CTF1-51, a truncated carboxyl-terminal fragment of amyloid precursor protein, suppresses the effects of Aβ42-lowering γ-secretase modulators

The pathogenesis of Alzheimer's disease (AD) is correlated with the toxicity of amyloid β-peptide (Aβ), especially Aβ42. γ-Secretase modulators (GSMs) are compounds that alter production of Aβ42 without interfering with the physiological function of γ-secretase. Aβ42-lowering GSMs have been studied with the hope of using them as therapeutic or prophylactic drugs for AD. However, the mechanism of action of GSMs is not well defined. We examined the effect of Aβ42-lowering GSMs on model cells producing large amounts of Aβ42: CHO cells expressing CTF1-51, a precursor peptide of Aβ that is mainly cleaved into Aβ42. Our results indicate that the effect of GSM in the model was weak. Thus, we conclude that CTF1-51 cleavage mainly yields Aβ42 and suppresses the effects of some GSMs, a phenomenon that may be related to their mechanism of action.     

Tissue plasminogen activator arrests Alzheimer's disease pathogenesis

The progressive deposition of amyloid-β (Aβ) in the brain is a pathologic feature of Alzheimer's disease (AD). This study was aimed to determine whether endogenous tissue plasminogen activator (tPA) modulates the pathogenic process of AD. tPA expression and activity developed around amyloid plaques in the brains of human amyloid precursor protein–overexpressing Tg2576 mice, which were weakened by the genetic ablation of tPA. Although the complete loss of tPA was developmentally fatal to Tg2576 mice, tPA-heterozygous Tg2576 mice expressed the more severe degenerative phenotypes than tPA wild-type Tg2576 mice, including abnormal and unhealthy growth, shorter life spans, significantly enhanced Aβ levels, and the deposition of more and larger amyloid plaques in the brain. In addition, the expression of synaptic function–associated proteins was significantly reduced, which in turn caused a more severe impairment in learning and memory performance in Tg2576 mice. Thus, endogenous tPA, preferentially its aggregate form, could degrade Aβ molecules and maintain low levels of brain Aβ, resulting in the delay of AD pathogenesis.     

Solubilized cystatin C amyloid is cytotoxic to cultured human cerebrovascular smooth muscle cells

Cerebral amyloid angiopathy (CAA) is characterized by the accumulation of amyloid within arteries of the cerebral cortex and leptomeninges. This condition is age related, especially prevalent in Alzheimer's disease (AD) and the main feature of certain hereditary disorders. The vascular smooth muscle cells (VSMC) appear to play a vital role in the development of CAA and have been found to produce the amyloid beta precursor protein (AbetaPP) and process it to Abeta the major component of most CAA amyloid. Moreover, synthesized Abeta has proven to be toxic to cerebral VSMC in culture possibly explaining the disintegration and disappearance of the muscle cells from affected cerebral blood vessels seen in CAA. An aggressive and extremely rare form of CAA, known as Hereditary Cerebral Hemorrhage With Amyloidosis-Icelandic Type (HCHWA-I), exhibits this withdrawal of VSMC as amyloid accumulates in the vessel wall. However, the amyloid in HCHWA-I is made from a variant of cystatin C (L68Q) instead of the more common Abeta. To evaluate possible cytotoxicity in this condition solubilized cystatin C amyloid extracted from HCHWA-I leptomeninges was applied to cerebral smooth muscle cells in culture and was found to kill the cells.     

High prevalence of pancreatic islet amyloid in patients with end-stage renal failure on dialysis treatment

Islet amyloid polypeptide (IAPP) is the main proteinaceous component of pancreatic islet amyloid, which is a characteristic feature of type 2 diabetes. The factors responsible for amyloid deposition are unclear. Patients with end-stage renal failure (ESRF) on dialysis treatment have increased insulin resistance which is associated with hypersecretion of beta-cell products. Furthermore, elevated concentrations of circulating IAPP are found in these patients due to reduced renal clearance of IAPP. To determine the prevalence of islet amyloid in this group of patients, pancreas was examined from 23 non-diabetic [aged 62 (29-79) years, median and range] and four type 2 diabetic [aged 67 (56-72) years] patients with ESRF on dialysis treatment. Pancreatic specimens from 30 non-diabetic control subjects [aged 67.5 (56-86) years] and 14 type 2 diabetic subjects without renal disease [aged 69 (48-86) years] were used as control groups. Islet amyloid was present in all type 2 diabetic patients with ESRF and in 12 out of 14 type 2 diabetic control subjects (86 per cent). Amyloid deposits were found in 8 out of 23 non-diabetic patients with ESRF (35 per cent), which was a higher prevalence than that found in non-diabetic control subjects (3 per cent) (P<0.01). This may be related to undiagnosed (pre)diabetes. Elevated secretion rates of IAPP due to insulin resistance and high circulation IAPP concentrations as a result of severely reduced renal clearance of IAPP will cause high pericellular concentrations of IAPP. This condition is likely to enhance amyloid fibril formation in pancreatic islets similar to that observed in type 2 diabetes.     

Calcium regulates the interaction of amyloid precursor protein with Homer3 protein

Ca²⁺ dysregulation is an important factor implicated in Alzheimer's disease pathogenesis. The mechanisms mediating the reciprocal regulation of Ca²⁺ homeostasis and amyloid precursor protein (APP) metabolism, function, and protein interactions are not well known. We have previously shown that APP interacts with Homer proteins, which inhibit APP processing toward amyloid-β. In this study, we investigated the effect of Ca²⁺ homeostasis alterations on APP/Homer3 interaction. Influx of extracellular Ca²⁺ upon treatment of HEK293 cells with the ionophore A23187 or addition of extracellular Ca²⁺ in cells starved of calcium specifically reduced APP/Homer3 but not APP/X11a interaction. Endoplasmic reticulum Ca²⁺ store depletion by thapsigargin followed by store-operated calcium entry also decreased the interaction. Interestingly, application of a phospholipase C stimulator, which causes inositol 1,4,5-trisphosphate-induced endoplasmic reticulum Ca²⁺ release, caused dissociation of APP/Homer3 complex. In human neuroblastoma cells, membrane depolarization also disrupted the interaction. This is the first study showing that changes in Ca²⁺ homeostasis affect APP protein interactions. Our results suggest that Ca²⁺ and Homers play a significant role in the development of Alzheimer's disease pathology.     

Long-term exposure to magnetic fields and the risks of Alzheimer's disease and breast cancer: Further biological research

Objective: Extremely low frequency (ELF) and radio frequency (RF) magnetic fields (MFs) pervade our environment. Whether or not these magnetic fields are associated with increased risk of serious diseases, e.g., cancers and Alzheimer's disease, is thus important when developing a rational public policy. The Bioinitiative Report was an effort by internationally recognized scientists who have spent significant time investigating the biological consequences of exposures to these magnetic fields to address this question. Our objective was to provide an unbiased review of the current knowledge and to provide our general and specific conclusions. Results: The evidence indicates that long-term significant occupational exposure to ELF MF may certainly increase the risk of both Alzheimer's disease and breast cancer. There is now evidence that two relevant biological processes (increased production of amyloid beta and decreased production of melatonin) are influenced by high long-term ELF MF exposure that may lead to Alzheimer's disease. There is further evidence that one of these biological processes (decreased melatonin production) may also lead to breast cancer. Finally, there is evidence that exposures to RF MF and ELF MF have similar biological consequences. Conclusion: It is important to mitigate ELF and RF MF exposures through equipment design changes and environmental placement of electrical equipment, e.g., AC/DC transformers. Further research related to these proposed and other biological processes is required.     

Classification of amyloid deposits in diagnostic cardiac specimens by immunofluorescence

BackgroundAt least 12 distinct forms of amyloidosis are known to involve the heart or great vessels. Patient treatment regimens require proper subtyping of amyloid deposits in small diagnostic cardiac specimens. A growing lack of confidence in immunohistochemical staining for subtyping amyloid has arisen primarily as a result of studies utilizing immunoperoxidase staining of formalin-fixed paraffin-embedded tissue. Immunofluorescence staining on fresh frozen tissue is generally considered superior to immunoperoxidase staining for subtyping amyloid; however, this technique has not previously been reported in a series of cardiac specimens.MethodsAmyloid deposits were subtyped in 17 cardiac specimens and 23 renal specimens using an immunofluorescence panel.ResultsAmyloid deposits were successfully subtyped as AL, AH, or AA amyloid by immunofluorescence in 82% of cardiac specimens and 87% of renal specimens. In all cases, the amyloid classification was in good agreement with available clinical and laboratory assessments. A cross-study analysis of 163 cases of AL amyloidosis reveals probable systemic misdiagnosis of cardiac AL amyloidosis by the immunoperoxidase technique, but not by the immunofluorescence technique.ConclusionsAmyloid deposits can be reliably subtyped in small diagnostic cardiac specimens using immunofluorescence. The practical aspects of implementing an immunofluorescence approach are compared with those of other approaches for subtyping amyloid in the clinical setting.     

No association of psychosis in Alzheimer disease with neurodegenerative pathway genes

Psychotic symptoms occur in approximately 40% of subjects with Alzheimer disease (AD with psychosis; AD + P) and identify a subgroup with more rapid cognitive decline. We evaluated in 867 AD subjects the association of AD + P with genes which may modify the pathological process via effects on the accumulation of amyloid beta (Aβ) protein and/or hyperphosphorylated microtubule-associated protein tau (MAPT): amyloid precursor protein (APP), beta-site amyloid precursor protein cleaving enzyme (BACE1), sortilin-related receptor (SORL1), and MAPT. Each gene was thoroughly interrogated with tag single-nucleotide polymorphisms (SNPs), and gene-based tests were used to enhance power. We found no association of these genes with AD + P.     

DMXB对β淀粉样蛋白所致痴呆小鼠的空间记忆及回避记忆的影响

目的:探讨选择性激动α7-N型乙酰胆碱受体(α7n ACh R)对痴呆小鼠空间参考记忆和被动回避记忆功能的影响。方法:小鼠45只随机分为3组:对照组、模型组、干预组。对照组侧脑室内注射生理盐水,模型组侧脑室内注射Aβ25-35,干预组连续14天腹腔注射α7n ACh R激动剂DMXB,采用Morris水迷宫和Y型电迷宫观察3组小鼠的行为学表现,电生理检测脑片海马CA1区突触长时程增强(LTP)以观察小鼠的学习记忆功能。结果:水迷宫训练第5天,模型组小鼠与对照组相比逃逸潜伏期增加,目标象限探索距离百分比、目标象限探索时间百分比、穿越原平台位置次数显著减少,干预组与对照组小鼠之间各项指标无显著性差异。与对照组相比,模型组小鼠在Y型电迷宫训练第5天的错误反应次数和全天总反应时间明显增加,干预组与模型组小鼠之间无显著性差异。侧脑室内注射Aβ25-35损害海马LTP诱导,DMXB的干预可恢复LTP。结论:Aβ25-35介导的神经毒性作用诱导AD小鼠,空间参考记忆和被动回避记忆明显减退,选择性激动α7n ACh R可以改善小鼠空间参考记忆,对被动回避记忆没有影响。     

17β-Estradiol regulates insulin-degrading enzyme expression via an ERβ/PI3-K pathway in hippocampus: Relevance to Alzheimer's prevention

Insulin-degrading enzyme (IDE), an enzyme that primarily degrades insulin, has recently been demonstrated to play a significant role in the catabolism of amyloid β (Aβ) protein in the brain. Reduced IDE expression and/or activity have been associated with the etiology and development of Alzheimer's disease (AD). Using three model systems, the present investigation provides the first documentation indicating that estrogen robustly regulates the expression of IDE in normal, menopausal and early-stage AD brains. In vitro analyses in primary cultures of rat hippocampal neurons revealed that 17β-estradiol (17β-E2) increased IDE in both mRNA and protein levels in a time-dependent manner. Further pharmacological analyses indicated that 17β-E2-induced IDE expression was dependent upon estrogen receptor (ER) β and required activation of phosphatidylinositol 3-kinase (PI3-K). In vivo analyses in adult female rats revealed a brain region-specific responsive profile. Ovariectomy (OVX) induced a significant decline in IDE expression in the hippocampus, which was prevented by 17β-E2. Neither OVX nor 17β-E2 affected IDE expression in the cerebellum. In vivo analyses in triple transgenic AD (3xTg-AD) female mice revealed an inverse correlation between the age-related increase in Aβ load and the decrease in IDE expression in the hippocampal formation. Treatment with 17β-E2 attenuated Aβ accumulation/plaque formation and elevated hippocampal IDE expression in 12-month-old 3xTg-AD OVX mice. Collectively, these findings indicate that 17β-E2 regulates IDE expression in a brain region-specific manner and such a regulatory role in the hippocampus, mediated by an ERβ/PI3-K pathway, could serve as a direct mechanism underlying estrogen-mediated preventative effect against AD when initiated at the onset of menopause.     

Amyloid-beta precursor protein mediates neuronal toxicity of amyloid beta through Go protein activation

Amyloid beta (Aβ) is a metabolic product of amyloid-β precursor protein (APP). Deposition of Aβ in the brain and neuronal degeneration are characteristic hallmarks of Alzheimer's disease (AD). Aβ induces neuronal degeneration, but the mechanism of neurotoxicity remains elusive. Here we show that overexpression of APP renders hippocampal neurons vulnerable to Aβ toxicity. Deletion of the extracellular Aβ sequence of APP prevents binding of APP to Aβ, and abolishes toxicity. Aβ toxicity is also abrogated by deletion of the cytoplasmic domain of APP, or by deletions comprising the Go protein-binding sequence of APP. Treatment with Pertussis toxin (PTX) abrogates APP-dependent toxicity of Aβ. Overexpression of PTX-insensitive Gα-o subunit, but not Gα-i subunit, of G protein restores Aβ toxicity in the presence of PTX, and this requires the integrity of APP-binding site for Go protein. Altogether, these experiments indicate that interaction of APP with toxic Aβ-species promotes toxicity in hippocampal neurons by a mechanism that involves APP-mediated Go protein activation, revealing an Aβ-receptor-like function of APP directly implicated in neuronal degeneration in AD.     

Cryptotanshinone,a compound from Salvia miltiorrhiza modulates amyloid precursor protein metabolism and attenuates β-amyloid deposition through upregulating α-secretase in vivo and in vitro

The amyloid precursor protein (APP) is cleaved enzymatically by non-amyloidogenic and amyloidogenic pathways. α-Secretase cleaves APP within β-amyloid protein (Aβ) sequence, resulting in the release of a secreted fragment of APP (sAPPα) and precluding Aβ generation. Cryptotanshinone (CTS), an active component of the medicinal herb Salvia miltiorrhiza, has been shown to improve learning and memory in several pharmacological models of Alzheimer's disease (AD). However, the effects of CTS on the Aβ plaque pathology and the APP processing in AD are unclear. Here we reported that CTS strongly attenuated amyloid plaque deposition in the brain of APP/PS1 transgenic mice. In addition, CTS significantly improved spatial learning and memory in APP/PS1 mice assessed by the Morris water maze testing. To define the exact molecular mechanisms involved in the beneficial effects of CTS, we investigated the effects of the CTS on APP processing in rat cortical neuronal cells overexpressing Swedish mutant human APP695. CTS was found to decrease Aβ generation in concentration-dependent (0–10 μM) manner. Interestingly, the N-terminal APP cleavage product, sAPPα was markedly increased by CTS. Further study showed that α-secretase activity was increased by CTS. Taken together, our results suggested CTS improved the cognitive ability in AD transgenic mice and promoted APP metabolism toward the non-amyloidogenic products pathway in rat cortical neuronal cells. CTS shows a promising novel way for the therapy of AD.     

Abnormal cleavage of APP impairs its functions in cell adhesion and migration

Amyloid precursor protein (APP) is expressed ubiquitously but its wrong cleavage only occurs in central nervous system. In this research, overexpression of wild type human APP695 was found to stimulate the adhesion and migration of N2a cells. In the cells co-transfected by familial Alzheimer’s disease (FAD)-linked Swedish mutant of APP695 gene plus E9 deleted presenilin1 gene (N2a/Swe.9), however, this stimulating function was impaired compared to that in the cells co-transfected by Swedish mutant of APP695 gene plus dominant negative mutant of presenilin1 D385A gene (N2a/Swe.385). Furthermore, it was also found that the phosphorylation of FAK Tyr-861 and GSK-3β Ser-9 was reduced in N2a/Swe.Δ9 cells, which can be possibly taken as a reasonable explanation for the underlying mechanism. Our results suggest that impaired cell adhesion and migration induced by abnormal cleavage of APP could contribute to the pathological effects in FAD brain.     

Amyloid Enhancing Factor (AEF) Isolation and Biochemical and Pathological Characteristics

Neutrophils and spleens were prepared from mice after treatment to induce amyloid deposition. The deposition of amyloid was accelerated in normal recipients by intravenous injection of more than 2x10⁴ neutrophils, and intraperitoneal injection of supernatants obtained by homogenization and centrifugation of the neutrophils and spleens. The supernatants were subjected individually to DEAE ion–exchange chromatography. Amyloid enhancing factor (AEF) activity was present in peaks eluted at an NaCl concentration of 0.17 M. The fractions containing AEF were subjected to high-performance liquid chromatography (HPLC), and AEF was eluted at a position corresponding to about 15 KDa. Purified AEF was analyzed by amino acid sequencing and gas chromatography. The N-terminal amino acid was blocked, and AEF contained some saccharides including glucose, mannose, galactosamine and sialic acid, and an undefined substance (probably derived from certain proteins). Immunoelectron microscopy by the preembedding method using an anti-AEF antiserum demonstrated that the cytosol, but not primary and specific granules in neutrophils from the spleen of amyloid-laden mice, reacted with the antiserum. These findings suggest that AEF is a glycoprotein associated with neutrophils.     

Amyloid enhancing factor (AEF) in the aging mouse

Summary Amyloid enhancing factor (AEF) activity was tested in spleen homogenates from 52, 28, and 9-week-old untreated mice. Strong AEF activity was present in the supernatant of spleen homogenate from the 52-week-old mice. Only weak activity was observed in the supernatant from the 28-week-old mice, and none was seen in that from the 9-week-old mice. Crude spleen homogenates from the 52-week-old mice were subjected to Sephacryl S-300 gel filtration. AEF activity existed in the first gel filtration peak. These findings suggest that AEF increases in the spleens of aged mice and that AEF may be a substance of high molecular weight.     

The pathogenesis and biochemistry of amyloidosis

The transformation of serum proteins into Congo red-sensitive fibrillar material is requisite for the onset and progression of amyloid disease. All the mechanisms which lead to the disease itself have not been elucidated, but our knowledge has increased significantly. It is apparent that in all types of amyloid fibrils, three common features are displayed by the major protein constituents. These are that the fibril protein has a serum precursor, a high degree of anti-parallel beta-sheet conformation and a distinctive ultrastructure on electron microscopy. In the AL and AA forms of amyloidosis, the putative precursors appear to undergo limited degradation to form the protein component of amyloid fibrils. It has been suggested that there may be certain primary structural characteristics inherent in precursor molecules which make them amyloidogenic, thus predisposing them to amyloid fibril formation. This would include certain subtypes of immunoglobulin light chains, possibly kappa I and lambda VI, in the AL type of amyloidosis and one of the polymorphic SAA species, SAA2, which has been identified as the predominating isotype found in AA amyloid fibrils. In AH amyloidosis, the mechanism of amyloid fibril formation appears to be simply a concentration phenomenon where elevated concentrations of B2-M are not handled normally and amyloid deposition is the result. Amyloidogenesis in the hereditary form of systemic amyloidosis may involve other factors in addition to the presence of a variant precursor prealbumin as indicated by the delayed onset of the disease. It is evident that the elucidation of the mechanism(s) which governs the onset and progression of the amyloidoses will allow future regulation and treatment of these all too often complex disorders.     

Suspected non-Alzheimer's pathology – Is it non-Alzheimer's or non-amyloid?

Neurodegeneration, the progressive loss of neurons, is a major process involved in dementia and age-related cognitive impairment. It can be detected clinically using currently available biomarker tests. Suspected Non-Alzheimer Pathology (SNAP) is a biomarker-based concept that encompasses a group of individuals with neurodegeneration, but no evidence of amyloid deposition (thereby distinguishing it from Alzheimer's disease (AD)). These individuals may often have a clinical diagnosis of AD, but their clinical features, genetic susceptibility and progression can differ significantly, carrying crucial implications for precise diagnostics, clinical management, and efficacy of clinical drug trials.SNAP has caused wide interest in the dementia research community, because it is still unclear whether it represents distinct pathology separate from AD, or whether in some individuals, it could represent the earliest stage of AD. This debate has raised pertinent questions about the pathways to AD, the need for biomarkers, and the sensitivity of current biomarker tests.In this review, we discuss the biomarker and imaging trials that first recognized SNAP. We describe the pathological correlates of SNAP and comment on the different causes of neurodegeneration. Finally, we discuss the debate around the concept of SNAP, and further unanswered questions that are emerging.