plaque-like deposits in the thalamus of rats subjected to transient middle cerebral artery occlusion (MCAO). Here, we investigated the underlying molecular effects of MCAO on APP processing and expression profiles of Aβ degrading enzymes in the cortex adjacent to the infarct (penumbra) and ipsilateral thalamus 2, 7 and 30 days after ischemic insult. Enhanced β-amyloidogenic processing of APP and altered insulin degrading enzyme and neprilysin expression were observed in the thalamus, but not the penumbral cortex, 7 and 30 days after MCAO coinciding with increased calcium levels and β-secretase (BACE) activity. Consecutively, increased BACE activity associated with depletion of BACE trafficking protein GGA3, suggesting a post-translational stabilization of BACE. These results demonstrate that focal cerebral ischemia leads to complex pathogenic events in the thalamus long after the initial insult.

Novel Role of RanBP9 in BACE1 Processing of Amyloid Precursor Protein and Amyloid β Peptide Generation

Accumulation of the amyloid beta (Abeta) peptide derived from the proteolytic processing of amyloid precursor protein (APP) is the defining pathological hallmark of Alzheimer disease. We previously demonstrated that the C-terminal 37 amino acids of lipoprotein receptor-related protein (LRP) robustly promoted Abeta generation independent of FE65 and specifically interacted with Ran-binding protein 9 (RanBP9). In this study we found that RanBP9 strongly increased BACE1 cleavage of APP and Abeta generation. This pro-amyloidogenic activity of RanBP9 did not depend on the KPI domain or the Swedish APP mutation. In cells expressing wild type APP, RanBP9 reduced cell surface APP and accelerated APP internalization, consistent with enhanced beta-secretase processing in the endocytic pathway. The N-terminal half of RanBP9 containing SPRY-LisH domains not only interacted with LRP but also with APP and BACE1. Overexpression of RanBP9 resulted in the enhancement of APP interactions with LRP and BACE1 and increased lipid raft association of APP. Importantly, knockdown of endogenous RanBP9 significantly reduced Abeta generation in Chinese hamster ovary cells and in primary neurons, demonstrating its physiological role in BACE1 cleavage of APP. These findings not only implicate RanBP9 as a novel and potent regulator of APP processing but also as a potential therapeutic target for Alzheimer disease.

Brain Traffic

Brain Traffic

Lysosomal modulation ameliorates Alzheimer‐type pathology: Evidence of protective cleavage of Aβ1‐42 by modulated cathepsins

Alzheimer's disease (AD) involves protein accumulation and progressive synaptic deterioration. Cathepsin proteases exhibit up‐regulation in response to accumulating proteins including Aβ1‐42, and they have been implicated in clearance of AD‐related deposits. Lysosomal modulation with Z‐Phe‐Ala‐diazomethylketone (PADK) involves 3‐8‐fold increases in cathepsin B in APPSwInd and APPswe/PS1dE9 mice, while neprilysin, IDE, and α‐secretase were unchanged. The PADK treatment reduced intracellular Aβ staining, decreased soluble Aβx‐42, and reduced extracellular‐enriched Aβ levels in the two AD models. The corresponding increase in Aβ1‐38 peptide as Aβ1‐42 was reduced indicates that intracellular proteolysis in lysosomes can detoxify peptides, likely through cathepsin cleavage. Associated with its effect on Aβ clearance, deficits in synaptic markers were eliminated in both mouse models by the lysosomal modulator. In addition, behavioral deficits were attenuated, reaching performance scores similar to those of age‐matched non‐transgenic mice. These findings indicate that intracellular processing of Aβ by lysosomes can be targeted to effectively offset the disruption of synaptic integrity and brain function. Lysosomal modulators represent an effective strategy for treating AD and other disorders involving pathogenic accumulations.

Aβ46 Is Processed to Aβ40 and Aβ43, but Not to Aβ42, in the Low Density Membrane Domains

Gamma-secretase cleaves the transmembrane domain of beta-amyloid precursor protein at multiple sites referred to as gamma-, epsilon-, and zeta-cleavage sites. We previously showed that N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), a potent dipeptide gamma-secretase inhibitor, causes differential accumulation of longer amyloid beta-proteins (Abetas) within Chinese hamster ovary cells co-expressing beta C-terminal fragment and wild-type presenilin 1 (C99/wtPS1 cells). In this study, we used sucrose density gradient centrifugation to fractionate the membranes from C99/wtPS1 cells that had been pretreated with DAPT. We found that accumulating Abeta46 localized exclusively to low density membrane (LDM) domains. Incubating the Abeta46-accumulating LDM domains at 37 degrees C produced Abeta40, Abeta42, Abeta43, and beta-amyloid precursor protein intracellular domain. The addition of L685,458 completely prevented beta-amyloid precursor protein intracellular domain generation and resulted in a large decrease in the level of Abeta46 and the concomitant appearance of Abeta40 and Abeta43 but not Abeta42. Further addition of DAPT suppressed the production of Abeta40/43 and abolished the decrease in the amount of Abeta46. These data indicate that preaccumulated Abeta46 is processed by gamma-secretase to Abeta40/43 but not to Abeta42 in the LDM domains. The amount of newly produced Abeta40 and Abeta43 was roughly equivalent to the decrease in the amount of Abeta46. Temporal profiles did not show a maximal concentration for Abeta43, suggesting that Abeta46 is processed to Abeta40 and Abeta43 through a nonsuccessive process.

Insulin Resistance Alzheimer's Disease: Pathophysiology and Treatment

Insulin and insulin resistance likely play a significant role in the pathophysiology and cognitive decline associated with Alzheimer's disease (AD). Insulin, insulin receptors, and insulin-sensitive glucose transporters are selectively localized the brain, including medial temporal areas that support memory. Raising brain insulin levels can facilitate memory and increase cerebrospinal fluid levels of β-amyloid (Aβ) and inflammatory markers. Insulin's effects on cognition may reflect normal regulation of glucose metabolism, long-term potentiation, and neurotransmitter levels. Consequently, insulin abnormalities may disrupt normal memory functioning and promote pathophysiological processes observed in patients with neurodegenerative disorders. Conversely, restoring normal insulin activity may exert a beneficial effect on pathophysiological processes. For example, peroxisome proliferator-activated receptor (PPAR)-gamma agonists (insulin sensitizing agents used to treat type 2 diabetes mellitus) modulate neuronal cell survival, inflammatory responses, mitochondrial functioning, and possibly Aβ processing and deposition. One PPAR-gamma agonist, rosiglitazone, facilitates memory and modulates plasma Aβ levels in patients with AD. Likewise, a healthy diet and regular exercise may improve insulin sensitivity and decrease the risk for both AD. Furthermore, intranasal insulin administration rapidly delivers insulin to the brain without altering plasma insulin or glucose levels. Studies to date suggest that this procedure can facilitate memory and modulate plasma Aβ levels in memory-impaired adults. Interestingly, the adverse effects of insulin abnormalities and the beneficial effects of improving insulin sensitivity may differ by apolipoprotein E (APOE) genotype, an established risk factor for AD. Patients who do carry lower doses of the APOE e4 allele have an enhanced risk for insulin abnormalities and are also more responsive to the memory enhancing effects of both rosiglitazone and intranasal insulin administration, relative to other patients. Therefore, future therapeutic trials should consider the moderating effects of APOE genotype.

Biogenesis of γ-secretase early in the secretory pathway

γ-Secretase is responsible for proteolytic maturation of signaling and cell surface proteins, including amyloid precursor protein (APP). Abnormal processing of APP by γ-secretase produces a fragment, Aβ42, that may be responsible for Alzheimer's disease (AD). The biogenesis and trafficking of this important enzyme in relation to aberrant Aβ processing is not well defined. Using a cell-free reaction to monitor the exit of cargo proteins from the endoplasmic reticulum (ER), we have isolated a transient intermediate of γ-secretase. Here, we provide direct evidence that the γ-secretase complex is formed in an inactive complex at or before the assembly of an ER transport vesicle dependent on the COPII sorting subunit, Sec24A. Maturation of the holoenzyme is achieved in a subsequent compartment. Two familial AD (FAD)–linked PS1 variants are inefficiently packaged into transport vesicles generated from the ER. Our results suggest that aberrant trafficking of PS1 may contribute to disease pathology.

P1-308: Association of variants in Aβ processing genes with late onset Alzheimer’s disease (LOAD)

P1-308: Association of variants in Aβ processing genes with late onset Alzheimer’s disease (LOAD)

Degradation of the Alzheimer Disease Amyloid β-Peptide by Metal-dependent Up-regulation of Metalloprotease Activity

Biometals play an important role in Alzheimer disease, and recent reports have described the development of potential therapeutic agents based on modulation of metal bioavailability. The metal ligand clioquinol (CQ) has shown promising results in animal models and small phase clinical trials; however, the actual mode of action in vivo has not been determined. We now report a novel effect of CQ on amyloid beta-peptide (Abeta) metabolism in cell culture. Treatment of Chinese hamster ovary cells overexpressing amyloid precursor protein with CQ and Cu(2+) or Zn(2+) resulted in an approximately 85-90% reduction of secreted Abeta-(1-40) and Abeta-(1-42) compared with untreated controls. Analogous effects were seen in amyloid precursor protein-overexpressing neuroblastoma cells. The secreted Abeta was rapidly degraded through up-regulation of matrix metalloprotease (MMP)-2 and MMP-3 after addition of CQ and Cu(2+). MMP activity was increased through activation of phosphoinositol 3-kinase and JNK. CQ and Cu(2+) also promoted phosphorylation of glycogen synthase kinase-3, and this potentiated activation of JNK and loss of Abeta-(1-40). Our findings identify an alternative mechanism of action for CQ in the reduction of Abeta deposition in the brains of CQ-treated animals and potentially in Alzheimer disease patients.

Compounds That Bind APP and Inhibit Aβ Processing in Vitro Suggest a Novel Approach to Alzheimer Disease Therapeutics

Extracellular deposits of aggregated amyloid-beta (Abeta) peptides are a hallmark of Alzheimer disease; thus, inhibition of Abeta production and/or aggregation is an appealing strategy to thwart the onset and progression of this disease. The release of Abeta requires processing of the amyloid precursor protein (APP) by both beta- and gamma-secretase. Using an assay that incorporates full-length recombinant APP as a substrate for beta-secretase (BACE), we have identified a series of compounds that inhibit APP processing, but do not affect the cleavage of peptide substrates by BACE1. These molecules also inhibit the processing of APP and Abeta by BACE2 and selectively inhibit the production of Abeta(42) species by gamma-secretase in assays using CTF99. The compounds bind directly to APP, likely within the Abeta domain, and therefore, unlike previously described inhibitors of the secretase enzymes, their mechanism of action is mediated through APP. These studies demonstrate that APP binding agents can affect its processing through multiple pathways, providing proof of concept for novel strategies aimed at selectively modulating Abeta production.

Therapeutic strategies for Alzheimer's disease based on new molecular mechanisms

Background and objective: Alzheimer’s disease (AD) – the main cause of dementia – is characterized by the presence of neuritic plaques containing the amyloid-β peptide (Aβ) and an intraneuronal accumulation of tubule-associated protein called tau. The current and future therapeutic strategies for AD will be discussed. Currently available treatment used in AD is based on acetylcholinesterase inhibitors, since in the course of AD there is a substantial loss in cholinergic neurons. Another registered drug used in more severe AD is NMDA antagonist – memantine. Available strategies for AD include vitamin supplementation for reducing homocysteine levels, statins and non-steroidal anti-inflammatory drugs. The big hope of the last few years – vitamin E and estrogen supplementation have not been proved efficient, but more studies are needed. There are several strategies aimed at acting directly on Aβ or amyloid precursor protein (APP) processing: vaccination with Aβ peptide, Aβ passive immunization, beta and gamma secretases inhibitors. Nerve growth factors and neurotrophines could also be targeted by new therapies. Conclusions: a better understanding of the role of APP processing and folate and homocysteine in neuronal homeostasis throughout life consist revealing novel and relatively inexpensive approaches for preventing and treating AD.

Hereditary cerebral hemorrhage with amyloidosis dutch type (AβPP 693): decreased plasma amyloid-β 42 concentration

Hereditary cerebral hemorrhage with amyloidosis—Dutch type (HCHWA-D) is a rare autosomal dominant disorder caused by an amyloid-β precursor protein (AβPP) 693 mutation that clinically leads to recurrent hemorrhagic strokes and dementia. The disease is pathologically characterised by the deposition of Aβ in cerebral blood vessels and as plaques in the brain parenchyma. This study measured the Aβ40 and Aβ42 concentration in plasma of Dutch AβPP693 mutation carriers and controls. We found that the Aβ40 concentration was not different between AβPP693 mutation carriers and controls. However, the Aβ42 concentration was significantly decreased in the mutation carriers. No correlation exists between the APOE ε4 allele and the plasma of Aβ40 and Aβ42 levels in HCHWA-D patients. This finding contrasted with the increased concentrations found in Alzheimer's disease. Therefore it is suggested that the Dutch AβPP693 mutation located within the Aβ coding region of the AβPP gene has a different effect not only on clinical and pathological expression but also on Aβ processing.

A Novel Approach for Studying Endogenous Aβ Processing Using Cultured Primary Neurons Isolated from APP Transgenic Mice

The central component of senile amyloid plaques in Alzheimer's disease (AD) is the β-amyloid peptide (Aβ), derived from proteolytic processing of the amyloid precursor protein (APP). In this study, we developed an in vitro model to measure and identify soluble Aβ from primary cortical neurons. Neurons were isolated from mice transgenic for human APP695 containing the K670N, M671L double mutation. We characterized soluble Aβ using Western blot and ELISA assays. We found that the Aβ levels in conditioned media from these neurons were readily detectable and almost five times higher than in CSF. The majority of Aβ in the media was Aβ1-40; however, Aβ1–42 was also detectable. When the neurons were exposed to Phorbol 12-myristate 13-acetate (PMA), α1-antichymotrypsin, or α1-antitrypsin, the alterations of soluble Aβ levels were consistent with other models reported. Most importantly, the soluble Aβ in our model was remarkably stable, and aliquots were unchanged after prolonged incubations or repeated freeze/thaw cycles. The Aβ appeared to be monomeric by Western blot analysis. Soluble Aβ coimmunoprecipitated with endogenous mouse apolipoprotein E from the primary cultures. Taken together, our data demonstrated that using a Western blot assay to detect soluble Aβ from transgenic mouse overexpressing APP695 is sensitive, specific, and reliable and provides an accessible model for examining the neuronal metabolism of APP and Aβ.

Positive–negative epitope-tagging of β amyloid precursor protein to identify inhibitors of Aβ processing

In this report, a novel positive–negative epitope tagging approach was developed to study the cellular processing of β amyloid precursor protein (βAPP). Amino acids centered around the α-secretase cleavage site within the Aβ sequence were replaced with residues comprising an epitope for which high-affinity monoclonal antibodies are commercially available. The resulting mutant βAPP cDNAs were expressed in human embryonic kidney cells (HEK 293). Cleavage of labeled βAPP by β- and γ-secretase(s) results in the release of an epitope-tagged Aβ peptide, whereas cleavage by α-secretase results in destruction of the epitope. Highly sensitive and specific immunoassays were developed to study processing of this labeled βAPP via the amyloidogenic pathway. Secretion of epitope-tagged Aβ was prevented by MDL 28170, a previously described γ-secretase inhibitor. Confocal microscopic studies revealed that processing and cellular trafficking of epitope-tagged βAPP was not different from wild-type βAPP. These results suggest that positive–negative epitope-tagged βAPP is normally processed within the cell and may be used to identify secretase inhibitors as therapeutics for Alzheimer’s disease.

Immunohistochemical localization of amyloid β-protein with amino-terminal aspartate in the cerebral cortex of patients with Alzheimer's disease

We investigated immunohistochemically the localization of amyloid β-protein (Aβ) with amino-terminal aspartate (N1[D]) in brains of patients with Alzheimer's disease, diffuse Lewy body disease and Down's syndrome. A monoclonal antibody, 4G8, which recognizes the middle portion of Aβ, was used as a reference antibody to label the total Aβ deposits. Double staining with anti-Aβ(N1[D]) and 4G8 revealed that Aβ deposits in the subiculum and the neocortical deep layers often lacked N1[D] immunoreactivity, indicating N-terminal truncation of Aβ in these deposits. Aβ deposits in the neocortical superficial layers and the presubicular parvopyramidal layer always contained Aβ with N1[D]. Such regional as well as laminar differences in the distribution of Aβ beginning at N1[D] suggest that some local factors influence N-terminal processing of Aβ deposited in the brain.

Amyloid β-Protein (Aβ) 1–40 But Not Aβ1–42 Contributes to the Experimental Formation of Alzheimer Disease Amyloid Fibrils in Rat Brain

Two major C-terminal variants ending at Val40 and Ala42 constitute the majority of amyloid beta-protein (Abeta), which undergoes postsecretory aggregation and deposition in the Alzheimer disease (AD) brain. To probe the differential pathobiology of the two Abeta variants, we used an in vivo paradigm in which freshly solubilized Abeta1-40 or Abeta1-42 was injected into rat brains, followed by examination using Congo red birefringence, Abeta immunohistochemistry, and electron microscopy. In the rat brain, soluble Abeta 1-40 and Abeta1-42 formed aggregates, and the Abeta1-40 but not the Abeta1-42 aggregates showed Congo red birefringence. Electron microscopy revealed that the Abeta1-40 aggregates contained fibrillar structures similar to the amyloid fibrils of AD, whereas the Abeta1-42 aggregates contained nonfibrillar amorphous material. Preincubation of Abeta1-42 solution in vitro led to the formation of birefringent aggregates, and after injection of the preincubated Abeta1-42, the aggregates remained birefringent in the rat brain. Thus, a factor or factors might exist in the rat brain that inhibit the fibrillar assembly of soluble Abeta1-42. To analyze the postsecretory processing of Abeta, we used the same in vivo paradigm and showed that Abeta1-40 and Abeta1-42 were processed at their N termini to yield variants starting at pyroglutamate, and at their C termini to yield variants ending at Val40 and at Val39. Thus the normal rat brain could produce enzymes that mediate the conversion of Abeta 1-40/1-42 into processed variants similar to those in AD. This experimental paradigm may facilitate efforts to elucidate mechanisms of Abeta deposition evolving into amyloid plaques in AD.