207441247
Pharmacogenetic features of cathepsin B inhibitors that improve memory deficit and reduce β-amyloid related to Alzheimer's disease
Abstract Beta-amyloid (Aβ) in the brain is a major factor involved in Alzheimer's disease (AD) that results in severe memory deficit. Our recent studies demonstrate pharmacogenetic differences in the effects of inhibitors of cathepsin B to improve memory and reduce Aβ in different mouse models of AD. The inhibitors improve memory and reduce brain Aβ in mice expressing the wild-type (WT) β-secretase site of human APP, expressed in most AD patients. However, these inhibitors have no effect in mice expressing the rare Swedish (Swe) mutant amyloid precursor protein (APP). Knockout of the cathepsin B decreased brain Aβ in mice expressing WT APP, validating cathepsin B as the target. The specificity of cathepsin B to cleave the WT β-secretase site, but not the Swe mutant site, of APP for Aβ production explains the distinct inhibitor responses in the different AD mouse models. In contrast to cathepsin B, the BACE1 β-secretase prefers to cleave the Swe mutant site. Discussion of BACE1 data in the field indicate that they do not preclude cathepsin B as also being a β-secretase. Cathepsin B and BACE1 could participate jointly as β-secretases. Significantly, the majority of AD patients express WT APP and, therefore, inhibitors of cathepsin B represent candidate drugs for AD.
37876059
Mutant genes in familial Alzheimer's disease and transgenic models.
The most common cause of dementia occurring in mid- to late-life is Alzheimer's disease (AD). Some cases of AD, particularly those of early onset, are familial and inherited as autosomal dominant disorders linked to the presence of mutant genes that encode the amyloid precursor protein (APP) or the presenilins (PS1 or PS2). These mutant gene products cause dysfunction/death of vulnerable populations of nerve cells important in memory, higher cognitive processes, and behavior. AD affects 7-10% of individuals > 65 years of age and perhaps 40% of individuals > 80 years of age. For the late-onset cases, the principal risk factors are age and apolipoprotein (apoE) allele type, with apoE4 allele being a susceptibility factor. In this review, we briefly discuss the clinical syndrome of AD and the neurobiology/neuropathology of the disease and then focus attention on mutant genes linked to autosomal dominant familial AD (FAD), the biology of the proteins encoded by these genes, and the recent exciting progress in investigations of genetically engineered animal models that express these mutant genes and develop some features of AD.
22498656
Genetically altered transgenic models of Alzheimer's disease.
Abnormal processing and aggregation of synaptic proteins might play an important role in the pathogenesis of neurodegenerative disorders. Among them, amyloid precursor protein (APP) has been clearly associated with Alzheimer's disease (AD) and various transgenic (tg) animal models have been developed where mutant APP is overexpressed under the regulatory control of neuronal promoters. These studies have shown that AD-like pathology (namely plaques and synapse damage) begins to develop at 6-8 months of age in mice expressing human APP under Thy1, platelet-derived growth factor (B-chain) or protease-resistant prion protein promoters, provided that levels of APP are higher than 5-7 fold of endogenous levels. None of these models have shown the presence of tangles; however, tau-immunoreactive neurites in plaques and astroglial/microglial activation are observed after 12 months of age. Neuronal loss and alterations of synaptic function and connectivity are found in the CA1 region in the PDAPP tg mice lacking the Swiss Webster background. Co-expression of other genes associated with AD modify this phenotype, for example, mutant presenilin 1 accelerates the onset of plaque formation, transforming growth factor beta enhances vascular amyloidosis, and apolipoprotein E decreases amyloid deposition. In conclusion, tg mice which are capable of mimicking some aspects of AD (provided that high enough levels of expression are achieved) can potentially be used to test novel drugs for the treatment of neurodegenerative disorders.
6312237
Does my mouse have Alzheimer's disease?
Small animal models that manifest many of the characteristic neuropathological and behavioral features of Alzheimer's disease (AD) have been developed and have proven of great value for studying the pathogenesis of this disorder at the molecular, cellular and behavioral levels. The great progress made in our understanding of the genetic factors that either cause or contribute to the risk of developing AD has prompted many laboratories to create transgenic (tg) mice that overexpress specific genes which cause familial forms of the disease. Several of these tg mice display neuropathological and behavioral features of AD including amyloid β‐peptide (Aβ) and amyloid deposits, neuritic plaques, gliosis, synaptic alterations and signs of neurodegeneration as well as memory impairment. Despite these similarities, important differences in neuropathology and behavior between these tg mouse models and AD have also been observed, and to date no perfect animal model has emerged. Moreover, ascertaining which elements of the neuropathological and behavioral phenotype of these various strains of tg mice are relevant to that observed in AD continues to be a challenge. Here we provide a critical review of the AD‐like neuropathology and behavioral phenotypes of several well‐known and utilized tg mice that express human APP transgenes.
5171124
Inhibitors of Cathepsin B Improve Memory and Reduce β-Amyloid in Transgenic Alzheimer Disease Mice Expressing the Wild-type, but Not the Swedish Mutant, β-Secretase Site of the Amyloid Precursor Protein*
Elucidation of Aβ-lowering agents that inhibit processing of the wild-type (WT) β-secretase amyloid precursor protein (APP) site, present in most Alzheimer disease (AD) patients, is a logical approach for improving memory deficit in AD. The cysteine protease inhibitors CA074Me and E64d were selected by inhibition of β-secretase activity in regulated secretory vesicles that produce β-amyloid (Aβ). The regulated secretory vesicle activity, represented by cathepsin B, selectively cleaves the WT β-secretase site but not the rare Swedish mutant β-secretase site. In vivo treatment of London APP mice, expressing the WT β-secretase site, with these inhibitors resulted in substantial improvement in memory deficit assessed by the Morris water maze test. After inhibitor treatment, the improved memory function was accompanied by reduced amyloid plaque load, decreased Aβ40 and Aβ42, and reduced C-terminal β-secretase fragment derived from APP by β-secretase. However, the inhibitors had no effects on any of these parameters in mice expressing the Swedish mutant β-secretase site of APP. The notable efficacy of these inhibitors to improve memory and reduce Aβ in an AD animal model expressing the WT β-secretase APP site present in the majority of AD patients provides support for CA074Me and E64d inhibitors as potential AD therapeutic agents.
20903931
Genetic cathepsin B deficiency reduces beta-amyloid in transgenic mice expressing human wild-type amyloid precursor protein.
Neurotoxic beta-amyloid (Abeta) peptides participate in Alzheimer's disease (AD); therefore, reduction of Abeta generated from APP may provide a therapeutic approach for AD. Gene knockout studies in transgenic mice producing human Abeta may identify targets for reducing Abeta. This study shows that knockout of the cathepsin B gene in mice expressing human wild-type APP (hAPPwt) results in substantial decreases in brain Abeta40 and Abeta42 by 67% and decreases in levels of the C-terminal beta-secretase fragment (CTFbeta) derived from APP. In contrast, knockout of cathepsin B in mice expressing hAPP with the rare Swedish (Swe) and Indiana (Ind) mutations had no effect on Abeta. The difference in reduction of Abeta in hAPPwt mice, but not in hAPPSwe/Ind mice, shows that the transgenic model can affect cathepsin B gene knockout results. Since most AD patients express hAPPwt, these data validate cathepsin B as a target for development of inhibitors to lower Abeta in AD.
13798730
An Overview of APP Processing Enzymes and Products
The generation of amyloid β-peptide (Aβ) by enzymatic cleavages of the β-amyloid precursor protein (APP) has been at the center of Alzheimer’s disease (AD) research. While the basic process of β- and γ-secretase-mediated generation of Aβ is text book knowledge, new aspects of Aβ and other cleavage products have emerged in recent years. Also our understanding of the enzymes involved in APP proteolysis has increased dramatically. All of these discoveries contribute to a more complete understanding of APP processing and the physiologic and pathologic roles of its secreted and intracellular protein products. Understanding APP processing is important for any therapeutic strategy aimed at reducing Aβ levels in AD. In this review, we provide a concise description of the current state of understanding the enzymes involved in APP processing, the cleavage products generated by different processing patterns, and the potential functions of those cleavage products.
54308213
Identification of a Novel Aspartic Protease (Asp 2) as β-Secretase
Abstract The Alzheimer's disease β-amyloid peptide (Aβ) is produced by excision from the type 1 integral membrane glycoprotein amyloid precursor protein (APP) by the sequential actions of β- and then γ-secretases. Here we report that Asp 2, a novel transmembrane aspartic protease, has the key activities expected of β-secretase. Transient expression of Asp 2 in cells expressing APP causes an increase in the secretion of the N-terminal fragment of APP and an increase in the cell-associated C-terminal β-secretase APP fragment. Mutation of either of the putative catalytic aspartyl residues in Asp 2 abrogates the production of the fragments characteristic of cleavage at the β-secretase site. The enzyme is present in normal and Alzheimer's disease (AD) brain and is also found in cell lines known to produce Aβ. Asp 2 localizes to the Golgi/endoplasmic reticulum in transfected cells and shows clear colocalization with APP in cells stably expressing the 751-amino-acid isoform of APP.
4413359
Purification and cloning of amyloid precursor protein β-secretase from human brain
Proteolytic processing of the amyloid precursor protein (APP) generates amyloid β (Aβ) peptide, which is thought to be causal for the pathology and subsequent cognitive decline in Alzheimer's disease. Cleavage by β-secretase at the amino terminus of the Aβ peptide sequence, between residues 671 and 672 of APP, leads to the generation and extracellular release of β-cleaved soluble APP, and a corresponding cell-associated carboxy-terminal fragment. Cleavage of the C-terminal fragment by γ-secretase(s) leads to the formation of Aβ. The pathogenic mutation K670M671 → N670L671 at the β-secretase cleavage site in APP, which was discovered in a Swedish family with familial Alzheimer's disease, leads to increased β-secretase cleavage of the mutant substrate. Here we describe a membrane-bound enzyme activity that cleaves full-length APP at the β-secretase cleavage site, and find it to be the predominant β-cleavage activity in human brain. We have purified this enzyme activity to homogeneity from human brain using a new substrate analogue inhibitor of the enzyme activity, and show that the purified enzyme has all the properties predicted for β-secretase. Cloning and expression of the enzyme reveals that human brain β-secretase is a new membrane-bound aspartic proteinase.
4320087
Membrane-anchored aspartyl protease with Alzheimer's disease β-secretase activity
Mutations in the gene encoding the amyloid protein precursor (APP) cause autosomal dominant Alzheimer's disease. Cleavage of APP by unidentified proteases, referred to as β- and γ-secretases, generates the amyloid β-peptide, the main component of the amyloid plaques found in Alzheimer's disease patients. The disease-causing mutations flank the protease cleavage sites in APP and facilitate its cleavage. Here we identify a new membrane-bound aspartyl protease (Asp2) with β-secretase activity. The Asp2 gene is expressed widely in brain and other tissues. Decreasing the expression of Asp2 in cells reduces amyloid β-peptide production and blocks the accumulation of the carboxy-terminal APP fragment that is created by β-secretase cleavage. Solubilized Asp2 protein cleaves a synthetic APP peptide substrate at the β-secretase site, and the rate of cleavage is increased tenfold by a mutation associated with early-onset Alzheimer's disease in Sweden. Thus, Asp2 is a new protein target for drugs that are designed to block the production of amyloid β-peptide peptide and the consequent formation of amyloid plaque in Alzheimer's disease.
26168632
Human aspartic protease memapsin 2 cleaves the b-secretase site of b-amyloid precursor protein
The cDNAs of two new human membrane-associated aspartic proteases, memapsin 1 and memapsin 2, have been cloned and sequenced. The deduced amino acid sequences show that each contains the typical pre, pro, and aspartic protease regions, but each also has a C-terminal extension of over 80 residues, which includes a single transmembrane domain and a C-terminal cytosolic domain. Memapsin 2 mRNA is abundant in human brain. The protease domain of memapsin 2 cDNA was expressed in Escherichia coli and was purified. Recombinant memapsin 2 specifically hydrolyzed peptides derived from the b-secretase site of both the wild-type and Swedish mutant b-amyloid precursor protein (APP) with over 60-fold increase of catalytic efficiency for the latter. Expression of APP and memapsin 2 in HeLa cells showed that memapsin 2 cleaved the b-secretase site of APP intracellularly. These and other results suggest that memapsin 2 fits all of the criteria of b-secretase, which catalyzes the rate-limiting step of the in vivo production of the b-amyloid (Ab) peptide leading to the progression of Alzheimer’s disease. Recombinant memapsin 2 also cleaved a peptide derived from the processing site of presenilin 1, albeit with poor kinetic efficiency. Alignment of cleavage site sequences of peptides indicates that the specificity of memapsin 2 resides mainly at the S1* subsite, which prefers small side chains such as Ala, Ser, and Asp.
13286371
BACE knockout mice are healthy despite lacking the primary beta-secretase activity in brain: implications for Alzheimer's disease therapeutics.
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by accumulation of amyloid plaques and neurofibrillary tangles in the brain. The major components of plaque, beta-amyloid peptides (Abetas), are produced from amyloid precursor protein (APP) by the activity of beta- and gamma-secretases. beta-secretase activity cleaves APP to define the N-terminus of the Abeta1-x peptides and, therefore, has been a long- sought therapeutic target for treatment of AD. The gene encoding a beta-secretase for beta-site APP cleaving enzyme (BACE) was identified recently. However, it was not known whether BACE was the primary beta-secretase in mammalian brain nor whether inhibition of beta-secretase might have effects in mammals that would preclude its utility as a therapeutic target. In the work described herein, we generated two lines of BACE knockout mice and characterized them for pathology, beta-secretase activity and Abeta production. These mice appeared to develop normally and showed no consistent phenotypic differences from their wild-type littermates, including overall normal tissue morphology and brain histochemistry, normal blood and urine chemistries, normal blood-cell composition, and no overt behavioral and neuromuscular effects. Brain and primary cortical cultures from BACE knockout mice showed no detectable beta-secretase activity, and primary cortical cultures from BACE knockout mice produced much less Abeta from APP. The findings that BACE is the primary beta-secretase activity in brain and that loss of beta-secretase activity produces no profound phenotypic defects with a concomitant reduction in beta-amyloid peptide clearly indicate that BACE is an excellent therapeutic target for treatment of AD.
32419070
Correlative Memory Deficits, Aβ Elevation, and Amyloid Plaques in Transgenic Mice
Transgenic mice overexpressing the 695-amino acid isoform of human Alzheimer β-amyloid (Aβ) precursor protein containing a Lys670 → Asn, Met671 → Leu mutation had normal learning and memory in spatial reference and alternation tasks at 3 months of age but showed impairment by 9 to 10 months of age. A fivefold increase in Aβ(1–40) and a 14-fold increase in Aβ(1–42/43) accompanied the appearance of these behavioral deficits. Numerous Aβ plaques that stained with Congo red dye were present in cortical and limbic structures of mice with elevated amounts of Aβ. The correlative appearance of behavioral, biochemical, and pathological abnormalities reminiscent of Alzheimer's disease in these transgenic mice suggests new opportunities for exploring the pathophysiology and neurobiology of this disease.
10848698
Novel epoxysuccinyl peptides A selective inhibitor of cathepsin B, in vivo
New derivatives of E‐64 (compound CA‐030 and CA‐074) were tested in vitro and in vivo for selective inhibition of cathepsin B. They exhibited 10000–30000 times greater inhibitory effects on purified rat cathepsin B than on cathepsin H and L; their initial K 1 values for cathepsin B were about 2–5 nM, like that of E‐64‐c, whereas their initial K 1 values for cathepsins H and L were about 40–200 μM. In in vivo conditions, such us intraperitoneal injection of compound CA‐030 or CA‐074 into rats, compound CA‐074 is an especially potent selective inhibitor of cathepsin B, whereas compound CA‐030 does not show selectivity for cathepsin B, although both compounds CA‐030 and CA‐074 show complete selectivity for cathepsin B in vitro.
34701497
Crystallization and preliminary X-ray study of the cathepsin B complexed with CA074, a selective inhibitor.
Cathepsin B from bovine spleen has been purified and crystallized as a complex with a specific inhibitor CA074 [N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-L- isoleucyl-L-proline], using the hanging-drop method. The complex crystals obtained from 50 mM-citrate buffer (pH 3.5) belong to the tetragonal space group P4(1) (or P4(3)) with a = 73.06 A and c = 141.59 A, and diffract beyond 2.2 A resolution. There are two complex molecules per asymmetric unit giving a packing density of 3.37 A3/Da and indicating a high solvent content of 63.5%.
1280064
Antiamyloidogenic and Neuroprotective Functions of Cathepsin B: Implications for Alzheimer's Disease
Alzheimer's disease (AD) may result from the accumulation of amyloid-beta (Abeta) peptides in the brain. The cysteine protease cathepsin B (CatB) is associated with amyloid plaques in AD brains and has been suspected to increase Abeta production. Here, we demonstrate that CatB actually reduces levels of Abeta peptides, especially the aggregation-prone species Abeta1-42, through proteolytic cleavage. Genetic inactivation of CatB in mice with neuronal expression of familial AD-mutant human amyloid precursor protein (hAPP) increased the relative abundance of Abeta1-42, worsening plaque deposition and other AD-related pathologies. Lentivirus-mediated expression of CatB in aged hAPP mice reduced preexisting amyloid deposits, even thioflavin S-positive plaques. Under cell-free conditions, CatB effectively cleaved Abeta1-42, generating C-terminally truncated Abeta peptides that are less amyloidogenic. Thus, CatB likely fulfills antiamyloidogenic and neuroprotective functions. Insufficient CatB activity might promote AD; increasing CatB activity could counteract the neuropathology of this disease.
201122165
Inhibition of cathepsin B reduces β-amyloid production in regulated secretory vesicles of neuronal chromaffin cells: evidence for cathepsin B as a candidate β-secretase of Alzheimer's disease
1 Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, CA 92093, USA 2 Department of Pathology, Stanford University, Palo Alto, CA 94305, USA 3 Department of Pharmaceutical Chemistry, School of Pharmacy, University of California at San Francisco, San Francisco, CA 94143, USA 4 Harvard University Microchemistry and Proteomics Analysis Facility, Cambridge, MA 02138, USA 5 American Life Science Pharmaceuticals, Inc., San Diego, CA 92121, USA
20342640
In vitro and in vivo inhibition of cysteine proteinases by EST, a new analog of E-64.
E-64 isolated from a culture of Aspergillus japonicus is a specific inhibitor of cysteine proteinases. E-64-c, a synthetic analog of E-64, was effective in model animals of muscular dystrophy only when it was given intraperitoneally and by means of osmotic minipump. It showed no effects due to its low absorbability from intestine when it was administered orally. EST, the ethyl ester of E-64-c, was expected to be readily absorbed through intestinal membrane, since it is more lipophilic than E-64-c. Both EST and E-64-c have a high specificity to cysteine proteinase similar to E-64 but E-64-c was 100 to 1000 times stronger than EST in in vitro cathepsin inhibition. However, EST was stronger than E-64-c in cathepsin inhibition when given orally. The cathepsin B&L activities (whole activities of cathepsins B and L) in the skeletal muscle, heart and liver of hamsters were strongly inhibited soon after oral administration of 100 mg/kg body weight of EST. The inhibition continued for at least 3 h and then disappeared gradually. E-64-c was found in plasma of hamster treated with EST, but unchanged EST was not found. These results suggested that EST was converted to E-64-c, a more active form, during the permeation through intestinal membrane. The conversion of EST to E-64-c was also indicated by the absorption experiment using in situ loop method. EST was thus shown to be useful as an oral drug and expected to be effective in therapeutic trials using model animals.
15150253
The Amyloid Hypothesis of Alzheimer's Disease: Progress and Problems on the Road to Therapeutics
It has been more than 10 years since it was first proposed that the neurodegeneration in Alzheimer's disease (AD) may be caused by deposition of amyloid β-peptide (Aβ) in plaques in brain tissue. According to the amyloid hypothesis, accumulation of Aβ in the brain is the primary influence driving AD pathogenesis. The rest of the disease process, including formation of neurofibrillary tangles containing tau protein, is proposed to result from an imbalance between Aβ production and Aβ clearance.
36632341
Alzheimer's disease: molecular understanding predicts amyloid-based therapeutics.
Degenerative diseases of the brain were long considered among the most obscure and intractable of human maladies. However, recent advances in understanding their mechanisms have brought us to the verge of potential disease-modifying agents. This progress is perhaps best exemplified by the case of Alzheimer's disease. The application of molecular pathology and genetics has led to the recognition that the four genes implicated to date in familial Alzheimer's disease all chronically elevate cerebral levels of the amyloid beta-protein (Abeta). Accordingly, small molecule inhibitors of the beta- and gamma-secretases, the proteases that generate Abeta from its precursor, are under active development, and some have shown in vivo efficacy in mouse models. An alternative approach, active or passive immunization against Abeta, has received extensive pre-clinical validation in mice, but an effective preparation free of significant side effects in humans is still awaited. Several other potential therapies are also reviewed here. If one or more of these varied approaches is ultimately proven to slow or prevent dementia, Alzheimer's disease will become a salient example of the successful application of reductionist biology to the most complex of organs, the human cerebral cortex.
24034737
Two distinct intracellular pathways transport secretory and membrane glycoproteins to the surface of pituitary tumor cells
The pituitary cell line, AtT-20, synthesizes adrenocorticotropic hormone (ACTH) as a glycoprotein precursor that is cleaved into mature hormones during packaging into secretory granules. The cells also produce an endogenous leukemia virus (MuLV) that is glycosylated after translation similar to the glycosylation of the ACTH precursor. Our evidence suggests that the envelope glycoprotein and some precursor ACTH get to the cell surface in a vesicle different from the mature ACTH secretory granule. Viral glycoproteins and ACTH precursor are released from the cells much sooner after synthesis than mature ACTH. Isolated secretory granules do not contain significant amounts of the envelope glycoprotein or ACTH precursor. Exposing cells to 8Br-cAMP stimulates release of mature ACTH four to five fold, but has little effect on the release of the ACTH precursor or the viral glycoproteins. We propose that the viral glycoproteins and some of the ACTH precursor are transported by a constitutive pathway, while mature ACTH is stored in secretory granules where its release is enhanced by stimulation.
38191385
Release of amyloid beta-protein precursor derivatives by electrical depolarization of rat hippocampal slices.
Proteolytic processing of the beta-amyloid protein precursor (APP) is regulated by cell-surface receptors. To determine whether neurotransmitter release in response to neuronal activation regulates APP processing in brain, we electrically depolarized superfused rat hippocampal slices and measured soluble APP derivatives released into the superfusate. Electrical depolarization caused a rapid increase in the release of both neurotransmitters and amino-terminal APP cleavage products. These derivatives lacked the APP carboxyl terminus and were similar to those found in both cell culture media and human cerebrospinal fluid. Superfusate proteins including lactate dehydrogenase were not changed by electrical depolarization. The release of amino-terminal APP derivatives increased with increasing stimulation frequencies from 0 to 30 Hz. The increased release was inhibited by the sodium-channel antagonist tetrodotoxin, suggesting that action-potential formation mediates the release of large amino-terminal APP derivatives. These results suggest that neuronal activity regulates APP processing in the mammalian brain.
7418845
Regulation of Amyloid Precursor Protein Secretion by Glutamate Receptors in Human Ntera 2 Neurons (NT2N)*
The amyloid precursor protein (APP) can be cleaved by a β-secretase to generate a β-amyloid peptide, which has been implicated in the pathogenesis of Alzheimer’s disease. However, APP can also be cleaved by an α-secretase to form a non-amyloidogenic secreted form of APP (APP-S). APP-S secretion can be physiologically regulated. This study examined the glutamatergic regulation of APP in the human neuronal Ntera 2 (NT2N) cell line. Metabotropic glutamate receptor subtypes 1α/β and 5α were identified in the NT2N neurons by reverse transcription-polymerase chain reaction. Stimulation of these phosphatidylinositol-linked receptors with glutamate or specific receptor agonists resulted in a dose- and time-dependent increase in the secretion of the amyloid precursor protein (APP-S), measured by the immunoprecipitation of APP-S from the medium of [35S]methionine-labeled NT2N neurons. The glutamate-induced APP-S secretion was maximal at 30 min and at a concentration of 1 mm glutamate. Glutamate-induced APP-S secretion required activation of phospholipase C, which resulted in inositol 1,4,5-trisphosphate production, as shown by the rapid glutamate-induced accumulation of inositol 1,4,5-trisphosphate. Glutamate also caused an increase in intracellular Ca2+. The protein kinase C activator phorbol 12-myristate 13-acetate, a phorbol ester, as well as 1-oleoyl-2-acetoyl-3-glycerol, a cell-permeable diacylglycerol analog, also stimulated APP-S secretion. These findings suggest that APP-S secretion from NT2N neurons can be regulated by the activation of phosphatidylinositol-linked metabotropic glutamate receptor signaling pathway.
6693980
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.
2073520
Release of nontransmembrane full-length Alzheimer's amyloid precursor protein from the lumenar surface of chromaffin granule membranes.
We previously demonstrated the presence of a soluble form of full-length Alzheimer's amyloid precursor protein (APP) in the lumen of adrenal medullary chromaffin granules (CG). Furthermore, full-length APP is released from CG membranes in vitro at pH 9.0 by an enzymatic mechanism, sensitive to protease inhibitors [Vassilacopoulou et al. (1995) J. Neurochem. 64, 2140-2146]. In this study, we found that when intact CG were subjected to exogenous trypsin, a fraction of APP was not digested, consistent with an intragranular population of APP. To examine the substrate-product relationship between membrane and soluble full-length APP, we labeled CG transmembrane APP with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID), a lipophilic probe, specific for membrane-spanning domains of proteins. APP released from the membranes at pH 9.0 was not labeled with [125I]TID. In addition, this APP was not biotinylated in intact CG. Combined, the results indicate that APP released from CG membranes derives from a unique nontransmembrane population of membrane-associated APP, located in the lumenal side of CG membranes. Dithiobis(succinimidylpropionate) (DSP) cross-linking indicated that APP in CG is situated in close proximity with other proteins, possibly with APP itself. APP complexes were also detected under nonreducing conditions, without DSP cross-linking. These results, combined with our previous studies, indicate that full-length APP within CG exists as three different populations: (I) transmembrane, (II) membrane-associated/nontransmembrane, and (III) soluble. The existence of nontransmembrane populations suggests that putative gamma-secretase cleavage sites of APP, assumed to be buried within the lipid bilayer, could be accessible to proteolysis in a soluble intravesicular environment.
84162059
Cysteine protease inhibitors reduce brain β-amyloid and β-secretase activity in vivo and are potential Alzheimer's disease therapeutics
Abstract β-Secretase inhibitors that lower brain β-amyloid peptides (Aβ) are likely to be effective for treating Alzheimer's disease (AD). Irreversible epoxysuccinyl cysteine protease inhibitors are known to reduce brain Aβ and β-secretase activity in the guinea pig model of human Aβ production. In this study, acetyl-l-leucyl-l-valyl-l-lysinal (Ac-LVK-CHO) is also shown to significantly reduce brain Aβ and β-secretase activity and brain Aβ in the same model. Ac-LVK-CHO is structurally distinct from the epoxysuccinyl inhibitors and is a reversible cysteine protease inhibitor. The results suggest that cysteine protease inhibitors generally, and reversible cysteine protease inhibitors specifically, have potential for development as AD therapeutics.
24454089
The two sides of enzyme-substrate specificity: lessons from the aspartic proteinases.
Like most proteolytic enzymes, the aspartic proteinases bind substrates and most inhibitors within an extended active site cleft. Bound ligands typically adopt a beta-strand conformation. Interactions with groups on both sides of the cleft determine the primary as well as secondary specificity of the enzymes. We have pursued the discovery of the sometimes subtle distinctions between members of the aspartic proteinase family by two routes. In the first case, we have constructed sets of oligopeptide substrates with systematic variation in each position to assess interactions at one position at a time. In the second type of experiment, we have altered residues of the enzymes in order to test theories of selectivity. The combination of the two approaches has provided a better understanding of the forces involved in determining specificity of enzyme action.
25211427
Alternative pathways for production of β-amyloid peptides of Alzheimer's disease
Abstract This highlight article describes three Alzheimer's disease (AD) studies presented at the 5th General Meeting of the International Proteolysis Society that address enzymatic mechanisms for producing neurotoxic β-amyloid (Aβ) peptides. One group described the poor kinetics of BACE 1 for cleaving the wild-type (WT) β-secretase site of APP found in most AD patients. They showed that cathepsin D displays BACE 1-like specificity and cathepsin D is 280-fold more abundant in human brain than BACE 1. Nevertheless, as BACE 1 and cathepsin D show poor activity towards the WT β-secretase site, they suggested continuing the search for additional β-secretase(s). The second group reported cathepsin B as an alternative β-secretase possessing excellent kinetic efficiency and specificity for the WT β-secretase site. Significantly, inhibitors of cathepsin B improved memory, with reduced amyloid plaques and decreased Aβ(40/42) in brains of AD animal models expressing amyloid precursor protein containing the WT β-secretase site. The third group addressed isoaspartate and pyroglutamate (pGlu) posttranslational modifications of Aβ. Results showed that cathepsin B, but not BACE 1, efficiently cleaves the WT β-secretase isoaspartate site. Furthermore, cyclization of N-terminal Glu by glutaminyl cyclase generates highly amyloidogenic pGluAβ(3–40/42). These presentations suggest cathepsin B and glutaminyl cyclase as potential new AD therapeutic targets.
12461387
BACE1 inhibition reduces endogenous Abeta and alters APP processing in wild‐type mice 2
Accumulation of amyloid beta peptide (Abeta) in brain is a hallmark of Alzheimer's disease (AD). Inhibition of beta‐site amyloid precursor protein (APP)‐cleaving enzyme‐1 (BACE1), the enzyme that initiates Abeta production, and other Abeta‐lowering strategies are commonly tested in transgenic mice overexpressing mutant APP. However, sporadic AD cases, which represent the majority of AD patients, are free from the mutation and do not necessarily have overproduction of APP. In addition, the commonly used Swedish mutant APP alters APP cleavage. Therefore, testing Abeta‐lowering strategies in transgenic mice may not be optimal. In this study, we investigated the impact of BACE1 inhibition in non‐transgenic mice with physiologically relevant APP expression. Existing Abeta ELISAs are either relatively insensitive to mouse Abeta or not specific to full‐length Abeta. A newly developed ELISA detected a significant reduction of full‐length soluble Abeta 1–40 in mice with the BACE1 homozygous gene deletion or BACE1 inhibitor treatment, while the level of x‐40 Abeta was moderately reduced due to detection of non‐full‐length Abeta and compensatory activation of alpha‐secretase. These results confirmed the feasibility of Abeta reduction through BACE1 inhibition under physiological conditions. Studies using our new ELISA in non‐transgenic mice provide more accurate evaluation of Abeta‐reducing strategies than was previously feasible.
24606862
Partial Reduction of BACE1 Has Dramatic Effects on Alzheimer Plaque and Synaptic Pathology in APP Transgenic Mice*
The aspartyl protease β-site amyloid precursor protein cleaving enzyme 1 (BACE1) initiates processing of amyloid precursor protein (APP) into amyloid β (Aβ) peptide, the major component of Alzheimer disease (AD) plaques. To determine the role that BACE1 plays in the development of Aβ-driven AD-like pathology, we have crossed PDAPP mice, a transgenic mouse model of AD overexpressing human mutated APP, onto mice with either a homozygous or heterozygous BACE1 gene knockout. Analysis of PDAPP/BACE(-/-) mice demonstrated that BACE1 is absolutely required for both Aβ generation and the development of age-associated plaque pathology. Furthermore, synaptic deficits, a neurodegenerative pathology characteristic of AD, were also reversed in the bigenic mice. To determine the extent of BACE1 reduction required to significantly inhibit pathology, PDAPP mice having a heterozygous BACE1 gene knock-out were evaluated for Aβ generation and for the development of pathology. Although the 50% reduction in BACE1 enzyme levels caused only a 12% decrease in Aβ levels in young mice, it nonetheless resulted in a dramatic reduction in Aβ plaques, neuritic burden, and synaptic deficits in older mice. Quantitative analyses indicate that brain Aβ levels in young APP transgenic mice are not the sole determinant for the changes in plaque pathology mediated by reduced BACE1. These observations demonstrate that partial reductions of BACE1 enzyme activity and concomitant Aβ levels lead to dramatic inhibition of Aβ-driven AD-like pathology, making BACE1 an excellent target for therapeutic intervention in AD.