Drug Target For Alzheimer's Disease Research Articles

Calcium Signaling and Neurodegeneration

Neurodegenerative disorders, such as Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), and spinocerebellar ataxias (SCA) are very important both for fundamental science and for practical medicine. Despite extensive research into the causes of these diseases, clinical researchers have had very limited progress and, as of now, there is still no cure for any of these diseases. One of the main obstacles in the way of creating treatments for these disorders is the fact that their etiology and pathophysiology still remain unclear. This paper reviews results that support the so-called "calcium hypothesis of neurodegenerative diseases." The calcium hypothesis states that the atrophic and degenerative processes in the neurons of AD, PD, ALS, HD, and SCA patients are accompanied by alterations in calcium homeostasis. Moreover, the calcium hypothesis states that this deregulation of calcium signaling is one of the early-stage and key processes in the pathogenesis of these diseases. Based on the results we reviewed, we conclude that the calcium channels and other proteins involved in the neuronal calcium signaling system are potential drug targets for AD, PD, ALS, HD, and SCA therapy.

Neuronal cholesterol esterification by ACAT1 in Alzheimer's disease

Cholesterol has been implicated in various neurodegenerative diseases. Here we review the connection between cholesterol and Alzheimer's disease (AD), focusing on a recent study that links neuronal cholesterol esterification with biosynthesis of 24(S)-hydroxycholesterol and the fate of human amyloid precursor protein in a mouse model of AD. We also briefly evaluate the potential of ACAT1 as a drug target for AD.

Transmembrane Domain 9 of Presenilin Determines the Dynamic Conformation of the Catalytic Site of γ-Secretase

One of the most prominent drug targets for the treatment of Alzheimer disease is gamma-secretase, a multi-protein complex responsible for the generation of the amyloid-beta peptide. The catalytic core of the complex lies on presenilin, a multi-spanning membrane protease, the activity of which depends on two aspartate residues located in transmembrane domains 6 and 7. We have recently shown by cysteine-scanning mutagenesis that these aspartates are facing a water-filled cavity in the lipid bilayer, demonstrating how proteolytic cleavage of the substrates can be taking place within the membrane. Here, we demonstrate that transmembrane domain 9 and hydrophobic domain VII in the large cytoplasmic loop of presenilin are dynamic structural parts of this cavity. Hydrophobic domain VII is associated with transmembrane domain 7 in the membrane, probably facilitating the entrance of water molecules in the catalytic site. Transmembrane domain 9, on the other hand, exhibits a highly flexible structure, potentially involved in the transport of substrates to the catalytic site, as well as in the binding of gamma-secretase inhibitors. The conserved proline-alanine-leucine motif at the cytoplasmic part of this domain is extremely close to the catalytic Asp257 and is crucial for conformational changes leading to the activation of the catalytic site. We, also, identify a unique mutant in this domain (I437C) that specifically blocks amyloid-beta peptide production without affecting the processing of the physiologically indispensable Notch substrate. Our data are finally combined to propose a model for the architectural organization and activation of the catalytic site of presenilin.

O3-03-03: Apolipoprotein E4 and its fragment impair mitochondrial dynamics in neuronal cultures

Apolipoprotein (apo) E4 has a gene-dose effect on the risk and age of onset of Alzheimer's disease (AD), but the molecular mechanisms are unclear. ApoE undergoes proteolytic cleavage into C-terminal-truncated fragments found in AD brains. The fragments, which are neurotoxic in vitro and cause neurodegeneration and behavioral deficits in transgenic mice, escape the secretory pathway and locate to mitochondria in neuronal cells, leading to mitochondrial dysfunction and loss of synaptic spines. The regulation of mitochondrial distribution is essential to maintaining distinct axonal and dendritic domains in response to local metabolic demand, morphologic plasticity, and synapse formation. We studied the effects of apoE4 and its predominant fragment on mitochondrial dynamics in neurites of differentiated PC12 cells. Time-lapse recordings demonstrated that apoE (7.5 μg/ml, 24 h) alters mitochondrial motility in an isoform-specific manner (apoE4 fragment > apoE4 > apoE3 > apoE2). Exogenous apoE4 and its fragment significantly decreased the percentage of motile mitochondria by 28±7% and 37±5%, respectively, their average speed by 37±10% and 57±6%, and the distance traveled by 60±7% and 73±4%. Similar effects were seen in PC12 cells stably expressing much lower levels of apoE. The apoE4 fragment reduced the activity-dependent response of mitochondria to KCl depolarization to only 45% of that in control cells. In contrast, when neuronal activity was blocked with 1 μM tetrodotoxin, the apoE isoforms had similar effects, suggesting that the detrimental effect of apoE4 and its fragment on mitochondrial dynamics involves an activity-dependent mechanism. The impaired mitochondrial motility was rescued by an R61T mutation in apoE4, which abolishes domain interaction and decreases susceptibility to proteolysis. These results demonstrate that apolipoprotein E4 and its fragment impair mitochondrial dynamics in neuronal cultures and reveal domain interaction as a potentially important drug target in apoE4-associated AD.

P-195: Inhibitory effect of EGCG on beta-amyloid and lipopolysaccharide induced-cognitive dysfunction and secretase activity

Alzheimer's disease (AD) is characterized by the extracellular deposition of beta-amyloid peptide (Aβ) in cerebral plaques. Aβ is derived from the β-amyloid precursor protein (APP) by the enzymes, β and γ-secretase. Recently, secretase is an attractive drug target for AD. Compounds that alter the proteolytic cleavage of APP, including those that inhibit β- or γ-secretase activity, can reduce the production of Aβ peptides and may have therapeutic potential in the treatment of AD. We investigated possible effects of (-)-epigallocatechin-3-gallate (EGCG) on memory dysfunction caused by lipopolysaccharide (LPS) and Aβ1–42 through inhibition of secretase activities and Aβ1–42 aggregation. In vitro study To determine whether EGCG affects Aβ fibrillogenesis, we measured in vitro aggregation of Aβ in the absence and presence of EGCG by the thioflavin T method. The inhibitory effect of EGCG on the Aβ aggregation was also found by the observation of Aβ aggregation under electronic microscope. In vivo study EGCG (1 and 2%; 1.5 and 3 mg/kg) was added into the drinking water and were accessed by mouse (25∼30 g) for 3 weeks ad libitum before the induction of memory impairment mice model. The inducers (Aβ1–42 of 0.23 ng/mouse and 1 μg/mouse of LPS) and vehicle (saline) were administrated intracerebroventricularly, and the behavioral tests were started 1 day after injection. Leaning and memory capacity was determined by passive avoidance and water maze tests. Activities of β- and γ-secretase were assessed by using commercially available assay kit. EGCG reversed the LPS and Aβ1–42-induced memory dysfunction in dose dependent manner (Figure 1A and B). EGCG also dose-dependently attenuated LPS and Aβ-induced β and γ-secretase activities in cortex and hippocampus of the mice brain. These inhibitory effects are accorded well with the inhibitory effect of EGCG on the Aβ aggregation in vitro (ID50: 6.18 μg/ml) (Figure 2). Our current study shows that EGCG has recovery effect against LPS and Aβ1–42-induced memory dysfunction through inhibition of secretase activity and Aβ aggregation in addition to anti-oxidant mechanism. This study therefore suggests that EGCG may be useful agent for prevention of development or progression of AD.

Pathological activity of familial Alzheimer’s disease-associated mutant presenilin can be executed by six different γ-secretase complexes

gamma-Secretase is a protease complex, which catalyzes the final of two subsequent cleavages of the beta-amyloid precursor protein (APP) to release the amyloid-beta peptide (Abeta) implicated in Alzheimer's disease (AD) pathogenesis. In human cells, six gamma-secretase complexes exist, which are composed of either presenilin (PS) 1 or 2, the catalytic subunit, nicastrin, PEN-2, and either APH-1a (as S or L splice variants) or its homolog APH-1b. It is not known whether and how different APH-1 species contribute to the pathogenic activity of gamma-secretase complexes with familial AD (FAD)-associated mutant PS. Here we show that all known gamma-secretase complexes are active in APP processing and that all combinations of APH-1 variants with either FAD mutant PS1 or PS2 support pathogenic Abeta(42) production. Since our data suggest that pathogenic gamma-secretase activity cannot be attributed to a discrete gamma-secretase complex, we propose that all gamma-secretase complexes have to be explored and evaluated for their potential as AD drug target.

Phenotypic and Biochemical Analyses of BACE1- and BACE2-deficient Mice

Beta-secretase (BACE1) is the rate-limiting protease for the generation of the amyloid beta-peptide (Abeta) in Alzheimer disease. Mice in which the bace1 gene is inactivated are reported to be healthy. However, the presence of a homologous gene encoding BACE2 raises the possibility of compensatory mechanisms. Therefore, we have generated bace1, bace2, and double knockout mice. We report here that BACE1 mice display a complex phenotype. A variable but significant number of BACE1 offspring died in the first weeks after birth. The surviving mice remained smaller than their littermate controls and presented a hyperactive behavior. Electrophysiologically, subtle alterations in the steady-state inactivation of voltage-gated sodium channels in BACE1-deficient neurons were observed. In contrast, bace2 knockout mice displayed an overall healthy phenotype. However, a combined deficiency of BACE2 and BACE1 enhanced the bace1-/- lethality phenotype. At the biochemical level, we have confirmed that BACE1 deficiency results in an almost complete block of Abeta generation in neurons, but not in glia. As glia are 10 times more abundant in brain compared with neurons, our data indicate that BACE2 could indeed contribute to Abeta generation in the brains of Alzheimer disease and, in particular, Down syndrome patients. In conclusion, our data challenge the general idea of BACE1 as a safe drug target and call for some caution when claiming that no major side effects should be expected from blocking BACE1 activity.

Presence of a “CAGA box” in the APP gene unique to amyloid plaque‐forming species and absent in all APLP ‐1/2 genes: implications in Alzheimer's disease

Potentially toxic amyloid beta-peptide (Abeta) in Alzheimer's disease (AD) is generated from a family of Abeta-containing precursor proteins (APP), which is regulated via the 5'-untranslated region (5'-UTR) of its mRNA. We analyzed 5'-UTRs of the APP superfamily, including amyloid plaque-forming and non-amyloid plaque-forming species, and of prions (27 different DNA sequences). A "CAGA" sequence proximal to the "ATG" start codon was present in a location unique to APP genes of amyloid plaque-forming species and absent in all other genes surveyed. This CAGA box is immediately upstream of an interleukin-1-responsive element (acute box). In addition, the proximal CAGA box is predicted to appear on a stem-loop structure in both human and guinea pig APP mRNA. This stem-loop is part of a predicted bulge-loop that encompasses a known iron regulatory element (IRE). Electrophoretic mobility shift with segments of the APP 5'-UTR showed that a region with the proximal CAGA sequence binds nuclear proteins, and this UTR fragment is active in a reporter gene functional assay. Thus, the 5'-UTR in the human APP but not those of APP-like proteins contains a specific region that may participate in APP regulation and may determine a more general model for amyloid generation as seen in AD. The 5'-UTR of human APP contains several interesting control elements, such as an acute box element, a CAGA box, an IRE, and a transforming growth factor-beta-responsive element, that could control APP expression and provide suitable and specific drug targets for AD.

Folding of Alzheimer's core PHF subunit revealed by monoclonal antibody 423

At present, the conformation-dependent monoclonal antibodies (mAb) provide the only information on folding of tau in the core PHF. Monoclonal antibody MN423 recognizes all and only those Alzheimer's disease (AD) core paired helical filaments (PHFs) subunits, which terminate at Glu391. Using recombinant analogs of the core PHF subunit corresponding to tau residues τ297–391, we found that the C-terminal pentapeptide 387DHGAE 391 represented only one component of the structure recognized by mAb 423. Therefore, deletion mutants of the core subunit were generated to identify assembled parts of this conformational structure. We localized two spatially close components in the region 306–325 ( 306VQIVYK 311 and 321KCGSL 325) contributing to formation of the structure identified by mAb 423. Thus, the spatial proximity of three subunit segments 306VQIVYK 311, 321KCGSL 325 and 387DHGAE 391 represents constraints for intramolecular folding of the core PHF subunit. Since PHF represents a compelling drug target in AD, structural knowledge presented could contribute to structure-based drug design.

BACE1: the beta-secretase enzyme in Alzheimer's disease.

Data that have accumulated for well over a decade have implicated the beta-amyloid (Abeta) peptide as a central player in the pathogenesis of Alzheimer's disease (AD). Amyloid plaques, composed primarily of Abeta progressively form in the brains of AD patients, and mutations in three genes (amyloid precursor protein [APP] and presenilin 1 and 2 [PS1 and PS2]) cause early-onset familial AD (FAD) by directly increasing production of the toxic, plaque-promoting Abeta42 peptide. Given the strong association between Abeta and AD, it is likely that therapeutic strategies to lower the levels of Abeta in the brain should prove beneficial for the treatment of AD. One such strategy could involve inhibiting the enzymes that generate Abeta. Abeta is a product of catabolism of the large type-I membrane protein APP. Two proteases, called beta- and gamma-secretase, endoproteolyze APP to liberate the Abeta peptide. Recently, the molecules responsible for these proteolytic activities have been identified. Several lines of evidence suggest that the PS1 and PS2 proteins are gamma-secretase, and the identity of beta-secretase has been shown to be the novel transmembrane aspartic protease, beta-site APP-cleaving enzyme 1 (BACE1; also called Asp2 and memapsin 2). BACE2, a protease homologous to BACE1, was also identified, and together the two enzymes define a new family of transmembrane aspartic proteases. BACE1 exhibits all the functional properties of beta-secretase, and as the key enzyme that initiates the formation of Abeta, BACE1 is an attractive drug target for AD. This review discusses the identification and initial characterization of BACE1 and BACE2, and summarizes recent studies of BACE1 knockout mice that have validated BACE1 as the authentic beta-secretase in vivo.

Dehydroepiandrosterone reduces expression and activity of BACE in NT 2 neurons exposed to oxidative stress

Recently, we showed that oxidative stress activates the expression and activity of the β-site AβPP-cleaving enzyme (BACE), an aspartyl protease responsible for the β-secretase cleavage of AβPP. The identification of compounds able to prevent the induction of this event is an important goal of therapeutic strategies for Alzheimer's disease (AD). Dehydroepiandrosterone (DHEA) is an adrenal steroid that improves a variety of functions in the central nervous system. Moreover, a series of evidence suggests that DHEA displays antioxidant properties in different experimental models. In the present paper we show that pretreatment with DHEA is able to rescue the increase of mRNA expression, protein levels, and activity of BACE, produced by oxidative stress in NT 2 neurons. BACE, being the enzyme that initiates the production of Aβ, is a drug target for AD. Our results imply that DHEA administration may slow down the AD pathological process, lowering Aβ accumulation.

Targeting Alzheimer's disease: Is there a light at the end of the tunnel?

AbstractThe prevalence of Alzheimer's disease (AD) is on the rise in developed nations as a consequence of longer human lifespan. Current costs to society are alarming, and are projected to become even more demanding on future health budgets. Considering the relative success of Parkinson's disease pharmacotherapy, the success of AD therapy has been disappointing. Quite a few novel and promising AD drug targets are presented in this special issue of Drug Development Research. These are built on countless research studies, by many bright minds, carried out over the last several decades. However, the answer to the growing AD threat must include reliable and accurate tools, presently lacking, for its early diagnosis in at‐risk individuals. Drug Dev. Res. 56:45–48, 2002. © 2002 Wiley‐Liss, Inc.

Advances in the Cellular and Molecular Biology of the Beta-Amyloid Protein in Alzheimer 's Disease

Alzheimer's disease (AD) is a progressive senile dementia characterized by deposition of a 4 kDa peptide of 39-42 residues known as amyloid beta-peptide (Abeta) in the form of senile plaques and the microtubule associated protein tau as paired helical filaments. Genetic studies have identified mutations in the Abeta precursor protein (APP) as the key triggers for the pathogenesis of AD. Other genes such as presenilins 1 and 2 (PS1/2) and apolipoprotein E (APOE) also play a critical role in increased Abeta deposition. Several biochemical and molecular studies using transfected cells and transgenic animals point to mechanisms by which Abeta is generated and aggregated to trigger the neurodegeneration that may cause AD. Three important enzymes collectively known as "secretases" participate in APP processing. An enzymatic activity, beta-secretase, cleaves APP on the amino side of Abeta producing a large secreted derivative, sAPPbeta, and an Abeta-bearing membrane-associated C-terminal derivative, CTFbeta, which is subsequently cleaved by the second activity, gamma-secretase, to release Abeta. Alternatively, a third activity, alpha-secretase, cleaves APP within Abeta to the secreted derivative sAPPalpha and membrane-associated CTFalpha. The predominant secreted APP derivative is sAPPalpha in most cell-types. Most of the secreted Abeta is 40 residues long (Abeta40) although a small percentage is 42 residues in length (Abeta42). However, the longer Abeta42 aggregates more readily and was therefore considered to be the pathologically important form. Advances in our understanding of APP processing, trafficking, and turnover will pave the way for better drug discovery for the eventual treatment of AD. In addition, APP gene regulation and its interaction with other proteins may provide useful drug targets for AD. The emerging knowledge related to the normal function of APP will help in determining whether or not the AD associated changes in APP metabolism affect its function. The present review summarizes our current understanding of APP metabolism and function and their relationship to other proteins involved in AD.

The beta-secretase, BACE: a prime drug target for Alzheimer's disease.

Evidence suggests that the beta-amyloid peptide (Abeta) is central to the pathophysiology of Alzheimer's disease (AD). Amyloid plaques, primarily composed of Abeta, progressively develop in the brains of AD patients, and mutations in three genes (APP, PS1, and PS2) cause early onset familial AD (FAD) by directly increasing synthesis of the toxic, plaque-promoting Abeta42 peptide. Given the strong association between Abeta and AD, therapeutic strategies to lower the concentration of Abeta in the brain should prove beneficial for the treatment of AD. One such strategy would involve inhibiting the enzymes that generate Abeta. Abeta is a product of catabolism of the large Typel membrane protein, amyloid precursor protein (APP). Two proteases, called beta- and gamma-secretase, mediate the endoproteolysis of APP to liberate the Abeta peptide. For over a decade, the molecular identities of these proteases were unknown. Recently, the gamma-secretase has been tentatively identified as the presenilin proteins, PS1 and PS2, and the identity of the beta-secretase has been shown to be the novel transmembrane aspartic protease, beta-site APP cleaving enzyme 1 (BACE1; also called Asp2 and memapsin2). BACE2, a novel protease homologous to BACE1, was also identified, and together the two enzymes define a new family of transmembrane aspartic proteases. BACE1 exhibits all the properties of the beta-secretase, and as the key rate-limiting enzyme that initiates the formation of Abeta, BACE1 is an attractive drug target for AD. Here, I review the identification and initial characterization of BACE1 and BACE2, and summarize our current understanding of BACE1 post-translational processing and intracellular trafficking. In addition, I discuss recent studies of BACE1 knockout mice and the BACE1 X-ray structure, and relate implications for BACE1 drug development.