Effects of thyroid hormone receptor gene disruption on myosin isoform expression in mouse skeletal muscles

Skeletal muscle is known to be a target for the active metabolite of thyroid hormone, i.e., 3,5,3'-triiodothyronine (T(3)). T(3) acts by repressing or activating genes coding for different myosin heavy chain (MHC) isoforms via T(3) receptors (TRs). The diverse function of T(3) is presumed to be mediated by TR-alpha(1) and TR-beta, but the function of specific TRs in regulating MHC isoform expression has remained undefined. In this study, TR-deficient mice were used to expand our knowledge of the mechanisms by which T(3) regulates the expression of specific MHC isoforms via distinct TRs. In fast-twitch extensor digitorum longus (EDL) muscle, TR-alpha(1)-, TR-beta-, or TR-alpha(1)beta-deficient mice showed a small but statistically significant decrease (P < 0.05) of type IIB MHC content and an increased number of type I fibers. In the slow-twitch soleus, the beta/slow MHC (type I) isoform was significantly (P < 0. 001) upregulated in the TR-deficient mice, but this effect was highly dependent on the type of receptor deleted. The lack of TR-beta had no significant effect on the expression of MHC isoforms. An increase (P < 0.05) of type I MHC was observed in the TR-alpha(1)-deficient muscle. A dramatic overexpression (P < 0.001) of the slow type I MHC and a corresponding downregulation of the fast type IIA MHC (P < 0.001) was observed in TR-alpha(1)beta-deficient mice. The muscle- and fiber-specific differences in MHC isoform expression in the TR-alpha(1)beta-deficient mice resembled the MHC isoform transitions reported in hypothyroid animals, i.e., a mild MHC transition in the EDL, a dramatic but not complete upregulation of the beta/slow MHC isoform in the soleus, and a variable response to TR deficiency in different soleus muscle fibers. Thus the consequences on muscle are similar in the absence of thyroid hormone or absence of thyroid hormone receptors, indicating that TR-alpha(1) and TR-beta together mediate the known actions of T(3). However, it remains unknown how thyroid hormone exerts muscle- and muscle fiber-specific effects in its action. Finally, although developmental MHC transitions were not studied specifically in this study, the absence of embryonic and fetal MHC isoforms in the TR-deficient mice indicates that ultimately the transition to the adult MHC isoforms is not solely mediated by TRs.     

Dominant inhibition of thyroid hormone action selectively in the pituitary of thyroid hormone receptor-beta null mice abolishes the regulation of thyrotropin by thyroid hormone

Thyroid hormones, T4 and T3, regulate their own production by feedback inhibition of TSH and TRH synthesis in the pituitary and hypothalamus when T3 binds to thyroid hormone receptors (TRs) that interact with the promoters of the genes for the TSH subunit and TRH. All TR isoforms are believed to be involved in the regulation of this endocrine axis, as evidenced by the massive dysregulation of TSH production in mice lacking all TR isoforms. However, the relative contributions of TR isoforms in the pituitary vs. the hypothalamus remain to be completely elucidated. Thus, to determine the relative contribution of pituitary expression of TR-alpha in the regulation of the hypothalamic-pituitary-thyroid axis, we selectively impaired TR-alpha function in TR-beta null mice (TR-beta-/-) by pituitary restricted expression of a dominant negative TR-beta transgene harboring a delta337T mutation. These animals exhibited 10-fold and 32-fold increase in T4 and TSH concentrations, respectively. Moreover, the negative regulation of TSH by exogenous T3 was completely absent and a paradoxical increase in TSH concentrations and TSH-beta mRNA was observed. In contrast, prepro-TRH expression levels in T3-treated TR-beta-/- were similar to levels observed in the delta337/TR-beta-/- mice, and ligand-independent activation of TSH in hypothyroid mice was equivalently impaired. Thus, isolated TR-beta deficiency in TRH paraventricular hypothalamic nucleus neurons and impaired function of all TRs in the pituitary recapitulate the baseline hormonal disturbances that characterize mice with complete absence of all TRs.     

Quantitation of rat tissue thyroid hormone binding receptor isoforms by immunoprecipitation of nuclear triiodothyronine binding capacity.

A panel of anti-thyroid hormone receptor (TR) antisera were generated to allow direct assay of the concentrations of the alpha 1 and beta 1 receptor isoforms in nuclear extracts from adult rat liver, kidney, brain and heart, and fetal brain. An antiserum, immunoglobulin G (IgG)-beta 1, raised against amino acid sequence 62-92 of the rat TR-beta 1 specifically precipitated only TR-beta 1 in vitro translation products. A second antiserum, IgG-alpha 1/beta, generated against a sequence that is identical in the ligand binding region of rat TR-alpha 1 and TR-beta isoforms immunoprecipitated both TR-alpha 1 and -beta 1 translation products. These IgG preparations were used to specifically immunoprecipitate thyroid hormone receptor binding activity from nuclear extracts. IgG-beta 1 cleared almost 80%, and the IgG-alpha 1/beta immunoprecipitated nearly all binding from hepatic nuclear extracts. This distribution of TR protein, 80% beta 1 and 20% alpha 1, is the same as previously reported for their respective mRNAs in liver. In heart, kidney, and brain IgG-beta 1 cleared 45, 43, and 28% of total binding, respectively, and IgG-alpha 1/beta cleared all T3 binding activity from these tissues. In agreement with an earlier study, marked variations in specific protein/mRNA ratios were noted among these tissues. Consistent with our earlier report of the presence of only very low levels of TR-beta 1 mRNA in fetal brain, IgG-beta 1 cleared just 5% of binding in this tissue. Studies using an antiserum (IgG-ch) generated against homologous segments of the hinge region in both TR-alpha 1 and -beta 1 yielded results which contrasted sharply with those of IgG-alpha 1/beta. Whereas IgG-ch could also immunoprecipitate virtually all binding from hepatic extracts it cleared only 40-50% of binding from the other tissues, including fetal brain in which TR-alpha 1 accounts for greater than 90% of binding protein. The data suggest the presence of posttranslational modification of the TR-alpha 1 protein in the hinge region, consistent with the presence in this segment of potential phosphorylation sites.     

Sick euthyroid syndrome is associated with decreased TR expression and DNA binding in mouse liver

Infection is associated with low serum thyroid hormones and thyrotropin levels. Here we demonstrate that infection also reduces thyroid hormone receptor (TR) expression. In gel shift experiments, retinoid X receptor (RXR)/TR DNA binding was reduced in mouse liver by 60 and 77%, respectively, 4 and 16 h after lipopolysaccharide (LPS) administration. Surprisingly, LPS did not decrease either TR-alpha or TR-beta protein levels at 4 h, but by 16 h TR-alpha(1), TR-alpha(2), and TR-beta levels were reduced by 55, 87, and 41%, respectively. We previously reported that LPS rapidly decreases RXR protein levels in liver. Therefore, we added RXR-beta to hepatic nuclear extracts prepared 4 h after LPS treatment, which restored RXR/TR DNA binding to a level comparable to that of controls. A similar experiment conducted on extracts prepared 16 h after LPS administration did not restore RXR/TR DNA binding. We propose that decreased RXR expression is limiting for RXR/TR DNA binding at 4 h, whereas the reduction in both TR and RXR levels results in further decreased binding at 16 h.     

Development and expression of cytoplasmic antigens in Purkinje cells recognized by monoclonal antibodies

Summary Five monoclonal antibodies reacting with intracellular constituents of Purkinje cells were investigated by means of indirect immunofluorescence on fresh-frozen sections of the cerebellum and retina from developing and adult normal and mutant mice. Antibodies PC1, PC2 and PC3, which recognize Purkinje cells, but no other cerebellar neuron type, label these cells from day 4 onward. PC4 antigen is expressed in addition to Purkinje cells also in granule cells and neurons of deep cerebellar nuclei and appears in Purkinje cells at day 4. M1 antigen (Lagenaur et al. 1980) is first detectable in Purkinje cell bodies by day 5; it is also detectable in deep cerebellar neurons. In the adult retina, only PC4 antigen is detectably expressed and is localized in the inner segments of photoreceptor cells.The neurological mutants weaver, reeler,jimpy and wobbler show detectable levels of these antigens in Purkinje cells. However, the mutants staggerer and Purkinje cell degeneration are abnormal in expression PC1, PC2, PC3, and M1 antigens. Staggerer never starts to express the antigens during development, whereas Purkinje cell degeneration first expresses the antigens, but then loses antigen expression after day 23. PC4 antigen is detectable in the remaining Purkinje cells in staggerer and Purkinje cell degeneration mice at all ages tested in this study. Deep cerebellar neurons are positive for both antigens, PC4 and M1, in all mutants and at all ages studied. In retinas of staggerer and Purkinje cell degeneration mutants, PC4 antigen is normally detectable in the inner segments of photoreceptor cells, even when these have started to degenerate in the case of Purkinje cell degeneration.     

G-protein alpha subunit interaction and guanine nucleotide dissociation inhibitor activity of the dual GoLoco motif protein PCP-2 (Purkinje cell protein-2)

Purkinje cell protein-2 (PCP-2; L7/GPSM4) is a GoLoco motif-containing protein that is specifically expressed in Purkinje and retinal ON bipolar cells. An alternative splice variant of PCP-2 has recently been isolated which contains two GoLoco motifs. Although the second GoLoco motif (GL2) of PCP-2 has been reported to interact with Galpha-subunits, a complete biochemical analysis of each individual motif of PCP-2 has not been performed. We demonstrate that the first GoLoco motif (GL1) of PCP-2 is equipotent as a guanine nucleotide dissociation inhibitor (GDI) towards Galphai1 and Galphai2, while it has sevenfold lower GDI activity for Galphai3 and greater than 20-fold lower GDI activity against Galphao. In contrast we found PCP-2 GL2 to be essentially equipotent as a GDI for all Galphai subunits, but it had negligible activity toward Galphao. Using co-immunoprecipitation from COS-7 cells, we found that PCP-2 was only able to interact with Galphai1 but not Galphao nor Galpha-subunits from other families (Galphas, Galphaq, or Galpha12). Mutational analysis of a non-canonical residue (glycine 24) in human PCP-2 GL1 provided evidence for heterogeneity in mechanisms of Galphai interactions with GoLoco motifs. Collectively, the data demonstrate that PCP-2 is a comparatively weak GoLoco motif protein that exhibits highest affinity interactions and GDI activity toward Galphai1, Galphai2, and Galphai3 subunits.     

Gene transfer into Purkinje cells using herpesviral amplicon vectors in cerebellar cultures

Purkinje cells play a crucial role in sensory motor coordination since they are the only output projection neurons in the cerebellar cortex and are affected in most spinocerebellar ataxias. They stand out in the central nervous system due to their large size and their profusely branched dendritic arbor. However, molecular and cellular studies on Purkinje cells are often hampered by the difficulty of maintaining these cells in culture. Here we report an easy, robust and reproducible method to obtain Purkinje-enriched mixed cerebellar cell cultures from day 16 mouse embryos using papain digestion and a semi-defined culture medium, being the composition of the culture approximately 20% Purkinje cells, 70% non-Purkinje neurons and 10% glial cells. We demonstrate that efficient gene transfer into Purkinje cells (as well as into other cerebellar populations) is possible using herpes simplex virus-1 (HSV-1)-derived vectors. Indeed, up to 50% of the Purkinje cells can be transduced and gene expression may persist for at least 14 days. As a result, this procedure permits functional gene expression studies to be carried out on cultured Purkinje neurons. To demonstrate this, we show that the expression of a dominant-negative form of glycogen synthase kinase-3 protects Purkinje neurons against cell death triggered by a chemical inhibitor of phosphatidylinositol-3 kinase. In summary, we have established reproducible and reliable cerebellar cell cultures enriched for Purkinje cells which enables gene transfer studies to be carried out using herpesviral vectors.     

Anti-Yo Antibody Uptake and Interaction with Its Intracellular Target Antigen Causes Purkinje Cell Death in Rat Cerebellar Slice Cultures: A Possible Mechanism for Paraneoplastic Cerebellar Degeneration in Humans with Gynecological or Breast Cancers

Anti-Yo antibodies are immunoglobulin G (IgG) autoantibodies reactive with a 62 kDa Purkinje cell cytoplasmic protein. These antibodies are closely associated with paraneoplastic cerebellar degeneration in the setting of gynecological and breast malignancies. We have previously demonstrated that incubation of rat cerebellar slice cultures with patient sera and cerebrospinal fluid containing anti-Yo antibodies resulted in Purkinje cell death. The present study addressed three fundamental questions regarding the role of anti-Yo antibodies in disease pathogenesis: 1) Whether the Purkinje cell cytotoxicity required binding of anti-Yo antibody to its intraneuronal 62 kDa target antigen; 2) whether Purkinje cell death might be initiated by antibody-dependent cellular cytotoxicity rather than intracellular antibody binding; and 3) whether Purkinje cell death might simply be a more general result of intracellular antibody accumulation, rather than of specific antibody-antigen interaction. In our study, incubation of rat cerebellar slice cultures with anti-Yo IgG resulted in intracellular antibody binding, and cell death. Infiltration of the Purkinje cell layer by cells of macrophage/microglia lineage was not observed until extensive cell death was already present. Adsorption of anti-Yo IgG with its 62 kDa target antigen abolished both antibody accumulation and cytotoxicity. Antibodies to other intracellular Purkinje cell proteins were also taken up by Purkinje cells and accumulated intracellularly; these included calbindin, calmodulin, PCP-2, and patient anti-Purkinje cell antibodies not reactive with the 62 kDa Yo antigen. However, intracellular accumulation of these antibodies did not affect Purkinje cell viability. The present study is the first to demonstrate that anti-Yo antibodies cause Purkinje cell death by binding to the intracellular 62 kDa Yo antigen. Anti-Yo antibody cytotoxicity did not involve other antibodies or factors present in patient serum and was not initiated by brain mononuclear cells. Purkinje cell death was not simply due to intraneuronal antibody accumulation.     

Effect of bax deletion on ethanol sensitivity in the neonatal rat cerebellum

The developing cerebellum is highly sensitive to ethanol during discrete neonatal periods. This sensitivity has been linked to ethanol-induced alterations in molecules of the Bcl-2 survival-regulatory gene family. Ethanol exposure during peak periods of cerebellar sensitivity, for example, results in increased expression of proapoptotic proteins of this family, while overexpression of the antiapoptotic Bcl-2 protein in the nervous system protects against ethanol neurotoxicity. For the present study, neonatal mice with a targeted deletion of the proapoptotic bax gene were used to determine whether elimination of this protein would mitigate ethanol toxicity. bax knock-out and wild-type mice pups were exposed to ethanol via vapor inhalation during the maximal period of neonatal cerebellar ethanol sensitivity and cerebellar tissue was subsequently assessed for Purkinje and granule cell number and ethanol-mediated generation of reactive oxygen species (ROS). The results revealed that: (1) ethanol exposure during the peak period of cerebellar vulnerability resulted in substantial loss of Purkinje cells in wild-type animals, but not in bax knock-outs; (2) granule cells in the bax gene-deleted animals were not similarly protected from ethanol effects; and (3) levels of ROS following acute ethanol exposure were appreciably enhanced in the wild-type animals but not in the bax knock-outs. These results imply that Bax is important to ethanol-induced Purkinje cell death during critical neonatal periods, but that ethanol effects on granule cells may function at least partially independent of this apoptosis agonist. Amelioration of ethanol-mediated increases in ROS production in the knock-outs may contribute to the observed effects.     

Disrupted cerebellar cortical development and progressive degeneration of Purkinje cells in SV40 T antigen transgenic mice.

SV40 T antigen (Tag) expression directed to cerebellar Purkinje cells resulted in the generation of three transgenic mouse lines that displayed ataxia, a neurological phenotype characteristic of cerebellar dysfunction. Onset of symptoms and cerebellar pathology, characterized by specific Purkinje cell degeneration, appeared to be directly dependent upon transgene copy number. The SV5 line (containing > 30 transgene copies), exhibited embryonic transgene expression that caused selective death of immature Purkinje cells and a subsequent block in cerebellar development and ataxia at 2 weeks. The developmental effect of the disruption of Purkinje cells in SV5 mice suggests that a normal complement of these cells is required for early development of the cerebellar cortex, especially granule cell proliferation and migration from external to internal layers. Transgene expression in a second line, SV4 (10 copies), was detectable during the second postnatal week. Death of mature Purkinje cells in the SV4 line resulted in onset of ataxia at 9 weeks. Ataxia in a third line, SV6 (2 copies), was detected after 15 weeks. The distinct cerebellar phenotypes of the SV4-6 lines correlate with specific Tag-induced Purkinje cell ablation as opposed to tumorigenesis.     

Neurotrophins promote the survival and development of neurons in the cerebellum of hypothyroid rats in vivo

The development of cerebellar cortex is strongly impaired by thyroid hormone (T3) deficiency, leading to altered migration, differentiation, synaptogenesis, and survival of neurons. To determine whether alteration in the expression of neurotrophins and/or their receptors may contribute to these impairments, we first analyzed their expression using a sensitive RNAse protection assay and in situ hybridization; second, we administered the deficient neurotrophins to hypothyroid animals. We found that early hypothyroidism disrupted the developmental pattern of expression of the four neurotrophins, leading to relatively higher levels of NGF and neurotrophin 4/5 mRNAs and to a severe deficit in NT-3 and brain-derived neurotrophic factor (BDNF) mRNA expression, without alteration in the levels of the full-length tyrosine kinase (trk) B and trkC receptor mRNAs. Grafting of P3 hypothyroid rats with cell lines expressing high levels of neurotrophin 3 (NT-3) or BDNF prevented hypothyroidism-induced cell death in neurons of the internal granule cell layer at P15. In addition, we found that NT-3, but not BDNF, induced the differentiation and/or migration of neurons in the external granule cell layer, stimulated the elaboration of the dendritic tree by Purkinje cells, and promoted the formation of the mature pattern of synaptic afferents to Purkinje cell somas. Thus, our results indicate that both granule and Purkinje neurons require appropriate levels of NT-3 for normal development in vivo and suggest that T3 may regulate the levels of neurotrophins to promote the development of cerebellum.     

Isometric force and endurance in soleus muscle of thyroid hormone receptor-alpha(1)- or -beta-deficient mice

The specific role of each subtype of thyroid hormone receptor (TR) on skeletal muscle function is unclear. We have therefore studied kinetics of isometric twitches and tetani as well as fatigue resistance in isolated soleus muscles of R-alpha(1)- or -beta-deficient mice. The results show 20-40% longer contraction and relaxation times of twitches and tetani in soleus muscles from TR-alpha(1)-deficient mice compared with their wild-type controls. TR-beta-deficient mice, which have high thyroid hormone levels, were less fatigue resistant than their wild-type controls, but contraction and relaxation times were not different. Western blot analyses showed a reduced concentration of the fast-type sarcoplasmic reticulum Ca(2+)-ATPase (SERCa1) in TR-alpha(1)-deficient mice, but no changes were observed in TR-beta-deficient mice compared with their respective controls. We conclude that in skeletal muscle, both TR-alpha(1) and TR-beta are required to get a normal thyroid hormone response.     

Genetic factors influencing neurosensitivity to early phenobarbital administration in mice

This study was designed to assess the possible genetic determinants of neurosensitivity to early (neonatal) phenobarbital (PhB) administration and to conduct a strain comparison for the cerebellar histology of both inbred and outbred mice. HS/Ibg, C57BL/10 and DBA/1 pups were injected with 50 mg PhB/kg daily on neonatal days 2-21. On day 50, treated animals (B) of all strains had smaller brains than controls (C). Moreover, the cerebellar area was decreased in HS and C57 B mice but not in DBA mice, suggesting genotype-environment interaction. B mice from all strains had similar Purkinje cell losses. Strain comparison showed that control C57 mice had smaller brains than control HS, and DBA had smaller brains than both HS and C57. Similarly, C57 had smaller cerebellar layers than HS and DBA had smaller cerebellar layers than both HS and C57. DBA and C57 mice had fewer Purkinje cells than HS but did not differ from each other.     

Differential Expression of GABAA/Benzodiazepine Receptor Subunit mRNAs and Ligand Binding Sites in Mouse Cerebellar Neurons Following In Vivo Ethanol Administration: An Autoradiographic Analysis

Abstract: The γ-aminobutyric acid_A (GABA_A)/benzodiazepine (BZ) receptor is a pentamer composed of subunits belonging to several classes (α_1–6, β_1–4, γ_1–4, δ, and ρ₁ and ρ₂). In situ hybridization, radioligand autoradiography, and immunocytochemistry were used to examine GABA_A/BZ receptor α₁, α₆, β₂, β₃, and γ₂ subunit expression in murine Purkinje, granule, and deep cerebellar neurons after in vivo ethanol exposure. Chronic ethanol treatment resulted in decreased α₁ subunit mRNA expression in each cell type, whereas the expression of α₆ and γ₂ subunit mRNA levels increased; no changes were observed in the expression of β₂ and β₃ subunit mRNA. GABA and BZ agonist binding and antibody staining paralleled the changes in mRNA levels. Acute ethanol injection resulted in increased expression of α₁ and β₃ mRNAs, whereas levels of α₆, β₂, and γ₂ mRNAs remained stable. Our results indicate that, in cerebellar neurons, the expression of specific GABA_A/BZ receptor subunit mRNAs, polypeptides, and binding sites is independently regulated by in vivo administration of alcohol. The observed changes were not restricted to any one cerebellar cell type, because subunit expression in Purkinje, granule, and deep cerebellar cells was similarly affected.     

多巴胺受体1在皖西白鹅小脑皮质发育中的表达

采用普通染色及免疫组化SABC染色法研究皖西白鹅小脑皮质的发育和多巴胺受体1(DRD1)阳性细胞在其发育中的表达.结果表明,小脑皮质在胚龄13 d(E13)由外向内分为外颗粒层(EGL)、浦肯野细胞层(PCL)和内颗粒层(IGL),E19由外向内分为EGL、分子层(ML)、PCL和IGL.随发育天数的增加,EGL的厚度和细胞层次呈先升后降的变化趋势,细胞密度逐渐下降;ML厚度逐渐增大,在E24到E28时增值最大;浦肯野细胞(PC)在E13、E19、E24和E28时随胚龄增大逐渐增大,在E28后趋于稳定,细胞密度随着发育天数的增加逐渐下降,在小脑皮质发育中还发现有一部分PC呈多层排列,且细胞层次逐渐变少;IGL厚度呈先升后降的变化趋势,细胞密度呈上升趋势.外颗粒层和内颗粒层在E13、E19、E24和E28时有DRD1阳性细胞表达,分子层在E24、E28、日龄7 d(P7)和15d(P15)有阳性细胞表达,PC在所检测的6个时段均有阳性表达.研究表明,小脑皮质的发育主要与细胞增殖、迁移和凋亡有关,外颗粒层的逐渐消失是以细胞迁移和凋亡为主,多层PC逐渐退化成单层是与细胞凋亡和正常突触联系的建立有关;DRD1在皖西白鹅小脑皮质发育中对外颗粒层细胞和PC起着重要作用.