Tissues Of Transgenic Mice Research Articles

αB‐crystallin (HspB5) in familial amyloidotic polyneuropathy

The small heat shock protein αB-crystallin (HspB5) is known to be overexpressed in several neurodegenerative disorders. In familial amyloidotic polyneuropathy (FAP), a neurodegenerative disorder characterized by extracellular deposition of mutated transthyretin (TTR), activation of heat shock factor 1 -HSF1- by extracellular TTR deposition has been shown as well as induction of the expression of heat shock proteins, HSP27 and HSP70. Here we investigate the expression of αB-crystallin in FAP. We first detected αB-crystallin in aggregates extracted from tissues of both FAP patients and transgenic mice for the human V30M mutant TTR; however, subsequent studies by confocal fluorescence microscopy did not confirm the association of αB-crystallin with TTR aggregates; thus the presence of αB-crystallin in aggregate extracts might derive from the extraction procedure. Increased levels of αB-crystallin were observed by immunohistochemistry in human FAP skin, as compared to normal skin. Furthermore, skin, stomach and dorsal root ganglia from V30M transgenic mice showed increased expression of αB-crystallin as compared to controls without deposition. A human neuroblastoma cell line incubated with TTR aggregates displayed increased expression of αB-crystallin. Overall, these results show that extracellular TTR deposits induce an intracellular response of αB-crystallin. This small heat shock protein (HSP), which is important for anti-apoptotic and chaperone properties, may have a protective role in FAP.

Glioma stem cells involved in tumor tissue remodeling in a xenograft model

Although tissue remodeling plays a crucial role in the tumorigenesis and progression of human gliomas, its mechanisms remain largely uncertain. In the current study, the authors investigated the potential role of human glioma stem cells (hGSCs) in the tissue remodeling of gliomas. Transgenic nude mice with ubiquitous green fluorescent protein (GFP) expression were obtained by crossing nontransgenic NC athymic nude mice with the GFP transgenic C57BL/6J mice. As a result, GFP was expressed in essentially all tissues in the offspring. Human glioma stem cells were then orthotopically implanted into the GFP nude mice in an effort to assess the hGSC-host brain interactions and thereby elucidate the roles of tissue remodeling during tumorigenesis and progression of human gliomas. All of the essential tissues in the GFP transgenic nude mice, including the brain, fluoresced green under an excitation light; therefore, tumor remodeling by hGSCs can be unambiguously distinguished from a bright green background composed of adjacent host GFP-expressing components. This technique enabled the authors to address the following concerns: 1) hGSCs were involved in the invasiveness of gliomas and adjacent stroma degradation of the host. 2) An in vivo study demonstrated that cell fusion occurred between hGSCs and host cells. 3) Vasculogenic mimicry--the formation of patterned, tubular networks of vascular channels by transdifferentiated hGSCs--could be observed. 4) Differentiation mimicry--namely, the differentiation direction of hGSCs bearing multidifferentiation potentials--seemed to be decided by the local host cellular microenviroment. The results of this study indicated that the GFP transgenic nude mice model with GFP expression in essentially all tissues could be obtained by crossing nontransgenic athymic nude mice with transgenic GFP mice. This model should greatly expand our knowledge of glioma-host interactions. The data indicated that hGSCs might play a decisive role in tissue remodeling of gliomas as well.

HIV-Associated Nephropathy: Role of Mammalian Target of Rapamycin Pathway

Both glomerular and tubular lesions are characterized by a proliferative phenotype in HIV-associated nephropathy. We hypothesized that mammalian target of rapamycin (mTOR) contributes to the development of the HIVAN phenotype. Both glomerular and tubular epithelial cells showed enhanced expression of phospho (p)-mTOR in HIV-1 transgenic mice (Tgs). In addition, renal tissues of transgenic mice (RT-Tg) showed enhanced phosphorylation of p70S6 kinase and an associated diminished phosphorylation of eEF2. Moreover, RT-Tgs showed enhanced phosphorylation of 4EBP1 and eIF4B; these findings indicated activation of the mTOR pathway in RT-Tgs. To test our hypothesis, age- and sex-matched control mice and Tgs were administered either saline or rapamycin (an inhibitor of the mTOR pathway) for 4 weeks. Tgs receiving rapamycin not only showed inhibition of the mTOR-associated downstream signaling but also displayed attenuated renal lesions. RT-Tgs showed enhanced expression of hypoxia-inducible factor-alpha and also displayed increased expression of vascular endothelial growth factor; on the other hand, rapamycin inhibited RT-Tg expression of both hypoxia-inducible factor-alpha and vascular endothelial growth factor. We conclude that the mTOR pathway contributes to the HIVAN phenotype and that inhibition of the mTOR pathway can be used as a therapeutic strategy to alter the course of HIVAN.

Regulation of Human MUC7 Mucin Gene Expression by Cigarette Smoke Extract or Cigarette Smoke and Pseudomonas aeruginosa Lipopolysaccharide in Human Airway Epithelial Cells and in MUC7 Transgenic Mice~!2010-04-05~!2010-06-09~!2010-07-14~!

Objective:The human MUC7 gene encodes a low-molecular-weight mucin glycoprotein that functions in lubrication/protection of epithelial surfaces of the oral cavity and respiratory tract. This study was designed to evaluate the effect of cigarette smoke extract (CSE), cigarette smoke (CS), and Pseudomonas aeruginosa lipopolysaccharide (LPS), either alone or in the combination, on MUC7 expression in vitro and in vivo.Materials and Methods:qRT-PCR was used to determine the levels of mucin gene transcription in the human lung carcinoma cell line NCI-H292 (in vitro) and MUC7 transgenic mouse tissues (in vivo). ELISA was used to assess mucin glycoprotein levels in the cell line, and immunohistochemistry to assess mucins in lung and trachea sections.Results:In vitro treatment of cells with LPS (10 (µg/ml) or CSE (0.5, 1, 2.5 and 5%) alone, resulted in a statistically significant increase of MUC7 transcripts only with 1%CSE (3.2-fold). The combined CSE/LPS treatment resulted in a synergistic increase of MUC7 with 0.5%CSE/LPS (4.4 fold). MUC7 glycoprotein levels increased only minimally, the highest increase was seen with the 0.5%CSE/LPS combination treatment (1.3-fold). In vivo exposure of MUC7 transgenic mice to CS, LPS or CS/LPS combination resulted in significant increase in MUC7 transcripts only with LPS treatment (in both trachea and lung). Immunohistochemistry indicated variable increase in MUC7 glycoprotein with CS and LPS treatment, both in the trachea and lungs, but CS/LPS exposure appeared to yield the highest increase.Conclusion:In vitro, CSE and a combination of CSE/LPS treatment upregulated MUC7 gene transcription. In vivo, LPS upregulated MUC7 transcription, and a combination of CS/LPS appeared to increase MUC7 glycoprotein.

Temporal and Spatial Resolution of Type I and III Interferon ResponsesIn Vivo

Although the action of interferons (IFNs) has been extensively studied in vitro, limited information is available on the spatial and temporal activation pattern of IFN-induced genes in vivo. We created BAC transgenic mice expressing firefly luciferase under transcriptional control of the Mx2 gene promoter. Expression of the reporter with regard to onset and kinetics of induction parallels that of Mx2 and is thus a hallmark for the host response. Substantial constitutive expression of the reporter gene was observed in the liver and most other tissues of transgenic mice, whereas this expression was strongly reduced in animals lacking functional type I IFN receptors. As expected, the reporter gene was induced not only in response to type I (alpha and beta) and type III (lambda) IFNs but also in response to a variety of IFN inducers such as double-stranded RNA, lipopolysaccharide (LPS), and viruses. In vivo IFN subtypes show clear differences with respect to their kinetics of action and to their spatial activation pattern: while the type I IFN response was strong in liver, spleen, and kidney, type III IFN reactivity was most prominent in organs with mucosal surfaces. Infection of reporter mice with virus strains that differ in their pathogenicity shows that the IFN response is significantly altered in the strength of IFN action at sites which are not primarily infected as well as by the onset and duration of gene induction.

Anesthetic Sevoflurane Causes Neurotoxicity Differently in Neonatal Naïve and Alzheimer Disease Transgenic Mice

Recent studies have suggested that children undergoing surgery under anesthesia could be at an increased risk for the development of learning disabilities, but whether anesthetics contribute to this learning disability is unclear. Therefore, the authors set out to assess the effects of sevoflurane, the most commonly used inhalation anesthetic, on caspase activation, apoptosis, beta-amyloid protein levels, and neuroinflammation in the brain tissues of neonatal naïve and Alzheimer disease (AD) transgenic mice. Six-day-old naïve and AD transgenic (B6.Cg-Tg[amyloid precursor protein swe, PSEN1dE9]85Dbo/J) mice were treated with sevoflurane. The mice were killed at the end of the anesthesia, and the brain tissues were harvested and then subjected to Western blot, immunocytochemistry, enzyme-linked immunosorbent assay, and real-time polymerase chain reaction. Herein, the authors show for the first time that sevoflurane anesthesia induced caspase activation and apoptosis, altered amyloid precursor protein processing, and increased beta-amyloid protein levels in the brain tissues of neonatal mice. Furthermore, sevoflurane anesthesia led to a greater degree of neurotoxicity in the brain tissues of the AD transgenic mice when compared with naïve mice and increased tumor necrosis factor-alpha levels in the brain tissues of only the AD transgenic mice. Finally, inositol 1,4,5-trisphosphate receptor antagonist 2-aminoethoxydiphenyl borate attenuated sevoflurane-induced caspase-3 activation and beta-amyloid protein accumulation in vivo. These results suggest that sevoflurane may induce neurotoxicity in neonatal mice. AD transgenic mice could be more vulnerable to such neurotoxicity. These findings should promote more studies to determine the potential neurotoxicity of anesthesia in animals and humans, especially in children.

Melphalan inhibits adenoma development through modulating the expression of K-ras-specific markers in K-ras Tg mice

In previous research, we focused on the discovery of K-ras biomarkers, and effects of genotoxic carcinogens on their expression were investigated in this study. It is well-known that mutated K-ras gene is involved in approximately 30% of human cancers such as lung cancer. To search for K-ras oncogene-induced modulators in lung tissues of K-ras transgenic mice, we analyzed K-ras-specific genes and proteins related to cancer development, signal transduction, inflammation as well as tumor suppression in a previous study. In this study, we investigated the modulating effects of genotoxic carcinogen treatment on expression of K-ras-dependent modulated genes and proteins in lung tissues of K-ras Tg mice. In order to evaluate candidate K-ras markers modulated by genotoxic stress and to investigate whether a genotoxic carcinogen would enhance or inhibit carcinogenesis in lung tissues of the K-ras Tg mice, the anti-cancer drug melphalan was intraperitoneally injected into K-ras Tg mice every two days for four weeks. RT-qPCR and proteomics analyses were performed in order to confirm whether K-ras-specific biomarkers would be modulated by melphalan treatment in K-ras Tg mice. The decreased adenomas were histopathologically observed and K-ras expression was suppressed in melphalan-treated K-ras Tg mice. Melphalan also recovered the expression of K-ras-dependent modulated biomarkers. These results suggest that melphalan inhibits carcinogenesis via modulating K-ras-specific genes and proteins expressed in the lung tissues of K-ras Tg mice.

Perilipin overexpression in mice protects against diet-induced obesity

Perilipin A is the most abundant phosphoprotein on adipocyte lipid droplets and is essential for lipid storage and lipolysis. Perilipin null mice exhibit diminished adipose tissue, elevated basal lipolysis, reduced catecholamine-stimulated lipolysis, and increased insulin resistance. To understand the physiological consequences of increased perilipin expression in vivo, we generated transgenic mice that overexpressed either human or mouse perilipin using the adipocyte-specific aP2 promoter/enhancer. Phenotypes of female transgenic and wild-type mice were characterized on chow and high-fat diets (HFDs). When challenged with an HFD, transgenic mice exhibited lower body weight, fat mass, and adipocyte size than wild-type mice. Expression of oxidative genes was increased and lipogenic genes decreased in brown adipose tissue of transgenic mice. Basal and catecholamine-stimulated lipolysis was decreased and glucose tolerance significantly improved in transgenic mice fed a HFD. Perilipin overexpression in adipose tissue protects against HFD-induced adipocyte hypertrophy, obesity, and glucose intolerance. Alterations in brown adipose tissue metabolism may mediate the effects of perilipin overexpression on body fat, although the mechanisms by which perilipin overexpression alters brown adipose tissue metabolism remain to be determined. Our findings demonstrate a novel role for perilipin expression in adipose tissue metabolism and regulation of obesity and its metabolic complications.

Over-expression of IL-33 leads to spontaneous pulmonary inflammation in mIL-33 transgenic mice

IL-33 plays an important role in inflammatory diseases including hypersensitive diseases like asthma, autoimmune diseases like rheumatoid arthritis, cardiovascular diseases like heart failure and neurodegenerative diseases like Alzheimer's disease. Here we reported the generation of an IL-33 transgenic mouse, in which mouse IL-33 full-length cDNA was controlled under the CMV promoter. The transgenic IL-33 was released as a cleaved form with molecular weight of 18 kDa in pulmonary, nephritic, cardiac and pancreatic tissues in transgenic mice and the p I of 18 kDa peptide was about pH 3–5 on the 2D PAGE which was similar with the activated peptide of IL-33. Histological analysis showed massive airway inflammation with infiltration of eosinophils around bronchi and small blood vessels, hyperplasia of goblet cells and accumulation of mucus-like material in pulmonary tissue of transgenic mice. An increase of IL-5, IL-8, IL-13 and IgE was detected in bronchoalveolar lavage fluid (BALF) of transgenic mice, which are inflammatory factors. These findings suggest transgenic IL-33 could be cleaved and secreted in an activated form and play an important role in the pathogenesis of pulmonary inflammation.

Abstract 174: Characterization of methylation-controlled DnaJD1 in Hep3B hepatoma cell line

Abstract The development of hepatocllular carcinoma (HCC) is a multistep process with accumulation of genetic and epigenetic alternations in regulatory genes, leading to activation of oncogenes and inactivation of tumor suppressor genes. For genome-wide DNA methylation study, we used homemade MCGI (mouse CpG islands) microarray to identify many genes that were hypermethylation in the liver tumor tissues of HBx-overexpressd transgenic mice. These selected hypermethylation genes were also confirmed in human hepatocellular carcinoma cell (HCC) lines. In this study, we focused on a methylation-controlled DNAJ family (MCJ) gene, DnaJD1. It has been observed highly frequent DnaJD1 gene methylation and silencing in ovarian cancer, and associated with increased resistance to chemotherapeutic. The DnaJD1 was found hypermethylation in the promoter region and followed reduced mRNA expression in Hep3B hepatoma cells, which contain HBV genome. After treated by 5-aza-2’-deoxycytidine (DAC) in Hep3B cells, the methylation level of DnaJD1 could be reduced and mRNA re-expressed. We over-expressed DnaJD1 in Hep3B cells to study the physiologic effects of DnaJD1 in cell proliferation, cell migration, and invasion ability in vitro. The tumorgenesis in xenotransplanted nude mice were also examined. Data suggested that DnaJD1 re-expressed in Hep3B cells that could affect the cell morphology and lost cell contact insensitive. In these results, we concluded that DnaJD1 could be the one of main role of HCC progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 174.

The delivery of HBcAg via Tat-PTD enhances specific immune response and inhibits Hepatitis B virus replication in transgenic mice

Recent studies have indicated that the therapeutic vaccine based on enhancement of HBV-specific cytotoxic T-lymphocyte (CTL) activity may lead to viral clearance in chronically infected individuals. It is demonstrated that protein transduction domains (PTD) from HIV-1-Tat protein is able to enter cells when combined with exogenous antigens and induce specific CTL responses. We have previously testified that the expressed and purified fusion protein containing Tat-PTD47-57 and HBcAg could enter cytoplasm of dendritic cells, and enhance T cells response to generate HBcAg-specific CTLs efficiently in vitro. In the present study, we evaluated HBcAg-specific immune responses of PTD-HBcAg fusion protein in BALB/c mice and antiviral immunity in HBV transgenic mice. The studies showed that PTD-HBcAg not only induced significantly higher antibody responses, but also increased production of cytokine (IFN-γ, IL-2, IL-4 and IL-10) compared to HBcAg alone and PBS. Moreover, PTD-HBcAg fusion protein increased significantly the percentages of IFN-γ+CD8+ T cells and HBcAg-specific (CTL) responses. Also, enhancement of immune response induced by fusion protein reduced HBV DNA and HBsAg levels and decreased the expression of HBsAg in liver tissue of HBV transgenic mice. In conclusion, PTD-HBcAg fusion protein could enhance not only cell immune response but also humoral immune response, and induce robust specific CTL activity and therapeutic effects in HBV transgenic mice.

Endogenous Proteolytic Cleavage of Disease-associated Prion Protein to Produce C2 Fragments Is Strongly Cell- and Tissue-dependent

The abnormally folded form of the prion protein (PrP(Sc)) accumulating in nervous and lymphoid tissues of prion-infected individuals can be naturally cleaved to generate a N-terminal-truncated fragment called C2. Information about the identity of the cellular proteases involved in this process and its possible role in prion biology has remained limited and controversial. We investigated PrP(Sc) N-terminal trimming in different cell lines and primary cultured nerve cells, and in the brain and spleen tissue from transgenic mice infected by ovine and mouse prions. We found the following: (i) the full-length to C2 ratio varies considerably depending on the infected cell or tissue. Thus, in primary neurons and brain tissue, PrP(Sc) accumulated predominantly as untrimmed species, whereas efficient trimming occurred in Rov and MovS cells, and in spleen tissue. (ii) Although C2 is generally considered to be the counterpart of the PrP(Sc) proteinase K-resistant core, the N termini of the fragments cleaved in vivo and in vitro can actually differ, as evidenced by a different reactivity toward the Pc248 anti-octarepeat antibody. (iii) In lysosome-impaired cells, the ratio of full-length versus C2 species dramatically increased, yet efficient prion propagation could occur. Moreover, cathepsin but not calpain inhibitors markedly inhibited C2 formation, and in vitro cleavage by cathepsins B and L produced PrP(Sc) fragments lacking the Pc248 epitope, strongly arguing for the primary involvement of acidic hydrolases of the endolysosomal compartment. These findings have implications on the molecular analysis of PrP(Sc) and cell pathogenesis of prion infection.

Fatal Transmissible Amyloid Encephalopathy: A New Type of Prion Disease Associated with Lack of Prion Protein Membrane Anchoring

Prion diseases are fatal neurodegenerative diseases of humans and animals characterized by gray matter spongiosis and accumulation of aggregated, misfolded, protease-resistant prion protein (PrPres). PrPres can be deposited in brain in an amyloid-form and/or non-amyloid form, and is derived from host-encoded protease-sensitive PrP (PrPsen), a protein normally anchored to the plasma membrane by glycosylphosphatidylinositol (GPI). Previously, using heterozygous transgenic mice expressing only anchorless PrP, we found that PrP anchoring to the cell membrane was required for typical clinical scrapie. However, in the present experiments, using homozygous transgenic mice expressing two-fold more anchorless PrP, scrapie infection induced a new fatal disease with unique clinical signs and altered neuropathology, compared to non-transgenic mice expressing only anchored PrP. Brain tissue of transgenic mice had high amounts of infectivity, and histopathology showed dense amyloid PrPres plaque deposits without gray matter spongiosis. In contrast, infected non-transgenic mice had diffuse non-amyloid PrPres deposits with significant gray matter spongiosis. Brain graft studies suggested that anchored PrPsen expression was required for gray matter spongiosis during prion infection. Furthermore, electron and light microscopic studies in infected transgenic mice demonstrated several pathogenic processes not seen in typical prion disease, including cerebral amyloid angiopathy and ultrastructural alterations in perivascular neuropil. These findings were similar to certain human familial prion diseases as well as to non-prion human neurodegenerative diseases, such as Alzheimer's disease.

Multilineage Differentiation of Dental Pulp Stem Cells from Green Fluorescent Protein Transgenic Mice

AimThe aim of this study was to confirm the multilineage differentiation ability of dental pulp stem cells (DPSCs) from green fluorescent protein (GFP) transgenic mice. The expression of GFP in DPSCs was also observed during differentiation.MethodologyDPSCs were harvested from the dental pulp tissue of transgenic nude mice, and then transferred to osteogenic, adipogenic, and chondrogenic media. The morphological characterization of induced cells was observed by microscopy and histological staining. The expression of marker genes was measured by RT-PCR.ResultsThe endogenous GFP and multilineage potential of transgenic DPSCs had no influence on each other. Moreover, the results of fluorescence microscopic imaging suggest that there was no significant decline of GFP expression during DPSCs differentiation.ConclusionAs the population of GFP labeled DPSCs can be easily identified, this will be a promising method for tracking DPSCs in vivo.

Epithelial Overexpression of SOCS-3 in Transgenic Mice Exacerbates Wound Inflammation in the Presence of Elevated TGF-β1

The suppressor of cytokine signaling (SOCS)-3 has been shown to impair proliferation and migration of keratinocytes. To assess the functional dependency among wound inflammation, SOCS-3 induction in keratinocytes, and the outcome of healing, we generated a transgenic mouse that specifically overexpresses SOCS-3 in keratinocytes. Acute wound healing in transgenic mice was severely impaired. Keratinocyte-specific overexpression of SOCS-3 led to atrophied wound-margin epithelia and augmented the inflammatory response of wound keratinocytes by an increase in chemokine (MIP-2) and inflammatory enzyme (COX-2 and iNOS) expression. In addition, wound tissue of transgenic mice showed a prolonged persistence of neutrophils and macrophages. Remarkably, impaired wounds showed elevated levels of transforming growth factor (TGF)-beta1, which appeared to interfere with healing, as its neutralization markedly improved wound closure in transgenic mice. Interestingly, administration of a TGF-beta-neutralizing antibody increased wound inflammation in nontransgenic mice but not in transgenic littermates. This study suggests that SOCS-3-driven disturbances in wound keratinocytes are sufficient to induce inflamed wound conditions that resemble characteristics of chronic wounds in mice.

Small intestinal glucose absorption in cystic fibrosis: a study in human and transgenic ΔF508 cystic fibrosis mouse tissues

Intestinal transport is disturbed in cystic fibrosis (CF), with both defective Cl- secretion and changes in absorption being reported. We have examined the effects of the disease on Na(+)-dependent glucose absorption by the small intestine. Active glucose absorption was monitored as changes in short-circuit current (SCC) in intact and stripped intestinal sheets from normal (Swiss) and transgenic CF (Cftr(tm1Eur) and Cftr(tm2Cam)) mice with the DeltaF508 mutation, and in jejunal biopsies from children with CF and normal controls. Na(+)-dependent glucose uptake at the luminal membrane was measured in brush-border membrane vesicles (BBMVs). Intact and stripped sheets of jejunum and midintestine from Swiss mice exhibited a concentration-dependent increase in SCC with glucose. Apparent Km values were similar in the two preparations, but the apparent Vmax was greater in stripped sheets. This difference was not due to a loss of neural activity in stripped sheets as tetrodotoxin did not influence the glucose-induced SCC in intact sheets. Similar results were observed in stripped sheets of jejunum and mid-intestine from wild-type Cftr(tm1Eur) mice, but in tissues from CF mice the apparent Vmax value was reduced significantly. A lower Vmax was also obtained in intact sheets of mid-intestine from CF (Cftr(tm2Cam)) mice. Jejunal biopsies from CF patients however, exhibited an enhanced glucose-dependent rise in SCC. Na(+)-dependent uptake by BBMVs from CF (Cftr(tm1Eur)) mice was not reduced compared with wild-type and Swiss BBMVs. It was concluded that, in contrast to human intestine, intestinal glucose absorption was reduced in transgenic mouse models of CF with the DeltaF508 mutation, but that this could not be detected in an isolated preparation of brush-border membranes. Transgenic mouse models of CF may not accurately reflect all aspects of intestinal dysfunction in the human disease.

Selective activation of PPARγ in skeletal muscle induces endogenous production of adiponectin and protects mice from diet-induced insulin resistance

The nuclear receptor peroxisome proliferator-activated receptor (PPAR)gamma plays a key role in regulating whole body glucose homeostasis and insulin sensitivity. Although it is expressed most highly in adipose, it is also present at lower levels in many tissues, including skeletal muscle. The role muscle PPARgamma plays in metabolic regulation and in mediating the antidiabetic effects of the thiazolidinediones is not understood. The goal of this work was to examine the molecular and physiological effects of PPARgamma activation in muscle cells. We found that pharmacological activation of PPARgamma in primary cultured myocytes, and genetic activation of muscle PPARgamma in muscle tissue of transgenic mice, induced the production of adiponectin directly from muscle cells. This muscle-produced adiponectin was functional and capable of stimulating adiponectin signaling in myocytes. In addition, elevated skeletal muscle PPARgamma activity in transgenic mice provided a significant protection from high-fat diet-induced insulin resistance and associated changes in muscle phenotype, including reduced myocyte lipid content and an increase in the proportion of oxidative muscle fiber types. Our findings demonstrate that PPARgamma activation in skeletal muscle can have a significant protective effect on whole body glucose homeostasis and insulin resistance and that myocytes can produce and secrete functional adiponectin in a PPARgamma-dependent manner. We propose that activation of PPARgamma in myocytes induces a local production of adiponectin that acts on muscle tissue to improve insulin sensitivity.

Degradation of Amyloid β Protein by Purified Myelin Basic Protein

The progressive accumulation of beta-amyloid (Abeta) in senile plaques and in the cerebral vasculature is the hallmark of Alzheimer disease and related disorders. Impaired clearance of Abeta from the brain likely contributes to the prevalent sporadic form of Alzheimer disease. Several major pathways for Abeta clearance include receptor-mediated cellular uptake, blood-brain barrier transport, and direct proteolytic degradation. Myelin basic protein (MBP) is the major structural protein component of myelin and plays a functional role in the formation and maintenance of the myelin sheath. MBP possesses endogenous serine proteinase activity and can undergo autocatalytic cleavage liberating distinct fragments. Recently, we showed that MBP binds Abeta and inhibits Abeta fibril formation (Hoos, M. D., Ahmed, M., Smith, S. O., and Van Nostrand, W. E. (2007) J. Biol. Chem. 282, 9952-9961; Hoos, M. D., Ahmed, M., Smith, S. O., and Van Nostrand, W. E. (2009) Biochemistry 48, 4720-4727). Here we show that Abeta40 and Abeta42 peptides are degraded by purified human brain MBP and recombinant human MBP, but not an MBP fragment that lacks autolytic activity. MBP-mediated Abeta degradation is inhibited by serine proteinase inhibitors. Similarly, Cos-1 cells expressing MBP degrade exogenous Abeta40 and Abeta42. In addition, we demonstrate that purified MBP also degrades assembled fibrillar Abeta in vitro. Mass spectrometry analysis identified distinct degradation products generated from Abeta digestion by MBP. Lastly, we demonstrate in situ that purified MBP can degrade parenchymal amyloid plaques as well as cerebral vascular amyloid that form in brain tissue of Abeta precursor protein transgenic mice. Together, these findings indicate that purified MBP possesses Abeta degrading activity in vitro.

Formic acid is essential for immunohistochemical detection of aggregated intraneuronal Aβ peptides in mouse models of Alzheimer's disease

The staining protocols so far applied to study intracellular Aβ accumulation in human tissue have been inconsistent with varying use of heat and formic acid (FA) for antigen retrieval. Microwave heat treatment has been reported to enhance the staining of intraneuronal Aβ as compared to no or enzymatic pretreatment. FA is widely used to increase the staining of plaque pathology in AD, yet the effect of FA on intraneuronal Aβ staining has been reported to be low and similar to the effect of heat or even to counteract the enhancing effect of heat pretreatment on intraneuronal Aβ immunohistochemical detection. To overcome these inconsistencies, there is a need for optimization of the staining protocol for intraneuronal Aβ detection and more knowledge is required concerning the effects of the different antigen retrieval methods. In the present work, we optimized the staining protocol for intraneuronal Aβ in paraffin-embedded sections in relation to heat and FA using four different mouse models known to accumulate intraneuronal Aβ peptides. It was found that FA is essential for the staining of highly aggregated intraneuronal Aβ peptides in AD transgenic mouse tissue.

Brain-derived Neurotrophic Factor Enhances the Basal Rate of Protein Synthesis by Increasing Active Eukaryotic Elongation Factor 2 Levels and Promoting Translation Elongation in Cortical Neurons

The constitutive and activity-dependent components of protein synthesis are both critical for neural function. Although the mechanisms controlling extracellularly induced protein synthesis are becoming clear, less is understood about the molecular networks that regulate the basal translation rate. Here we describe the effects of chronic treatment with various neurotrophic factors and cytokines on the basal rate of protein synthesis in primary cortical neurons. Among the examined factors, brain-derived neurotrophic factor (BDNF) showed the strongest effect. The rate of protein synthesis increased in the cortical tissues of BDNF transgenic mice, whereas it decreased in BDNF knock-out mice. BDNF specifically increased the level of the active, unphosphorylated form of eukaryotic elongation factor 2 (eEF2). The levels of active eEF2 increased and decreased in BDNF transgenic and BDNF knock-out mice, respectively. BDNF decreased kinase activity and increased phosphatase activity against eEF2 in vitro. Additionally, BDNF shortened the ribosomal transit time, an index of translation elongation. In agreement with these results, overexpression of eEF2 enhanced protein synthesis. Taken together, our results demonstrate that the increased level of active eEF2 induced by chronic BDNF stimulation enhances translational elongation processes and increases the total rate of protein synthesis in neurons.