Absence of Nitric Oxide Synthase 3 Increases Amyloid <i>β</i>-Protein Pathology in Tg-5xFAD Mice

Aim: The abnormal accumulation, assembly and deposition of the amyloid β-protein (Aβ) are prominent pathological features of patients with Alzheimer’s disease (AD) and related disorders. A number of factors in the brain can influence Aβ accumulation and associated pathologies. The aim of the present study was to determine the consequences of deleting nitric oxide synthase (NOS) 3, the endothelial form of NOS, in Tg-5xFAD mice, a model of parenchymal AD-like amyloid pathology. Methods: Tg-5xFAD mice were bred with NOS3-/- mice. Cohorts of Tg-5xFAD mice and bigenic Tg-5xFAD/NOS3-/- mice were aged to six months followed by collection of the blood and brain tissues from the mice for biochemical and pathological analyses. Results: ELISA analyses show that the absence of NOS3 results in elevated levels of cerebral and plasma Aβ peptides in Tg-5xFAD mice. Immunohistochemical analyses show that the absence of NOS3 increased the amount of parenchymal Aβ deposition and fibrillar amyloid accumulation in Tg-5xFAD mice. The elevated levels of Aβ were not due to changes in the expression levels of transgene encoded human amyloid precursor protein (APP), endogenous β-secretase, or increased proteolytic processing of APP. Conclusions: The results from this study suggest that the loss of NOS3 activity enhances Aβ pathology in Tg-5xFAD mice. These findings are similar to previous studies of NOS2 deletion suggesting that reduced NOS activity and NO levels enhance amyloid-associated pathologies in human APP transgenic mice.

Molecular basis of cross-interactions between Aβ and Tau protofibrils probed by molecular simulations

Amyloid β-protein(Aβ) and Tau, two common pathogenic proteins associated with Alzheimer’s disease(AD), cross-interact, and thus co-assemble into hybrid aggregates. However, molecular mechanism of the cross-interactions remains unclear. To explore the issue, docking and molecular dynamics(MD) simulations were coupled to study the cross-interactions between Aβ pentamer and Tau pentamer. Four stable hybrid decamer conformations including double layer, single layer, block, and part-in were obtained by protein-protein docking software HADDOCK 2.2. Then, MD simulations were used to explore the molecular mechanism of cross-interactions between Aβ pentamer and Tau pentamer. The results of MD simulations showed that the part-in structure was the most stable among all the above four representative ones. The binding energy between Aβ and Tau was about-759.77 kJ·mol-1in the part-in structure. Moreover, the part-in conformation would undergo conformational transition, which would improve its hydrophobicity and make the structure more compact. This work offers a structural understanding of cross-interactions between Aβ and Tau linked to AD.

β-淀粉样肽鼠脑室内灌注制作阿尔茨海默病动物模型的实验技术研究

目的 探讨 β -淀粉样肽鼠脑室内灌注制作阿尔茨海默病 (AD)模型过程中的技术问题。 方法 经颅骨穿刺进入鼠侧脑室 ,以微渗透泵持续灌注 β -淀粉样肽 (Aβ) ,使Aβ弥散入脑室周围及皮层 ,造成与神经元相关的Aβ沉积等类似AD的病理变化。 4周后鼠脑冠状位连续切片 ,在普通光学显微镜下观察脑导管进入侧脑室的痕迹。结果 大鼠有 92 % (5 5 /6 0 )导管准确进入侧脑室 ,接近进入或在皮层 5 % (3/6 0 ) ,小鼠仅 4 3% (2 3/5 4 )导管进入侧脑室 ,接近进入或在皮层 31% (17/5 4 )。结论 大鼠脑室内灌注Aβ的方法可作为AD动物模型的方法之一。

Huge primitive neuroectodermal tumor of the pancreas:Report of a case and review of the literature

Primitive neuroectodermal tumor (PNET) of the pancreas is an extremely rare tumor that usually occurs in children or young adults. We report a case of a 33-year-old male patient with an 18 cm × 18 cm × 16 cm mass arising from the pancreatic body and tail with a one- day history of abdominal pain. Initial CT scan showed no signs of metastatic tumor spread. The tumor caused intrabdominal bleeding and the patient underwent primary tumor resection including partial gastrectomy, left pancreatic resection and splenectomy. Diagnosis of PNET was confi rmed by histology, immunohistochemistry and FISH analysis. All neoplastic cells were stained positive for MIC2-protein (CD99). Approximately one month after surgery, several liver metastases were observed and the patient underwent chemotherapy according to the Euro- Ewing protocol. Subsequent relaparotomy excluded any residual hepatic or extrahepatic abdominal metastases. Although PNET in the pancreas is an extremely rare entity, it should be considered in the diffential diagnosis of pancreatic masses, especially in young patients. This alarming case particularly illustrates that PNET in the pancreas although in an advanced stage can present with only a short history of mild symptoms.

Schisandrin B protects PC12 cells by decreasing the expression of amyloid precursor protein and vacuolar protein sorting 35

PC12 cell injury was induced using 20 μM amyloid β-protein 25-35 to establish a model of Alzheimer＇s disease. The cells were then treated with 5, 10, and 25 μM Schisandrin B. Methylthiazolyldiphenyl-tetrazolium bromide assays and Hoechst 33342 staining results showed that with increasing Schisandrin B concentration, the survival rate of PC12 cells injured by amyloid β-protein 25-35 gradually increased and the rate of apoptosis gradually decreased. Reverse transcription-PCR, immunocytochemical staining and western blot results showed that with increasing Schisandrin B concentration, the mRNA and protein expression of vacuolar protein sorting 35 and amyloid precursor protein were gradually decreased. Vacuolar protein sorting 35 and amyloid precursor protein showed a consistent trend for change. These findings suggest that 5, 10, and 25 μM Schisandrin B antagonizes the cellular injury induced by amyloid β-protein 25-35 in a dose-dependent manner. This may be caused by decreasing the expression of vacuolar protein sorting 35 and amyloid precursor protein.

Head-to-tail cyclization of a heptapeptide eliminates its cytotoxicity and significantly increases its inhibition effect on amyloid β-protein fibrillation and cytotoxicity

Amyloid-β （Aβ） protein aggregation is the main hallmark of Alzheimer＇s disease （AD）. Inhibition of Aft fibrillation is thus a promising therapeutic approach to the prevention and treatment of AD. Recently, we designed a heptapeptide inhibitor, LVFFARK （LK7）. LK7 shows a promising inhibitory capability on Aft fibrillation, but is prone to self-assembling and displays high cytotoxicity, which would hinder its practical application. Herein, we modified LK7 by a head-to-tail cyclization and obtained a cyclic LK7 （cLK7）. cLK7 exhibits a different self-assembly behavior from LK7, and has higher stability against proteolysis than LK7 and little cytotoxicity to SH-SY5Y cells. Thermodynamic analysis revealed that both LK7 and cLK7 could bind to Aβ40 by electrostatic interactions, hydrogen bonding and hydrophobic interactions, but the binding affinity of cLK7 for Afl40 （KD = 4.96 μmol/L） is six times higher than that of LK7 （KD = 32.2 μmol/L）. The strong binding enables cLK7 to stabilize the secondary structure of Aβ40 and potently inhibit its nucleation, fibrillation and cytotoxicity at extensive concentration range, whereas LK7 could only moderately inhibit Aβ40 fibrillation and cytotoxicity at low concentrations. The findings indicate that the peptide cyclization is a promising approach to enhance the performance of peptide-based amyloid inhibitors.

基于“Aβ异常沉积”浅析中医“从痰论治”阿尔茨海默病

阿尔茨海默病(AD)是常见的神经退行性疾病,其典型的病理特征为老年斑和神经元纤维缠结,而Aβ异常沉积是老年斑形成的关键,是Aβ级联假说的中心环节,是AD病理改变的核心。因此,AD的治疗可从调节Aβ沉积入手,以Aβ为靶点的治疗给AD带来希望,但相关药物的研发均告失败。从中医的视角,'痰邪'是AD关键的病因,是AD病机的核心,'痰邪'与Aβ异常沉积密切相关,Aβ异常沉积可归属'痰邪'的范畴。从痰论治能减少Aβ沉积是AD有效治法,推测从痰论治可能通过靶向调节Aβ沉积取效,Aβ是其重要的治疗靶点,但这还需进一步的研究证实。

阿尔茨海默病突触功能障碍与Aβ的关系

阿尔茨海默病（AD）是不可逆、进行性脑病,缓慢损害记忆和认知功能,是最常见的痴呆类型,给患者、家属和社会带来沉重的经济和精神负担。年龄是最重要的危险因素。65岁以后每隔五年AD发病率翻番。AD也可在中年发病,早发型痴呆指65岁之前发病的痴呆类型,较少见,其病理表现与晚发型相似。临床上AD的确切发病时间难以确定,常常表现为轻微发作性记忆障碍，之后出现数月的缓慢进展，并出现其他认知功能损害，数年后出现显著的痴呆。

Intracellular distribution of amyloid beta peptide and its relationship to the lysosomal system

Background:Amyloid beta peptide(Aβ)is the main component of extraneuronal senile plaques typical of Alzheimer’s disease(AD)brains.Although Aβis produced by normal neurons,it is shown to accumulate in large amounts within neuronal lysosomes in AD.We have recently shown that under normal conditions the majority of Aβis localized extralysosomally,while oxidative stress significantly increases intralysosomal Aβcontent through activation of macroautophagy.It is also suggested that impaired Aβsecretion and resulting intraneuronal increase of Aβcan contribute to AD pathology.However,it is not clear how Aβis distributed inside normal neurons,and how this distribution is effected when Aβsecretion is inhibited.Methods:Using retinoic acid differentiated neuroblastoma cells and neonatal rat cortical neurons,we studied intracellular distribution of Aβby double immunofluorescence microscopy for Aβ40 or Aβ42 and different organelle markers.In addition,we analysed the effect of tetanus toxin-induced exocytosis inhibition on the intracellular distribution of Aβ.Results:Under normal conditions,Aβwas found in the small cytoplasmic granules in both neurites and perikarya.Only minor portion of Aβwas colocalized with trans-Golgi network,Golgi-derived vesicles,early and late endosomes,lysosomes,and synaptic vesicles,while the majority of Aβgranules were not colocalized with any of these structures.Furthermore,treatment of cells with tetanus toxin significantly increased the amount of intracellular Aβin both perikarya and neurites.Finally,we found that tetanus toxin increased the levels of intralysosomal Aβalthough the majority of Aβstill remained extralysosomally.Conclusion:Our results indicate that most Aβis not localized to Golgi-related structures,endosomes,lysosomes secretory vesicles or other organelles,while the suppression of Aβsecretion increases intracellular intra-and extralysosomal Aβ.

Magnesium ion influx reduces neuroinflammation in Aβ precursor protein/Presenilin 1 transgenic mice by suppressing the expression of interleukin-1β

Alzheimer＇s disease （AD） has been associated with magnesium ion （Mg2＋） deficits and interleukin-1β（IL-1β） elevations in the serum or brains of AD patients. However, the mechanisms regulating IL-1β expression during Mg2＋ dyshomeostasis in AD remain unknown. We herein studied the mechanism of IL-1β reduction using a recently developed compound, magnesium-L-threonate （MgT）. Using human glioblastoma A172 and mouse brain DIA glial cells as an in vitro model system, we delineated the signaling pathways by which MgT suppressed the expression of IL-1β in glial cells. In detail, we found that MgT incubation stimulated the activity of extracellular signal-regulated protein kinases I and 2 （ERK1/2） and peroxisome proliferator-activated receptor gamma （PPARγ） signaling pathways by phosphorylation, which resulted in IL-1β suppression. Simultaneous inhibition of the phosphorylation of ERK1/2 and PPARγ, induced IL-1β upregulation in MgT-stimulated glial cells. In accordance with our in vitrodata, the intracerebroventricular （i.c.v） injection of MgT into the ventricles of APP/PS1 transgenic mice and treatment of Aβ precursor protein （APP）/PS1 brain slices suppressed the mRNA and protein expression of IL-1β. These in vivo observations were further supported by the oral administration of MgT for 5 months. Importantly, Mg2＋ influx into the ventricles of the mice blocked the effects of IL-1β or amyloid p-protein oligomers in the cerebrospinal fluid. This reduced the stimulation of IL-1β expression in the cerebral cortex of APP/PS1 transgenic mice, which potentially contributed to the inhibition of neuroinflammation.

Atomistic characterization of binding modes and affinity of peptide inhibitors to amyloid-β protein

Abstract The aggregation of amyloid β-protein （Aβ） is tightly linked to the pathogenesis of Alzheimer＇s disease. Previous studies have found that three peptide inhibitors （i.e., KLVFF, VVIA, and LPFFD） can inhibit Aβ aggregation and alleviate Aβ-induced neurotoxicity. How- ever, atomic details of binding modes and binding affinities between these peptide inhibitors and Aβ have not been revealed. Here, using molecular dynamics simulations and molecular mechanics Poisson Boltzmann surface area （MM/PBSA） analysis, we examined the effect of three peptide inhibitors （KLVFF, VVIA, and LPFFD） on their sequence-specific interactions with Aβ and the molecular basis of their inhibition. All inhibitors exhibit varied binding affinity to Aβ, in which KLVFF has the highest binding affinity, whereas LPFFD has the least. MM/PBSA analysis further revealed that different peptide inhibitors have different modes of interaction with Aβ, consequently hotspot binding residues, and underlying driving forces. Specific residue-based interactions between inhibitors and Aβ were determined and compared for illustrating different binding and inhibition mechanisms. This work provides structure-based binding information for further modifica- tion and optimization of these three peptide inhibitors to enhance their binding and inhibitory abilities against Aβ aggregation.

The role of amyloid beta clearance in cerebral amyloid angiopathy:more potential therapeutic targets

Cerebral amyloid angiopathy(CAA)is characterized by the deposition of amyloid β-protein(Aβ)in the leptomeningeal and cortical blood vessels,which is an age-dependent risk factor for intracerebral hemorrhage(ICH),ischemic stroke and contributes to cerebrovascular dysfunction leading to cognitive impairment.However clinical prevention and treatment of the disease is very difficult because of its occult onset and severity of the symptoms.In recent years,many anti-amyloid β immunotherapies have not demonstrated clinical efficacy in subjects with Alzheimer’s disease(AD),and the failure may be due to the deposition of Aβ in the cerebrovascular export pathway resulting in further damage to blood vessels and aggravating CAA.So decreased clearance of Aβ in blood vessels plays a crucial role in the development of CAA and AD,and identification of the molecular pathways involved will provide new targets for treatment.In this review,we mainly describe the mechanisms of Aβ clearance through vessels,especially in terms of some proteins and receptors involved in this process.