Alterations in striatal synaptic transmission are consistent across genetic mouse models of Huntington's disease

Since the identification of the gene responsible for HD (Huntington''s disease), many genetic mouse models have been generated. Each employs a unique approach for delivery of the mutated gene and has a different CAG repeat length and background strain. The resultant diversity in the genetic context and phenotypes of these models has led to extensive debate regarding the relevance of each model to the human disorder. Here, we compare and contrast the striatal synaptic phenotypes of two models of HD, namely the YAC128 mouse, which carries the full-length huntingtin gene on a yeast artificial chromosome, and the CAG140 KI (knock-in) mouse, which carries a human/mouse chimaeric gene that is expressed in the context of the mouse genome, with our previously published data obtained from the R6/2 mouse, which is transgenic for exon 1 mutant huntingtin. We show that striatal MSNs (medium-sized spiny neurons) in YAC128 and CAG140 KI mice have similar electrophysiological phenotypes to that of the R6/2 mouse. These include a progressive increase in membrane input resistance, a reduction in membrane capacitance, a lower frequency of spontaneous excitatory postsynaptic currents and a greater frequency of spontaneous inhibitory postsynaptic currents in a subpopulation of striatal neurons. Thus, despite differences in the context of the inserted gene between these three models of HD, the primary electrophysiological changes observed in striatal MSNs are consistent. The outcomes suggest that the changes are due to the expression of mutant huntingtin and such alterations can be extended to the human condition.     

Triiodothyronine stimulates hepatocyte proliferation in two models of impaired liver regeneration

Abstract.  Objectives : Liver regeneration is attenuated in old age and is substantially slower after 90% than after 70% partial hepatectomy (PH). We have previously demonstrated that the proliferative response to a primary mitogen is intact in aged mice, indicating that impaired liver regeneration is not due to loss of proliferative capacity. Here, we have investigated whether mitogenic effects of triiodothyronine (T3) could reverse the impaired regeneration of ageing or 90% hepatectomy, in the rat. Materials and methods : T3 (20 µg/100 g body weight) was administered to 14-month-old rats subjected to 70% PH or to young rats subjected to 90% PH. Cell-proliferative capacity was determined by bromodeoxyuridine incorporation and microscopy and changes of cell cycle-related proteins were analysed by Western blot analysis. Results : Treatment of old intact rats with T3 increased cyclin D₁ expression that was followed by an enhanced proliferative response, the labelling index (LI), being 7.8% versus 1.3% of controls. T3 given before 70% PH stimulated regenerative response (LI was 10.8% versus 2.28%), and expression of cyclin D₁ and proliferating cell nuclear antigen (PCNA) 24 h after PH. Pre-treatment with T3 also improved the regenerative response of the liver after 90% hepatectomy (LI was 27.9% versus 14.2%). Conclusions : These findings show in principle that mitogen-induced hyperplasia could be applied to human therapy in patients with reduced regenerative capacity or massive loss of hepatocytes.     

Hepatitis B virus transgenic mouse model of chronic liver disease.

A model for hepatitis B virus-associated chronic liver disease has been made using cloned hepatitis B virus DNA as a transgene in a severe combined immunodeficient host. These mice consistently support virus gene expression and replication. After adoptive transfer of unprimed, syngeneic splenocytes, these mice cleared virus from liver and serum, and developed chronic liver disease. This model will permit identification of the host and virus contributions to chronic liver disease in the absence of tolerance.     

Tau reduction does not prevent motor deficits in two mouse models of Parkinson's disease

Many neurodegenerative diseases are increasing in prevalence and cannot be prevented or cured. If they shared common pathogenic mechanisms, treatments targeting such mechanisms might be of benefit in multiple conditions. The tau protein has been implicated in the pathogenesis of diverse neurodegenerative disorders, including Alzheimer's disease (AD) and Parkinson's disease (PD). Tau reduction prevents cognitive deficits, behavioral abnormalities and other pathological changes in multiple AD mouse models. Here we examined whether tau reduction also prevents motor deficits and pathological alterations in two mouse models of PD, generated by unilateral striatal injection of 6-hydroxydopamine (6-OHDA) or transgene-mediated neuronal expression of human wildtype α-synuclein. Both models were evaluated on Tau(+/+), Tau(+/-) and Tau(-/-) backgrounds in a variety of motor tests. Tau reduction did not prevent motor deficits caused by 6-OHDA and slightly worsened one of them. Tau reduction also did not prevent 6-OHDA-induced loss of dopaminergic terminals in the striatum. Similarly, tau reduction did not prevent motor deficits in α-synuclein transgenic mice. Our results suggest that tau has distinct roles in the pathogeneses of AD and PD and that tau reduction may not be of benefit in the latter condition.     

Intraclonal cell expansion and selection driven by B cell receptor in chronic lymphocytic leukemia

The mutational status of the immunoglobulin heavy-chain variable region (IGHV) genes utilized by chronic lymphocytic leukemia (CLL) clones defines two disease subgroups. Patients with unmutated IGHV have a more aggressive disease and a worse outcome than patients with cells having somatic IGHV gene mutations. Moreover, up to 30% of the unmutated CLL clones exhibit very similar or identical B cell receptors (BcR), often encoded by the same IG genes. These "stereotyped" BcRs have been classified into defined subsets. The presence of an IGHV gene somatic mutation and the utilization of a skewed gene repertoire compared with normal B cells together with the expression of stereotyped receptors by unmutated CLL clones may indicate stimulation/selection by antigenic epitopes. This antigenic stimulation may occur prior to or during neoplastic transformation, but it is unknown whether this stimulation/selection continues after leukemogenesis has ceased. In this study, we focused on seven CLL cases with stereotyped BcR Subset #8 found among a cohort of 700 patients; in six, the cells expressed IgG and utilized IGHV4-39 and IGKV1-39/IGKV1D-39 genes, as reported for Subset #8 BcR. One case exhibited special features, including expression of IgM or IgG by different subclones consequent to an isotype switch, allelic inclusion at the IGH locus in the IgM-expressing cells and a particular pattern of cytogenetic lesions. Collectively, the data indicate a process of antigenic stimulation/selection of the fully transformed CLL cells leading to the expansion of the Subset #8 IgG-bearing subclone.     

Isolation of mouse C-reactive protein from liver and serum.

Purified proteins have been isolated from the sera and livers of mice. These proteins are antigenically identical but share antigenic determinants in common with human C-reactive protein and do not share antigenic determinants in common with mouse or human immunoglobulins. The proteins interact with C-polysaccharide, are precipitated by calcium ions, migrate electrophoretically with gamma mobilities, and have isoelectric points of 4.8 and 5.62. Since these properties are characteristic of C-reactive protein of man, monkey, rabbit and dog, the pure proteins isolated from mice are designated mouse C-reactive protein.     

Secretion of serum amyloid protein and assembly of serum amyloid protein-rich high density lipoprotein in primary mouse hepatocyte culture

The serum amyloid protein (apo-SAA) is a unique high density lipoprotein apoprotein exhibiting dramatic increases in plasma concentration following host injury. The events involved in the secretion of apo-SAA and assembly of apo-SAA-rich lipoprotein particles were studied in primary, serum-free culture of adult BALB/c mouse hepatocytes harvested 3 h following administration of the potent apo-SAA inducer, bacterial endotoxin (50 micrograms of intraperitoneally administered Salmonella typhosa lipopolysaccharide). An approximately 3.5-fold increase in the initial rate of apo-SAA secretion was observed over that of hepatocytes isolated from control mice, whereas the rate of apo-A-I secretion was unchanged by endotoxin administration. Sodium dodecyl sulfate-gel electrophoresis and autoradiography of [35S]methionine-labeled cell products indicated the synthesis of both major mouse apo-SAA isotypes by hepatocytes. Essentially all of the secreted apo-SAA chromatographed in Sephadex G-150 with an elution volume corresponding to a molecular weight of approximately 12,000. Approximately 90% of the secreted apo-SAA was recovered in fractions (d greater than 1.21 g/ml) following ultracentrifugal fractionation. In media supplemented with human lipoproteins (100 micrograms/ml), approximately 50% of the secreted apo-SAA was recovered in the high density lipoprotein fraction. These results suggest that mouse apo-SAA is secreted in monomeric form and becomes associated with lipoproteins in the intravascular compartment.     

Alterations of cell volume regulation in the development of hepatocyte necrosis

Intracellular Na+ accumulation has been shown to contribute to hepatocyte death caused by anoxia or oxidative stress. In this study we have investigated the mechanism by which Na+ overload can contribute to the development of cytotoxicity. ATP depletion in isolated hepatocytes exposed to menadione-induced oxidative stress or to KCN was followed by Na+ accumulation, loss of intracellular K+, and cell swelling. Hepatocyte swelling occurred in two phases: a small amplitude swelling (about 15% of the initial size) with preservation of plasma membrane integrity and a terminal large amplitude swelling associated with cell death. Inhibition of Na+ accumulation by the use of a Na+-free medium prevented K+ loss, cell swelling, and cytotoxicity. Conversely, blocking K+ efflux by the addition of BaCl2 did not influence Na+ increase and small amplitude swelling, but greatly stimulated large amplitude swelling and cytotoxicity. Menadione or KCN killing of hepatocytes was also enhanced by inducing cell swelling in an hypotonic medium. However, increasing the osmolarity of the incubation medium did not protect against large amplitude swelling and cytotoxicity, since stimulated Na+ accumulation and K+ efflux. Altogether these results indicate that the impairment of volume regulation in response to the osmotic load caused by Na+ accumulation is critical for the development of cell necrosis induced by mitochondrial inhibition or oxidative stress.     

Secretion of acetylcholinesterase by a mouse hepatocyte X rat liver cell hybrid culture

Summary A hybrid cell line (E-2) that secretes the enzyme acetylcholinesterase (AChE) has been prepared. The E-2 cell was the product of a fusion between primary mouse hepatocytes and a chemically transformed rat liver cell line (FRL), neither of which expresses AChE activity. The enzyme was determined to be AChE on the basis of its susceptibility to inhibition by BW284c51 but not by iso-OMPA, as well as its substrate specificity. Although the secreted enzyme was salt soluble and its activity not modified by the addition of the nonionic detergent, Triton X-100, the activity of the cellular enzyme (derived from homogenates of E-2 cells) was greatly enhanced in the presence of the detergent. This work was supported by funds from the Chemical Research and Development Center, Aberdeen Proving Ground, MD. The opinions and assertions are the private ones of the authors and are not to be construed as official. These experiments were conducted according to the principles set forth in the “Guide for the Care and Use of Experimental Animals,” DHEW Publ. No. (NIH) 78-23.     

Embryonic liver cells and permanent lines as models for hepatocyte and bile duct cell differentiation

Analysis of liver cells during development is facilitated by the possibility of complementing in vivo analysis with experiments on cultured cells. In this review, we discuss results from several laboratories concerning bipotential hepatic stem cells from mouse (HBC-3, H-CFU-C, MMH and BMEL), rat (rhe14321) and primate (IPFLS) embryos. Several groups have used fluorescence-activated cell sorting to identify clonogenic bipotential cells; others have derived bipotential cell lines by plating liver cell suspensions and cloning. The bipotential cells, which probably originate from hepatoblasts, can differentiate as hepatocytes or bile duct cells, and undergo morphogenesis in culture. Disparities in differentiation can be explained by distinct medium compositions, extracellular matrix coated culture surfaces, and gene expression detection methods. Potential applications of these cell lines are discussed.     

The establishment and characterization of immortal hepatocyte cell lines from a mouse liver injury model

Hepatocytes are an important research tool used for numerous applications. However, a short life span and a limited capacity to replicate in vitro limit the usefulness of primary hepatocyte cultures. We have hypothesized that in vivo priming of hepatocyte could make them more susceptible to growth factors in the medium for continuous proliferation in vitro. Here, a novel approach used to establish hepatocyte cell lines that included hepatocyte priming in vivo prior to culture with a 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet was attempted. The cell line grew in a monolayer while maintaining a granular cytoplasm and a round nucleus. Electron microscopy displayed hepatocyte-like features including mitochondria, glycogen granules, and the presence of bile canaliculi. This cell line expressed many mature hepatocyte-specific genes including albumin, alpha1-antitrypsin, glucose 6-phosphatase, and tyrosine aminotransferase. Functional characteristic of hepatocytes like the ability to store glycogen, lipid, and synthesis of urea is well demonstrated by this cell line. These cells demonstrated anchorage dependent growth properties in soft agar and did not form tumors after transplantation into nude mice. This cell line can be sustained in culture for more than 100 passages (>1.5 years) without undergoing noticeable morphological changes or transformation. This novel method resulted in the establishment of an immortal, non-transformed hepatocyte cell line with functional characteristics that may aid research of cell metabolism, toxicology, and hepatocyte transplantation.     

A cell population analysis of the initial period of hepatocyte proliferation in the regenerating mouse liver

Morphological autoradiography was used to determine the hepatocyte percentage in the regenerating liver of mice in S-, G-, M-, post-M phases at various times after partial hepatectomy, namely at 24, 30, 36 and 42 hours. The total number of cells in the above mentioned phases turned out to be very little at 24 and 30 hours. At 36 h., the percentage of cells in S phase (labelled nuclei) as well as in G2 phase (unlabelled morphologically obvious premitotic nuclei) appeared to be high. The two alternative explanation of G2 hepatocytes emerging at 36 hours were verified to find out if they are a) that is called "a reserved G2 population", or b) a population characterized by a shorter (down to 2 or 3 h) time of S phase.     

Deciphering the role of GLUT4 N-glycosylation in adipocyte and muscle cell models

GLUT4 (glucose transporter 4) is responsible for the insulin-induced uptake of glucose by muscle and fat cells. In non-stimulated (basal) cells, GLUT4 is retained intracellularly, whereas insulin stimulation leads to its translocation from storage compartments towards the cell surface. How GLUT4 is retained intracellularly is largely unknown. Previously, aberrant GLUT4 N-glycosylation has been linked to increased basal cell-surface levels, while N-glycosylation-deficient GLUT4 was found to be quickly degraded. As recycling and degradation of GLUT4 are positively correlated, we hypothesized that incorrect N-glycosylation of GLUT4 might reduce its intracellular retention, resulting in an increased cell-surface recycling, in increased basal cell-surface levels, and in enhanced GLUT4 degradation. In the present study, we have investigated N-glycosylation-deficient GLUT4?in detail in 3T3-L1 preadipocytes, 3T3-L1 adipocytes and L6 myoblasts. We have found no alterations in retention, insulin response, internalization or glucose transport activity. Degradation of the mutant molecule was increased, although once present at the cell surface, its degradation was identical with that of wild-type GLUT4. Our findings indicate that N-glycosylation is important for efficient trafficking of GLUT4 to its proper compartments, but once the transporter has arrived there, N-glycosylation plays no further major role in its intracellular trafficking, nor in its functional activity.     

Characterization of the mouse liver cell line FL83B

The previously reported mouse liver cell line FL83B has been characterized more completely with respect to its light and electron microscopic appearance, chromosomal composition, and ability to secrete various serum proteins. These cells bear many striking morphological similarities to parenchymal liver cells. Chromosomal analysis showed that the cells were transformed. The ability of these cells to grow in a completely chemically defined medium permitted the unequivocal demonstration of the synthesis of at least 12 mouse serum proteins including albumin and high density lipoprotein.     

血清铁蛋白含量与HBV相关慢性肝病的临床相关性

目的 研究HBV相关慢性肝病患者血清铁蛋白（SF）含量及其与肝功能血清学指标的相关性,进一步探讨铁蛋白在诊断HBV相关慢性肝病中的价值。方法 选取2016年1~6月于安徽医科大学第二附属医院就诊的慢性乙型肝炎患者（CHB）27例、肝硬化代偿期患者（LC）17例、肝硬化失代偿期患者（DLC）30例及健康对照者30例。采用免疫比浊法分析患者SF含量,采用Spearman秩相关分析SF含量与血清丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）、白蛋白（ALB）、前白蛋白（PALB）、总胆红素水平（TBIL）的相关性,运用受试者工作特征（ROC）曲线评价SF能否作为鉴别诊断LC与DLC的临床指标。结果 与对照组相比,CHB患者与LC患者的SF水平明显升高,DLC患者SF水平明显低于CHB患者。与LC患者相比,DLC患者SF水平明显降低。CHB患者与LC患者间SF水平差异无统计学意义。ROC曲线显示,采用SF诊断LC与DLC的曲线下面积为0.816,敏感性为56.67%,特异性为94.12%,界值为66.3。Spearman分析显示,CHB患者SF水平与ALT、AST、TBIL水平呈显著正相关,与ALB、PALB水平呈显著负相关;LC患者SF水平与ALT、AST、TBIL水平呈显著正相关;DLC患者SF水平与ALB、PALB、TBIL水平呈显著正相关。结论 SF水平与HBV相关慢性肝病的进展具有相关性,可作为临床鉴别诊断LC与DLC的血清学指标和判断HBV相关肝病损伤的潜在指标。     

Synaptic plasticity deficits and mild memory impairments in mouse models of chronic granulomatous disease

Reactive oxygen species (ROS) are required in a number of critical cellular signaling events, including those underlying hippocampal synaptic plasticity and hippocampus-dependent memory; however, the source of ROS is unknown. We previously have shown that NADPH oxidase is required for N-methyl-D-aspartate (NMDA) receptor-dependent signal transduction in the hippocampus, suggesting that NADPH oxidase may be required for NMDA receptor-dependent long-term potentiation (LTP) and hippocampus-dependent memory. Herein we present the first evidence that NADPH oxidase is involved in hippocampal synaptic plasticity and memory. We have found that pharmacological inhibitors of NADPH oxidase block LTP. Moreover, mice that lack the NADPH oxidase proteins gp91(phox) and p47(phox), both of which are mouse models of human chronic granulomatous disease (CGD), also lack LTP. We also found that the gp91(phox) and p47(phox) mutant mice have mild impairments in hippocampus-dependent memory. The gp91(phox) mutant mice exhibited a spatial memory deficit in the Morris water maze, and the p47(phox) mutant mice exhibited impaired context-dependent fear memory. Taken together, our results are consistent with NADPH oxidase being required for hippocampal synaptic plasticity and memory and are consistent with reports of cognitive dysfunction in patients with CGD.