Oligomeric forms of amyloid-β peptide (Aβ) are thought to play a pivotal role in the pathogenesis of Alzheimer's disease (AD), but the mechanism involved is still unclear. Here, we generated induced pluripotent stem cells (iPSCs) from familial and sporadic AD patients and differentiated them into neural cells. Aβ oligomers accumulated in iPSC-derived neurons and astrocytes in cells from patients with a familial amyloid precursor protein (APP)-E693Δ mutation and sporadic AD, leading to endoplasmic reticulum (ER) and oxidative stress. The accumulated Aβ oligomers were not proteolytically resistant, and docosahexaenoic acid (DHA) treatment alleviated the stress responses in the AD neural cells. Differential manifestation of ER stress and DHA responsiveness may help explain variable clinical results obtained with the use of DHA treatment and suggests that DHA may in fact be effective for a subset of patients. It also illustrates how patient-specific iPSCs can be useful for analyzing AD pathogenesis and evaluating drugs.
In addition to its role as an inhibitory neurotransmitter, ␥-aminobutyric acid (GABA) is presumed to be involved in the development and plasticity of the nervous system. GABA is synthesized by glutamic acid decarboxylase (GAD), but the respective roles of its two isoforms (GAD65 and 67) have not been determined. The selective elimination of each GAD isoform by gene targeting is expected to clarify these issues. Recently we have produced GAD65 ؊͞؊ mice and demonstrated that lack of GAD65 does not change brain GABA contents or animal behavior, except for a slight increase in susceptibility to seizures. Here we report the production of GAD67 ؊͞؊ mice. These mice were born at the expected frequency but died of severe cleft palate during the first morning after birth. GAD activities and GABA contents were reduced to 20% and 7%, respectively, in the cerebral cortex of the newborn GAD67 ؊͞؊ mice. Their brain, however, did not show any discernible defects. Previous pharmacological and genetic investigations have suggested the involvement of GABA in palate formation, but this is the first demonstration of a role for GAD67-derived GABA in the development of nonneural tissue.
A new simple method of detecting calcium binding proteins in a protein mixture is described. A sample which might include calcium binding proteins was subjected to SDS-polyacrylamide gel electrophoresis and then electrophoretically transferred to a nitrocellulose membrane. The membrane was then incubated with 45Ca to detect calcium binding proteins as radioactive bands by autoradiography. Purified troponin-C, calmodulin, myosin DTNB light chain, and parvalbumin were clearly identified by this method. In the whole homogenate of chicken skeletal muscle, myosin DTNB light chain, troponin-C, and 55K calcium binding protein were found to be radioactive. In the frog skeletal muscle, small molecular weight proteins of approximately 13-15K and 70K protein appeared to be the calcium binding proteins. In the case of the carp skeletal muscle, small molecular weight proteins including parvalbumin and two proteins of about 80K seemed to bind calcium ion. Two high molecular weight calcium binding proteins were present in the scallop striated muscle. The procedure described can be completed within 24 h and can detect as little as 2 micrograms of calcium binding protein in the starting sample. Under appropriate conditions it was possible to detect only high affinity calcium binding proteins.
To gain insights into the significance of presenilins (PS) in the pathogenetic mechanisms of early-onset familial Alzheimer disease (FAD), we expressed cDNAs for wild-type PS2 and PS2 with the Volga German (N141I) mutation in cultured cells and then examined the metabolism of the transfected proteins and their effect on the C-terminal properties of secreted amyloid beta protein (A beta). PS2 was identified as a 50- to 55-kDa protein, which was cleaved to produce N-terminal fragments of 35-40 kDa and C-terminal fragments of 19-23 kDa. The Volga German (N141I) mutation did not cause any significant change in the metabolism of PS2. COS-1 cells doubly transfected with cDNAs for N141I mutant PS2 and human beta-amyloid precursor protein (betaAPP) or a C-terminal fragment thereof, as well as mouse Neuro2a neuroblastoma cells stably transfected with N141I mutant PS2 alone, secreted 1.5- to 10-fold more A beta ending at residues 42 (or 43) [A beta42(43)] compared with those expressing the wild-type PS2. These results strongly suggest that the PS2 mutation (N141I) linked to FAD alters the metabolism of A beta/betaAPP to foster the production of the form of A beta that most readily deposits in amyloid plaques. Thus, mutant PS2 may lead to AD by altering the metabolism of A beta/betaAPP.
The immune system plays a central role in orchestrating the tissue healing process. Hence, controlling the immune system to promote tissue repair and regeneration is an attractive approach when designing regenerative strategies. This review discusses the pathophysiology of both acute and chronic wounds and possible strategies to control the immune system to accelerate chronic wound closure and promote skin regeneration (scar-less healing) of acute wounds. Recent studies have revealed the key roles of various immune cells and immune mediators in skin repair. Thus, immune components have been targeted to promote chronic wound repair or skin regeneration and several growth factors, cytokines, and biomaterials have shown promising results in animal models. However, these novel strategies are often struggling to meet efficacy standards in clinical trials, partly due to inadequate drug delivery systems and safety concerns. Excess inflammation is a major culprit in the dysregulation of normal wound healing, and further limiting inflammation effectively reduces scarring. However, current knowledge is insufficient to efficiently control inflammation and specific immune cells. This is further complicated by inadequate drug delivery methods. Improving our understanding of the molecular pathways through which the immune system controls the wound healing process could facilitate the design of novel regenerative therapies. Additionally, better delivery systems may make current and future therapies more effective. To promote the entry of current regenerative strategies into clinical trials, more evidence on their safety, efficacy, and cost-effectiveness is also needed.
The estrogen receptor (ER) regulates the expression of target genes in a ligand-dependent manner. The ligand-dependent activation function AF-2 of the ER is located in the ligand binding domain (LBD), while the N-terminal A/B domain (AF-1) functions in a ligand-independent manner when isolated from the LBD. AF-1 and AF-2 exhibit cell type and promoter context specificity. Furthermore, the AF-1 activity of the human ERalpha (hERalpha) is enhanced through phosphorylation of the Ser(118) residue by mitogen-activated protein kinase (MAPK). From MCF-7 cells, we purified and cloned a 68-kDa protein (p68) which interacted with the A/B domain but not with the LBD of hERalpha. Phosphorylation of hERalpha Ser(118) potentiated the interaction with p68. We demonstrate that p68 enhanced the activity of AF-1 but not AF-2 and the estrogen-induced as well as the anti-estrogen-induced transcriptional activity of the full-length ERalpha in a cell-type-specific manner. However, it did not potentiate AF-1 or AF-2 of ERbeta, androgen receptor, retinoic acid receptor alpha, or mineralocorticoid receptor. We also show that the RNA helicase activity previously ascribed to p68 is dispensable for the ERalpha AF-1 coactivator activity and that p68 binds to CBP in vitro. Furthermore, the interaction region for p68 in the ERalpha A/B domain was essential for the full activity of hERalpha AF-1. Taken together, these findings show that p68 acts as a coactivator specific for the ERalpha AF-1 and strongly suggest that the interaction between p68 and the hERalpha A/B domain is regulated by MAPK-induced phosphorylation of Ser(118).
To identify the amyloid beta peptide (Abeta) 1-42-degrading enzyme whose activity is inhibited by thiorphan and phosphoramidon in vivo, we searched for neprilysin (NEP) homologues and cloned neprilysin-like peptidase (NEPLP) alpha, NEPLP beta, and NEPLP gamma cDNAs. We expressed NEP, phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PEX), NEPLPs, and damage-induced neuronal endopeptidase (DINE) in 293 cells as 95- to 125-kDa proteins and found that the enzymatic activities of PEX, NEPLP alpha, and NEPLP beta, as well as those of NEP and DINE, were sensitive to thiorphan and phosphoramidon. Among the peptidases tested, NEP degraded both synthetic and cell-secreted Abeta1-40 and Abeta1-42 most rapidly and efficiently. PEX degraded cold Abeta1-40 and NEPLP alpha degraded both cold Abeta1-40 and Abeta1-42, although the rates and the extents of the digestion were slower and less efficient than those exhibited by NEP. These data suggest that, among the endopeptidases whose activities are sensitive to thiorphan and phosphoramidon, NEP is the most potent Abeta-degrading enzyme in vivo. Therefore, manipulating the activity of NEP would be a useful approach in regulating Abeta levels in the brain.
Double-stranded DNA (dsDNA) derived from pathogen- or host-damaged cells triggers innate immune responses when exposed to cytoplasm. However, the machinery underlying the primary recognition of intracellular dsDNA is obscure. Here we show that the DNA damage sensor, meiotic recombination 11 homolog A (MRE11), serves as a cytosolic sensor for dsDNA. Cells with a mutation of MRE11 gene derived from a patient with ataxia-telangiectasia–like disorder, and cells in which Mre11 was knocked down, had defects in dsDNA-induced type I IFN production. MRE11 physically interacted with dsDNA in the cytoplasm and was required for activation of stimulator of IFN genes (STING) and IRF3. RAD50, a binding protein to MRE11, was also required for dsDNA responses, whereas NBS1, another binding protein to MRE11, was dispensable. Collectively, our results suggest that the MRE11–RAD50 complex plays important roles in recognition of dsDNA and initiation of STING-dependent signaling, in addition to its role in DNA-damage responses.
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