Libang Yang

Elevated β-secretase expression and enzymatic activity detected in sporadic Alzheimer disease [1]

Elevated β-secretase expression and enzymatic activity detected in sporadic Alzheimer disease

Inflammatory repertoire of Alzheimer's disease and nondemented elderly microglia in vitro

We have previously developed and characterized isolated microglia and astrocyte cultures from rapid (<4 h) brain autopsies of Alzheimers disease (AD) and nondemented elderly control (ND) patients. In the present study, we evaluate the inflammatory repertoire of AD and ND microglia cultured from white matter (corpus callosum) and gray matter (superior frontal gyrus) with respect to three major proinflammatory cytokines, three chemokines, a classical pathway complement component, a scavenger cell growth factor, and a reactive nitrogen intermediate. Significant, dose‐dependent increases in the production of pro‐interleukin‐1β (pro‐IL‐1β), interleukin‐6 (IL‐6), tumor necrosis factor‐α (TNF‐α), monocyte chemoattractant protein‐1 (MCP‐1), macrophage inflammatory peptide‐1α (MIP‐1α), IL‐8, and macrophage colony‐stimulating factor (M‐CSF) were observed after exposure to pre‐aggregated amyloid β peptide (1–42) (Aβ1–42). Across constitutive and Aβ‐stimulated conditions, secretion of complement component C1q, a reactive nitrogen intermediate, and M‐CSF was significantly higher in AD compared with ND microglia. Taken together with previous in situ hybridization findings, these results demonstrate unequivocally that elderly human microglia provide a brain endogenous source for a wide range of inflammatory mediators. GLIA 35:72–79, 2001.

Tumor Necrosis Factor Death Receptor Signaling Cascade Is Required for Amyloid-β Protein-Induced Neuron Death

Tumor necrosis factor type I receptor (TNFRI), a death receptor, mediates apoptosis and plays a crucial role in the interaction between the nervous and immune systems. A direct link between death receptor activation and signal cascade-mediated neuron death in brains with neurodegenerative disorders remains inconclusive. Here, we show that amyloid-β protein (Aβ), a major component of plaques in the Alzheimers diseased brain, induces neuronal apoptosis through TNFRI by using primary neurons overexpressing TNFRI by viral infection or neurons from TNFRI knock-out mice. This was mediated via alteration of apoptotic protease-activating factor (Apaf-1) expression that in turn induced activation of nuclear factor κB (NF-κB). Aβ-induced neuronal apoptosis was reduced with lower Apaf-1 expression, and little NF-κB activation was found in the neurons with mutated Apaf-1 or a deletion of TNFRI compared with the cells from wild-type (WT) mice. Our studies suggest a novel neuronal response of Aβ, which occurs through a TNF receptor signaling cascade and a caspase-dependent death pathway.

Estrogen Enhances Uptake of Amyloid β‐Protein by Microglia Derived from the Human Cortex

Abstract: In recent years, inflammatory mechanisms have been increasingly appreciated as important steps in the pathology of Alzheimers disease (AD). There are two pathological defects in AD: chronic inflammation and impaired clearance of amyloid β‐peptide (Aβ). In the periphery, estrogen both increases macrophage phagocytosis and has antiinflammatory effects. If estrogen had a similar effect in the CNS, it could reverse inflammatory defects in AD. Although microglia are a key component of the immune system and help clear Aβ deposits in the AD brain, little is known about the effects of estrogen on CNS microglia. Therefore, we sought to determine the relationship between estrogen treatment and internalization of Aβ by microglia by quantifying the internalization of aggregated Aβ by human cortical microglia. Aβ uptake was found to be dose‐ and time‐dependent in cultured microglia. Increased Aβ uptake was observed at 1.5 and 24 h after addition of aggregated Aβ (50, 100, or 1,000 nM Aβ), and this uptake was enhanced by pretreatment with estrogen. The expression of estrogen receptor (ER) β (ER‐β) was also up‐regulated by estrogen treatment. Cells cotreated with ICI 182,780, an ER antagonist, showed significantly reduced internalization of Aβ in cultured microglia. These results indicate that microglia express an ER‐β but that the effect of estrogen on enhancing clearance of Aβ may be related to the receptor‐independent action of estrogen or to nonclassical ER effects of estrogen. Thus, stimulation of the ER might contribute to the therapeutic action of estrogen in the treatment of AD.

Complement activation by neurofibrillary tangles in Alzheimer's disease

Brain inflammation is widely documented to occur in Alzheimers disease (AD), but its sources are still incompletely understood. Here, we present in vitro and in situ evidence that, like amyloid beta peptide (Abeta), tau, the major protein constituent of the neurofibrillary tangle, is a potent, antibody-independent activator of the classical complement pathway. Complement activation, in turn, is known to drive numerous inflammatory responses, including scavenger cell activation and cytokine production. Because Abeta deposits and extracellular tangles are present from early preclinical to terminal stages of AD, their ability to activate complement provides a ready mechanism for initiating and sustaining chronic, low-level inflammatory responses that may cumulate over the disease course.

Differential activation of tumor necrosis factor receptors distinguishes between brains from Alzheimer's disease and non-demented patients.

We reported that tumor necrosis factor receptor I (TNFRI) is required for neuronal death induced by amyloid-beta protein in the Alzheimers disease (AD) brain. However, whether TNF receptor subtypes are expressed and activated differentially in AD brains compared to non-demented brains remains unclear. Our studies on Western blot and ELISA measurements demonstrated that TNFRI levels are increased whereas TNFRII levels are decreased in AD brains compared to non-demented brains (p <0.05). Immunohistochemical results demonstrated that both TNFRI and TNFRII are expressed in neurons in AD and non-demented brains. However, in situ hybridization studies showed little change in the mRNA levels of either type of TNF receptor in the neurons of AD brains compared to non-demented brains. To examine whether different levels of TNF receptors in AD brains are correlated with the alteration of functional binding of TNF receptors, by using 125I-TNF-alpha binding technique, we found that, in AD brains, 125I-TNF-alpha binding affinity to TNFRI is increased, whereas binding affinity to TNFRII is decreased (p < 0.01). These studies reveal a novel observation of abnormal TNF receptor activation in AD brains. Differential TNF receptor protein levels and binding affinities suggest distinct pathogenic mechanisms of neurodegeneration in the AD brain.

Myocardial Tissue Engineering with Cells Derived from Human-Induced Pluripotent Stem Cells and a Native-Like, High-Resolution, 3-Dimensionally Printed Scaffold

Rationale: Conventional 3-dimensional (3D) printing techniques cannot produce structures of the size at which individual cells interact. Objective: Here, we used multiphoton-excited 3D printing to generate a native-like extracellular matrix scaffold with submicron resolution and then seeded the scaffold with cardiomyocytes, smooth muscle cells, and endothelial cells that had been differentiated from human-induced pluripotent stem cells to generate a human-induced pluripotent stem cell–derived cardiac muscle patch (hCMP), which was subsequently evaluated in a murine model of myocardial infarction. Methods and Results: The scaffold was seeded with ≈50 000 human-induced pluripotent stem cell–derived cardiomyocytes, smooth muscle cells, and endothelial cells (in a 2:1:1 ratio) to generate the hCMP, which began generating calcium transients and beating synchronously within 1 day of seeding; the speeds of contraction and relaxation and the peak amplitudes of the calcium transients increased significantly over the next 7 days. When tested in mice with surgically induced myocardial infarction, measurements of cardiac function, infarct size, apoptosis, both vascular and arteriole density, and cell proliferation at week 4 after treatment were significantly better in animals treated with the hCMPs than in animals treated with cell-free scaffolds, and the rate of cell engraftment in hCMP-treated animals was 24.5% at week 1 and 11.2% at week 4. Conclusions: Thus, the novel multiphoton-excited 3D printing technique produces extracellular matrix–based scaffolds with exceptional resolution and fidelity, and hCMPs fabricated with these scaffolds may significantly improve recovery from ischemic myocardial injury.

Induction of Tumorigenesis and Metastasis by the Murine Orthologue of Tumor Protein D52

Expression studies have consistently identified tumor protein D52 (TPD52) overexpression in tumor cells. Murine TPD52 (mD52) shares 86% identity with the human orthologue. To study a possible role for TPD52 in transformation, 3T3 fibroblasts were transfected with the full-length cDNA for mD52. Expression of mD52 was confirmed by reverse transcription-PCR (RT-PCR), real-time PCR, and Western blot analysis compared with 3T3 and vector-transfected 3T3 (3T3.V), and the resultant cell line was designated 3T3.mD52. At 4 weeks, 3T3.mD52 gained a 2-fold increase in growth rate, lost contact inhibition, and exhibited a marked phenotype change. Further characterization revealed an acquired ability for anchorage-independent cell growth. To determine whether 3T3.mD52 had become tumorigenic, naïve, healthy, immunocompetent syngeneic mice were inoculated subcutaneously with varying cell doses. Tumors measuring >1 cm2 were detected 60 days postinoculation with 3T3.mD52, and a 50% subcutaneous tumor incidence was obtained with as few as 5 × 105 3T3.mD52 cells. Remarkably, when lungs from 3T3.mD52 tumor-bearing mice were analyzed, numerous tumor nodules were observed, ranging from nodules less than 10 to nodules too numerous to count (inoculation with 1 × 105 and 5 × 106 cells, respectively). Further support for the metastatic capacity of 3T3.mD52 was the demonstration that transforming growth factor (TGF)-βR1 (receptor) expression decreased and TGF-β1 secretion increased in 3T3.mD52 compared with 3T3 controls. cDNA microarray analysis showed a gene expression pattern that further supported mD52-induced transformation and metastasis. Together, these data suggest that mD52 expression in 3T3 cells initiated cellular transformation, tumorigenesis, and progression to metastasis. (Mol Cancer Res 2007;5(2):133–44)

Isolation of living neurons from human elderly brains using the immunomagnetic sorting DNA-linker system.

Isolation and culture of mature neurons from affected brain regions during diseased states provide a well-suited in vitro model system to study age-related neurodegeneration under dynamic conditions at cellular levels. We have developed a novel technique to isolate living neurons from rapidly autopsied human elderly brains, and have succeeded in keeping them alive in vitro. Specifically, the parietal cortex blocks were fractionated by density gradients and further enriched for neurons by an immunomagnetic sorting DNA-linker technique. The postmortem interval averaged 2.6 hours. After isolation and purification of neurons using this technology, the cells were maintained in vitro for 2 weeks. Our evaluation revealed that 80% of the isolated cells were neurons and they exhibited neurotransmitter phenotypes (glutamate and gamma-aminobutyric acid) as well as glutamate receptors. Studies on cell viability and calcium influx suggest that these isolated living cortical neurons still retain their typical neuronal functions. Our present study demonstrates that neurons isolated from human elderly brain autopsies can survive in vitro and maintain their functional properties. Our study has opened an opportunity to apply such neurons to dynamic pharmacological studies of neurological disorders at the single-cell level.

What does complement do in Alzheimer's disease? Old molecules with new insights

Increasing evidence suggests that inflammatory and immune components in brain are important in Alzheimer’s disease (AD) and anti-inflammatory and immunotherapeutic approaches may be amenable to AD treatment. It is known that complement activation occurs in the brain of patients with AD, and contributes to a local inflammatory state development which is correlated with cognitive impairment. In addition to the complement’s critical role in the innate immune system recognizing and killing, or targeting for destruction, complement proteins can also interact with cell surface receptors to promote a local inflammatory response and contributes to the protection and healing of the host. On the other hand, complement activation also causes inflammation and cell damage as an essential immune function to eliminate cell debris and potentially toxic protein aggregates. It is the balance of these seemingly competing events that influences the ultimate state of neuronal function. Our mini review will be focusing on the unique molecular interactions happening in the AD development, the functional outcomes of those interactions, as well as the contribution of each element to AD.