Whether elevated ␤-secretase (BACE) activity is related to plaque formation or amyloid ␤ peptide (A␤) production in Alzheimer's disease (AD) brains remains inconclusive. Here, we report that we used sandwich enzyme-linked immunoabsorbent assay to quantitate various A␤ species in the frontal cortex of AD brains homogenized in 70% formic acid. We found that most of the A␤ species detected in rapidly autopsied brains (<3 h) with sporadic AD were A␤ 1-x and A␤ 1-42, as well as A␤x-42. To establish a linkage between A␤ levels and BACE, we examined BACE protein, mRNA expression and enzymatic activity in the same brain region of AD brains. We found that both BACE mRNA and protein expression is elevated in vivo in the frontal cortex. The elevation of BACE enzymatic activity in AD is correlated with brain A␤ 1-x and A␤1-42 production. To examine whether BACE elevation was due to mutations in the BACE-coding region, we sequenced the entire ORF region of the BACE gene in these same AD and nondemented patients and performed allelic association analysis. We found no mutations in the ORF of the BACE gene. Moreover, we found few changes of BACE protein and mRNA levels in Swedish mutated amyloid precursor protein-transfected cells. These findings demonstrate correlation between A␤ loads and BACE elevation and also suggest that as a consequence, BACE elevation may lead to increased A␤ production and enhanced deposition of amyloid plaques in sporadic AD patients. A lzheimer's disease (AD) is the most common cause of dementia in the population Ͼ60 years of age. Senile plaques and paired helical filaments are the two hallmarks of the brain pathology of AD (1-3). Amyloid ␤ peptide (A␤), a major protein component (4 kDa) of the senile plaque (4), is generated from amyloid precursor protein (APP) by enzymatic digestion involving ␤-secretase (BACE) and ␥-secretase activities.The mechanisms of A␤ accumulation in the majority of AD patients (sporadic AD) remain unclear although a minority of AD patients carry mutations in the APP and presenilin (PS) genes, which lead to an increase in A␤ production (5). A␤ is the cleavage product from APP by two enzymes: BACE and ␥-secretase. BACE is a transmembrane aspartyl protease and has recently been cloned and characterized (6-10). Overexpression of BACE in transfected cells increases the amount of C99 and C89, which are both BACE-cleavage products. More BACE-cleaved APP products were found in the Swedish mutation (APPsw) as compared with that in wild-type substrate (APPwt) (6). The BACE cleavage occurs at the known ␤-cleavage sites of APP, Asp 1, and Glu 11 (6-10). The role of BACE played in A␤ production in vitro might explain the higher production of A␤ peptide in AD brains and the early onset of Swedish familial AD. Recently, we and other investigators demonstrated higher BACE expression levels found in sporadic AD brains compared with healthy age-matched controls (11-13). A␤ accumulation in the AD brain is a chronic process and a small elevation of BACE might lead to a significant increase in ...

Rationale Conventional three-dimensional (3D) printing techniques cannot produce structures of the size at which individual cells interact. Objective Here, we used multiphoton-excited, 3-dimensional printing (MPE-3DP) to generate a native-like, extracellular matrix (ECM) scaffold with submicron resolution, and then seeded the scaffold with cardiomyocytes (CMs), smooth-muscle cells (SMCs), and endothelial cells (ECs) that had been differentiated from human induced-pluripotent stem cells (iPSCs) to generate a human, iPSC-derived cardiac muscle patch (hCMP), which was subsequently evaluated in a murine model of myocardial infarction (MI). Methods and Results The scaffold was seeded with ~50,000 human, iPSC-derived CMs, SMCs, and ECs (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 MI, 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 MPE-3DP technique produces ECM-based scaffolds with exceptional resolution and fidelity, and hCMPs fabricated with these scaffolds may significantly improve recovery from ischemic myocardial injury.

Tumor necrosis factor receptor-I (TNFRI) and TNFRII are two TNFR subtypes in the immune system, but their roles in the brain remain unclear. Here we present a novel interaction between TNFR subtypes and TNF-alpha in the brain. Our studies on target-depleted TNFR in mice show that TNF-alpha has little effect on hippocampal neurons in which TNFRI, containing an "intracellular death domain," is absent (TNFRI -/-), whereas neurons from TNFRII knock-out mice are vulnerable to TNF-alpha even at low doses. Moreover, little nuclear factor-kappaB (NF-kappaB) translocation is induced by TNF-alpha in neurons of TNFRI -/-, whereas NF-kappaB subunit p65 is still translocated from the cytoplasm into the nucleus in neurons from wild-type and TNFRII -/- mice. Furthermore, p38 mitogen-activated protein (MAP) kinase activity is upregulated in neurons from both wild-type and TNFRI -/-, but no alteration of p38 MAP kinase was found in neurons from TNFRII. Results from overexpression of TNF receptors further support the above findings. NT2 neuronal-like cells transiently transfected with TNFRI are very sensitive to TNF-alpha, whereas TNF-alpha is not toxic and even seems to be trophic to the cells with TNFRII overexpression. Last, our radioligand-binding experiments demonstrate that TNF-alpha binds TNFRI with high affinity (K(d) of 0.6 nm), whereas TNFRII shows lower binding affinity (K(d) of 1.14 nm) to TNF-alpha in NT2 transfected cells. Together, these studies reveal novel neuronal responses of TNF-alpha in mediating consequences of TNF receptor activation differently. Subsequent neuronal death or survival may ultimately depend on a particular subtype of TNF receptor that is predominately expressed in neurons of the brain during neural development or with neurological diseases.

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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-␤ protein (A␤), a major component of plaques in the Alzheimer's 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 B (NF-B). A␤-induced neuronal apoptosis was reduced with lower Apaf-1 expression, and little NF-B 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 A␤, which occurs through a TNF receptor signaling cascade and a caspase-dependent death pathway.

Complement defense 59 (CD59) is a cell surface glycophosphoinositol (GPI)-anchored protein that prevents complement membrane attack complex (MAC) assembly. Here, we present evidence from ELISA assays that CD59 protein levels are significantly decreased in the frontal cortex and hippocampus of Alzheimer's disease (AD) compared with nondemented elderly (ND) patients, whereas complement component 9, a final component to form MAC, is significantly increased. To further confirm the CD59 deficit, PI-specific phospholipase C (PIPLC) was used to cleave the CD59 GPI anchor at the cell surface in intact slices from AD and ND cortex. CD59 released by PIPLC cleavage was significantly reduced in AD compared with ND samples. By the use of a ribonuclease protection technique, amyloid ␤-peptide was found to downregulate CD59 expression at the mRNA level, suggesting a partial explanation of CD59 deficits in the AD brain.To evaluate the pathophysiological significance of CD59 alterations in neurons, we exposed cultured NT2 cells, which normally underexpress CD59, and NT2 cells transfected to overexpress CD59 to homologous human serum. Lactic acid dehydrogenase assays revealed significant complement-induced cell lysis in CD59-underexpressing NT2 cells and significant protection from such lysis in CD59-overexpressing NT2 cells. Moreover, cells expressing normal levels of CD59 showed no evidence of MAC assembly or damage after exposure to homologous serum, whereas pretreatment of these cells with a CD59-neutralizing antibody resulted in MAC assembly at the cell surface and morphological damage. Taken together, these data suggest that CD59 deficits may play a role in the neuritic losses characteristic of AD. Key words: neurodegeneration; Alzheimer; neuron death; amyloid protein; complement; inflammationThe activation of complement in Alzheimer's disease (AD) seems to occur via a unique, amyloid ␤-peptide (A␤)-mediated, antibodyindependent mechanism (Eikelenboom and Stam, 1982;McGeer et al., 1989;Rogers et al., 1992;Shen et al., 1998) and proceeds fully to formation of the terminal complement component C5b-9 or the membrane attack complex (MAC) (Rogers et al., 1992;Webster et al., 1997;Shen et al., 1998). The MAC is a macrocomplex made up of complement components C5, C6, C7, and C8 plus multiple C9 molecules. After being formed, the ring-like structure of the MAC opens a pore in the membrane of targeted cells, permitting massive Ca 2ϩ influx and subsequent cell lysis (Kim et al., 1987). The MAC is present in pathologically vulnerable areas of the AD brain and is highly colocalized with the A␤ deposit (McGeer et al., 1989;Rogers et al., 1992;Webster et al., 1997).One of the major defense mechanisms against MAC attack is that most cells express complement defense 59 (CD59) (membrane inhibitor of reactive lysis), a glycophosphoinositol (GPI)-anchored membrane protein that binds C8 or C9 and thereby prevents further assembly of the poly-C9 MAC and its full insertion into the cell membrane (Zalman et al., 1989;Meri et al., 1990;Ninomiya an...

We reported that tumor necrosis factor receptor I (TNFRI) is required for neuronal death induced by amyloid-β protein in the Alzheimer’s 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-α binding technique, we found that, in AD brains, 125I-TNF-α 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.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease, but the mechanisms driving progression remain incompletely defined. We previously reported that the IPF lung harbors fibrogenic mesenchymal progenitor cells (MPCs), which serve as a cell of origin for IPF fibroblasts. Proliferating IPF MPCs are located at the periphery of fibroblastic foci in an active cellular front at the interface between the myofibroblast-rich focus core and adjacent normal alveolar structures. Among a large set of genes that distinguish IPF MPCs from their control counterparts, we identified IL-8 as a candidate mediator of IPF MPC fibrogenicity and driver of fibrotic progression. IPF MPCs and their progeny displayed increased steady-state levels of IL-8 and its cognate receptor CXCR1 and secreted more IL-8 than did controls. IL-8 functioned in an autocrine manner promoting IPF MPC self-renewal and the proliferation and motility of IPF MPC progeny. Secreted IL-8 also functioned in a paracrine manner stimulating macrophage migration. Analysis of IPF lung tissue demonstrated codistribution of IPF MPCs with activated macrophages in the active cellular front of the fibroblastic focus. These findings indicate that IPF MPC-derived IL-8 is capable of expanding the mesenchymal cell population and recruiting activated macrophages cells to actively evolving fibrotic lesions.