Furin is an endogenous regulator of alpha-secretase associated APP processing

Constitutive and PKC-regulated alpha-secretase pathways have been reported to produce the secreted form of alpha-secretase-derived APP (sAPPalpha). Here, we examined putative role of furin in the regulation of alpha-secretase activity in vitro and in vivo. Overexpression of the prodomain of furin and infection with a furin-specific inhibitor significantly reduced the levels of sAPPalpha regardless of PKC activity, whereas total APP levels remained unchanged. Furin mRNA levels in the brains of AD patients and Tg2576 mice were significantly lower than those in controls, whereas ADAM10 and TACE mRNA levels were much alike between Tg2576 and littermate mice. Moreover, the injection of furin-adenovirus into Tg2576 mouse brains markedly increased alpha-secretase activity and reduced beta-amyloid protein (Abeta) production in infected brain regions. Our results suggest that furin enhances alpha-secretase activity via the cleavage of ADAM10 and TACE, and that attenuated furin activity is connected to the production of Abeta.     

Regulation of alpha- and beta-secretase activity by oxysterols: cerebrosterol stimulates processing of APP via the alpha-secretase pathway

Herpes simplex virus type-1 thymidine kinase (HSV-1TK) and Escherichia coli cytosine deaminase (CD) fusion protein was designed using InsightII software. The structural rationality of the fusion proteins incorporating a series of flexible linker peptide was analyzed, and a suitable linker peptide was chosen for further investigated. The recombinant plasmid containing the coding regions of HSV-1TK and CD cDNA connected by this linker peptide coding sequence was generated and subsequently transfected into the human embryonic kidney 293 cells (HEK293). The Western blotting indicated that the recombinant fusion protein existed as a dimer with a molecular weight of approximately 90 kDa. The toxicity of the prodrug on the recombinant plasmid-transfected human lung cancer cell line NCIH460 was evaluated, which showed that TKglyCD-expressing cells conferred upon cells prodrug sensitivities equivalent to that observed for each enzyme independently. Most noteworthy, cytotoxicity could be enhanced by concurrently treating TKglyCD-expressing cells with prodrugs GCV and 5-FC. The results indicate that we have successfully constructed a HSV-1TKglyCD fusion gene which might have a potential application for cancer gene therapy.     

APP intracellular domain formation and unaltered signaling in the presence of familial Alzheimer's disease mutations

One of the cardinal neuropathological findings in brains from Alzheimer's disease (AD) patients is the occurrence of amyloid beta-peptide (Abeta) deposits. The gamma-secretase-mediated intramembrane proteolysis event generating Abeta also results in the release of the APP intracellular domain (AICD), which may mediate nuclear signaling. It was recently shown that AICD starts at a position distal to the site predicted from gamma-secretase cleavage within the membrane. This novel site, the epsilon site, is located close to the inner leaflet of the membrane bilayer. The relationship between proteolysis at the gamma and epsilon sites has not been fully characterized. Here we studied AICD signaling in intact cells using a chimeric C99 molecule and a luciferase reporter system. We show that the release of AICD from the membrane takes place in a compartment downstream of the endoplasmic reticulum, is dependent on presenilin proteins, and can be inhibited by treatment with established gamma-secretase inhibitors. Moreover, we find that AICD signaling remains unaltered from C99 derivatives containing mutations associated with increased Abeta42 production and familial AD. These findings indicate that there are very similar routes for Abeta and AICD formation but that FAD-linked mutations in APP primarily affect gamma-secretase-mediated Abeta42 formation, and not AICD signaling.     

Calmodulin-binding domains in Alzheimer's disease proteins: extending the calcium hypothesis

The calcium hypothesis of Alzheimer's disease (AD) invokes the disruption of calcium signaling as the underlying cause of neuronal dysfunction and ultimately apoptosis. As a primary calcium signal transducer, calmodulin (CaM) responds to cytosolic calcium fluxes by binding to and regulating the activity of target CaM-binding proteins (CaMBPs). Ca(2+)-dependent CaMBPs primarily contain domains (CaMBDs) that can be classified into motifs based upon variations on the basic amphiphilic alpha-helix domain involving conserved hydrophobic residues at positions 1-10, 1-14 or 1-16. In contrast, an IQ or IQ-like domain often mediates Ca(2+)-independent CaM-binding. Based on these attributes, a search for CaMBDs reveals that many of the proteins intimately linked to AD may be calmodulin-binding proteins, opening new avenues for research on this devastating disease.     

A transgenic rat expressing human APP with the Swedish Alzheimer's disease mutation

In recent years, transgenic mice have become valuable tools for studying mechanisms of Alzheimer's disease (AD). With the aim of developing an animal model better for memory and neurobehavioural testing, we have generated a transgenic rat model of AD. These animals express human amyloid precursor protein (APP) containing the Swedish AD mutation. The highest level of expression in the brain is found in the cortex, hippocampus, and cerebellum. Starting after the age of 15 months, the rats show increased tau phosphorylation and extracellular Abeta staining. The Abeta is found predominantly in cerebrovascular blood vessels with very rare diffuse plaques. We believe that crossing these animals with mutant PS1 transgenic rats will result in accelerated plaque formation similar to that seen in transgenic mice.     

Alzheimer's disease: channeling APP to non-amyloidogenic processing

A good number of pharmacologic agents have over the years been touted as potentially beneficial in either preventing the onset or delay the progression of Alzheimer's disease. These include compounds such as non-steroidal anti-inflammatory drugs (NSAIDs) (HMG-CoA reductase inhibitors (statins)) and flavonoids. The underlying mechanisms for the beneficial effect of these agents are by and large attributed to their ability to reduce beta-amyloid (Abeta) production and amyloid load in the brain, via inhibition of amyloidogenic gamma-secretase activity. Recent reports have now provided mechanistic insights as to how non-amyloidogenic processing might also be enhanced by these seemingly unrelated treatments. Intriguingly, this appears to involve the inhibition of the activity of small GTPase Rho and its effector, the Rho-associated kinase, ROCK. Dietary caloric restriction (CR) also enhances non-amyloidogenic processing of APP, and this may be part of a more general anti-aging effect of CR mediated by gene expression changes downstream of the activity of the histone deacetylase SIRT1.     

BAG-1M is up-regulated in hippocampus of Alzheimer's disease patients and associates with tau and APP proteins

The accumulation of tau and amyloid beta proteins is the major molecular pathology of Alzheimer's disease (AD). The mechanisms leading to the accumulation of these proteins are not completely clear. Hsc-70/Hsp-70, a chaperone protein, has been shown to bind both these proteins and regulate their degradation. We have previously shown that the co-chaperone protein BAG-1 can inhibit the degradation of tau by forming a complex with Hsc-70 and tau. In this current work, we show that there is an increase in the BAG-1M isoform in the hippocampus of AD patients. In addition, BAG-1 binds to both tau and amyloid precursor protein physically, and is found highly expressed in the same neurons that contain intracellular tau or amyloid in hippocampal sections from AD patients. Over-expression of BAG-1M in cell culture also induced an increase in both tau and amyloid precursor protein levels. In conclusion, we report a specific increase of BAG-1M in human AD patients, which is both physically and functionally associated to the two major molecular markers of AD.     

APP mouse models for Alzheimer's disease preclinical studies

Animal models of human diseases that accurately recapitulate clinical pathology are indispensable for understanding molecular mechanisms and advancing preclinical studies. The Alzheimer's disease (AD) research community has historically used first‐generation transgenic (Tg) mouse models that overexpress proteins linked to familial AD (FAD), mutant amyloid precursor protein (APP), or APP and presenilin (PS). These mice exhibit AD pathology, but the overexpression paradigm may cause additional phenotypes unrelated to AD. Second‐generation mouse models contain humanized sequences and clinical mutations in the endogenous mouse App gene. These mice show Aβ accumulation without phenotypes related to overexpression but are not yet a clinical recapitulation of human AD. In this review, we evaluate different APP mouse models of AD, and review recent studies using the second‐generation mice. We advise AD researchers to consider the comparative strengths and limitations of each model against the scientific and therapeutic goal of a prospective preclinical study.     

Divergent roles of GSK3 and CDK5 in APP processing

Glycogen synthase kinase-3 (GSK3) and cyclin-dependent kinase 5 (CDK5) are related serine/threonine kinases that have been well studied for their role in tau hyperphosphorylation, however, little is known about their significance in APP processing. Here we report that GSK3 and CDK5 are involved in APP processing in a divergent manner. Specific inhibition of cellular GSK3 by lithium or GSK3beta antisense elicits a reduction in Abeta. Conversely, negative modulation of cellular CDK5 activity by CDK5 inhibitor, roscovitine, or CDK5 antisense stimulates Abeta production. Neither GSK3 nor CDK5 inhibition by these means significantly affected cellular APP levels or APP maturation. Moreover, oral administration of lithium significantly reduces Abeta production whereas direct ICV administration of roscovitine augmented Abeta production in the brains of PDAPP (APP(V717F)) mice. Our data support a function for both GSK3 and CDK5 in APP processing, further implicating these two kinases in the pathogenesis of Alzheimer's disease.     

Prion disease incubation time is not affected in mice heterozygous for a dynein mutation

A mechanism for transmission of the infectious prions from the peripheral nerve ends to the central nervous system is thought to involve neuronal anterograde and retrograde transport systems. Cytoplasmic dynein is the major retrograde transport molecular motor whose function is impaired in the Legs at odd angles (Loa) mouse due to a point mutation in the cytoplasmic dynein heavy chain subunit. Loa is a dominant trait which causes neurodegeneration and progressive motor function deficit in the heterozygotes. To investigate the role of cytoplasmic dynein in the transmission of prions within neurons, we inoculated heterozygous Loa and wild type littermates with mouse-adapted scrapie prions intracerebrally and intraperitonially, and determined the incubation period to onset of clinical prion disease. Our data indicate that the dynein mutation in the heterozygous state does not affect prion disease incubation time or its neuropathology in Loa mice.     

ADAM19 autolysis is activated by LPS and promotes non-classical secretion of cysteine-rich protein 2

ADAM family proteins are type I transmembrane, zinc-dependent metalloproteases. This family has multiple conserved domains, including a signal peptide, a pro-domain, a metalloprotease domain, a disintegrin (DI) domain, a cysteine-rich (Cys) domain, an EGF-like domain, a transmembrane domain, and a cytoplasmic domain. The Cys and DI domains may play active roles in regulating proteolytic activity or substrate specificity. ADAM19 has an autolytic processing activity within its Cys domain, and the processing is necessary for its proteolytic activity. To identify a new physiological function of ADAM19, we screened for associating proteins by using the extracellular domain of ADAM19 in a yeast two-hybrid system. Cysteine-rich protein 2 (CRIP2) showed an association with ADAM19 through its DI and Cys domains. Sequence analysis revealed that CRIP2 is a secretable protein without a classical signal. CRIP2 secretion was increased by overexpression of ADAM19 and decreased by suppression of ADAM19 expression. Moreover, CRIP2 secretion increased in parallel with the autolytic processing of ADAM19 stimulated by lipopolysaccharide. These findings suggest that ADAM19 autolysis is activated by lipopolysaccharide and that ADAM19 promotes the secretion of CRIP2.     

Dynamic change of Adamalysin 19 (ADAM19) in human placentas and its effects on cell invasion and adhesion in human trophoblastic cells

Human ADAM19 is a recently identified member of the ADAM family. It is highly expressed in human placentas, but its dynamic change and function at the human feto-maternal interface during placenta-tion remain to be elucidated. In this present study, the spatial and temporal expression and cellular localization of ADAM19 in normal human placentas were first demonstrated, and the effects of ADAM19 on trophoblast cell adhesion and invasion were further investigated by using a human choriocarcinoma cell line (JEG-3) as an in vitro model. The data demonstrated that ADAM19 was widely distributed in villous cytotrophoblast cells, syncytiotrophoblast cells, column trophoblasts, and villous capillary endothelial cells during early pregnancy. The mRNA and protein level of ADAM19 in placentas was high at gestational weeks 8—9, but diminished significantly at mid- and term pregnancy. In JEG-3 cells, the overexpression of ADAM19 led to diminished cell invasion, as well as increases in cell adhesiveness and the expression of E-cadherin, with no changes in b-catenin expression observed. These data in-dicate that ADAM19 may participate in the coordinated regulation of human trophoblast cell behaviors during the process of placentation.     

Meningoencephalitis associated with passive immunization of a transgenic murine model of Alzheimer's amyloidosis

Immunization against the Abeta peptide reverses the pathologic and behavioral manifestations of Alzheimer's disease in murine models. Since active immunization is associated with an autoimmune meningoencephalitis in a subset of humans, passive transfer of anti-Abeta immunoglobulin is being pursued as a potentially safer alternative. We have identified cases of meningoencephalitis subsequent to peripheral and intracerebral passive immunization of Tg2576 mice. The vasocentric mononuclear infiltrate localized only to brain regions affected by Abeta amyloid deposits suggesting that the inflammatory reaction was Abeta specific. This report indicates that current passive immunization in humans should proceed with careful regard for autoimmune complications.     

Visualization of beta-secretase cleavage in living cells using a genetically encoded surface-displayed [corrected] FRET probe

The human beta-secretase, BACE, plays a key role in the generation of pathogenic amyloid beta-peptide (Abeta) in Alzheimer's disease and has been identified as an ideal target for therapy. Previous studies reported the monitoring of BACE activity in vitro utilizing chemical synthesized sensors. Here we describe the first genetically encoded FRET probe that can detect BACE activity in vivo. The FRET probe was constructed with the BACE substrate site (BSS) and two mutated green fluorescent proteins. In living cell, the FRET probe was directed to the secretory pathway and anchored on the cell surface to measure BACE enzymatic activity. The results show that the FRET probe can be cleaved by BACE effectively in vivo, suggesting that the probe can be used for real-time monitoring of BACE activity. This assay provides a novel platform for BACE inhibitor screening in vivo.     

Detection of presenilin-1 homodimer formation in intact cells using fluorescent lifetime imaging microscopy

Presenilin-1 (PS1) is a multipass transmembrane domain protein, which is believed to be the catalytic component of the gamma-secretase complex. The complex is comprised of four major components: PS1, nicastrin, Aph-1, and Pen-2. The exact stoichiometric relationship between the four components remains unclear. It has been shown that gamma-secretase exists as high molecular weight complexes, suggesting the possibility of dimer/multimer formation. We combined a biochemical approach with a novel morphological microscopy assay to analyze PS1 dimer formation and subcellular distribution in situ, in intact mammalian cells. Both coimmunoprecipitation and fluorescent lifetime imaging microscopy approaches showed that wildtype PS1 molecules form dimers. Moreover, PS1 holoproteins containing the D257A mutation also come into close enough proximity to form a dimer, suggesting that cleavage within the loop is not necessary for dimer formation. Taken together these data suggest that PS1 dimerization occurs during normal PS1 function.     

alphaB-crystallin competes with Alzheimer's disease beta-amyloid peptide for peptide-peptide interactions and induces oxidation of Abeta-Met35

Alzheimer's disease (AD) is associated with plaque deposition in the brain of AD patients. The major component of the aggregate is a 39-42 long peptide termed beta-amyloid (Abeta). Except for Abeta, plaques contain several other components which co-precipitate together with Abeta. One such component is the small heat shock protein (sHSP) alphaB-crystallin. Instead of preventing the cell from the neurotoxicity of Abeta, alphaB-crystallin induces an increased neurotoxicity. We find - using solution state NMR spectroscopy - that alphaB-crystallin competes efficiently for Abeta monomer-monomer interactions. Interactions between Abeta and alphaB-crystallin involve the hydrophobic core residues 17-21 as well as residues 31-32 of Abeta, and thus the same chemical groups which are important for Abeta aggregation. In the presence of alphaB-crystallin, Met35 in Abeta becomes efficiently oxidized. In order to quantify the redox properties of the different complexes consisting of Abeta/alphaB-crystallin/copper, we suggest an NMR assay which allows to estimate the electrochemical properties indirectly by monitoring the rate of glutathion (GSH) auto-oxidation. The oxidation of the side chain Met35 in Abeta might account for the increased neurotoxicity and the inability of Abeta to form fibrillar structures, which has been observed previously in the presence of alphaB-crystallin [Stege, G.J. et al. (1999) The molecular chaperone alphaB-crystallin enhances amyloid-beta neurotoxicity. Biochem. Biophys. Res. Commun. 262, 152-156.].     

Alzheimer病患者APP及PS1基因表达量的研究

为了检测Alzheimer病（Alzheimer’s disease,AD）患者外周血中淀粉样前体蛋白（Amyloid Precursor Protein, APP）基因及早老素1（Presenilin 1, PS1）基因的表达情况,进而探讨APP及PS1基因的表达与AD的相关性,采用SYBRGreenⅠ的方法对45例AD患者、25例血管性痴呆（vascular dementia, VD）患者及60名正常对照组样本的mRNA进行绝对定量,检测得到APP基因及PS1基因在对照组中的表达水平分别为0.026±0.005 amol/μg cDNA和0.026±0.004 amol/μg cDNA;在AD患者组中的表达量分别为0.044±0.006 amol/μg cDNA和0.051±0.011 amol/μg cDNA;,在VD患者组中的表达水平分别为0.072±0.013 amol/μg cDNA和0.039±0.005 amol/μg cDNA 。经显著性检验,AD患者组APP基因的表达水平上调,t=2.639, P<0.01;PS1基因的表达水平同样呈上调趋势,t=2.173,P<0.05,差异均具有统计学意义。VD患者组APP基因的表达水平上调,t=3.028,P<0.01;PS1基因的表达水平也同样呈上调趋势,t=2.012,P<0.05,均有显著性差异。因此,APP及PS1基因的表达水平的增高并不一定与AD发生特异性关联,而可能与多种导致痴呆的脑部病变发生关联。