Immunohistochemical localization of insulin-like growth factor binding protein 2 in the central nervous system of SOD1<Superscript>G93A</Superscript> transgenic mice

In the present study, we performed immunohistochemical studies to investigate the changes of insulin-like growth factor binding protein 2 (IGFBP2) in the central nervous system of SOD1^G93A mutant transgenic mice as an in vivo model of amyotrophic lateral sclerosis (ALS). Decreased immunoreactivity for IGFBP2 was observed in the cerebral cortex, hippocampus and brainstem of SOD1^G93A transgenic mice. In the cerebral cortex, the number of IGFBP2-positive cells was decreased in the somatomotor area, somatosensory area, auditory area, visual area, entorhinal area, piriform area and prefrontal area. In the hippocampal formation, IGFBP2 immunoreactivity was significantly decreased in the CA1-3 areas and the dentate gyrus. In the brainstem, few IGFBP2-immunoreactive cells were observed in the medullary and pontine reticular formation, vestibular nucleus, trigeminal motor nucleus, facial nucleus, hypoglossal nucleus and raphe nucleus. In the spinal cord, IGFBP2 immunoreactivity was not significantly decreased in SOD1^G93A transgenic mice. This study showing decreased IGFBP2 in different brain regions of SOD1^G93A transgenic mice may provide clues for understanding differential susceptibility of neural structures in ALS. S. E. Sim and Y. H. Chung have contributed equally to this work.     

Electrophysiologic Biomarkers for Assessing Disease Progression and the Effect of Riluzole in SOD1 G93A ALS Mice

Objective

To compare electrical impedance myography (EIM) 50 kHz phase to weight, motor score, paw grip endurance (PGE), CMAP amplitude, and MUNE for the identification of disease progression and the effect of riluzole in the SOD1 G93A mouse.

Methods

Twenty-three animals received 8 mg/kg/day riluzole in the drinking water starting at 6 weeks of age; 22 animals served as controls. Weight, motor score, PGE, CMAP, MUNE, and EIM were performed weekly to evaluate disease progression.

Results

No difference in clinical disease onset or survival was found between treated and untreated groups. In addition, all methods failed to identify any beneficial effect of riluzole. Thus, data from all animals were combined for additional analyses. Of the 4 parameters, EIM phase showed the earliest change from baseline and the most linear decline throughout the entire measurement period. In addition, EIM phase correlated with PGE, CMAP amplitude, and MUNE (Spearman r = 0.92, 0.90, and 0.72, respectively, p<0.01 for all). The rate of EIM phase decline also correlated with individual animal survival (Spearman r = −0.31, p<0.05).

Conclusions

At this dose, riluzole is ineffective in slowing progression of ALS. However, EIM phase shows early linear declines, supporting its potential as a useful new biomarker for preclinical drug testing.     

Tempol Moderately Extends Survival in a hSOD1G93A ALS Rat Model by Inhibiting Neuronal Cell Loss,Oxidative Damage and Levels of Non-Native hSOD1G93A Forms

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive dysfunction and death of motor neurons by mechanisms that remain unclear. Evidence indicates that oxidative mechanisms contribute to ALS pathology, but classical antioxidants have not performed well in clinical trials. Cyclic nitroxides are an alternative worth exploring because they are multifunctional antioxidants that display low toxicity in vivo. Here, we examine the effects of the cyclic nitroxide tempol (4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl) on ALS onset and progression in transgenic female rats over-expressing the mutant hSOD1^G93A . Starting at 7 weeks of age, a high dose of tempol (155 mg/day/rat) in the rat´s drinking water had marginal effects on the disease onset but decelerated disease progression and extended survival by 9 days. In addition, tempol protected spinal cord tissues as monitored by the number of neuronal cells, and the reducing capability and levels of carbonylated proteins and non-native hSOD1 forms in spinal cord homogenates. Intraperitoneal tempol (26 mg/rat, 3 times/week) extended survival by 17 days. This group of rats, however, diverted to a decelerated disease progression. Therefore, it was inconclusive whether the higher protective effect of the lower i.p. dose was due to higher tempol bioavailability, decelerated disease development or both. Collectively, the results show that tempol moderately extends the survival of ALS rats while protecting their cellular and molecular structures against damage. Thus, the results provide proof that cyclic nitroxides are alternatives worth to be further tested in animal models of ALS.     

Amino Acids as biomarkers in the SOD1G93A mouse model of ALS

The development of therapies for Amyotrophic Lateral Sclerosis (ALS) has been hindered by the lack of biomarkers for both identifying early disease and for monitoring the effectiveness of drugs. The identification of ALS biomarkers in presymptomatic individuals might also provide clues to the earliest biochemical correlates of the disease. Previous attempts to use plasma metabolites as biomarkers have led to contradictory results, presumably because of heterogeneity in both the underlying genetics and the disease stage in the clinical population. To eliminate these two sources of heterogeneity we have characterized plasma amino acids and other metabolites in the SOD1^G93A transgenic mouse model for ALS. Presymptomatic SOD1^G93A mice have significant differences in concentrations of several plasma metabolites compared to wild type animals, most notably in the concentrations of aspartate, cystine/cysteine, and phosphoethanolamine, and in changes indicative of methylation defects. There are significant changes in amino acid compositions between 50 and 70 days of age in both the SOD1^G93A and wild type mice, and several of the age-related and disease-related differences in metabolite concentration were also gender-specific. Many of the SOD1^G93A-related differences could be altered by treatment of mice with methionine sulfoximine, which extends the lifespan of this mouse, inhibits glutamine synthetase, and modifies brain methylation reactions. These studies show that assaying plasma metabolites can effectively distinguish transgenic mice from wild type, suggesting that one or more plasma metabolites might be useful biomarkers for the disease in humans, especially if genetic and longitudinal analysis is used to reduce population heterogeneity.     

Simultaneous <Superscript>1</Superscript>H- or <Superscript>2</Superscript>H-, <Superscript>15</Superscript>N- and multiple-band-selective <Superscript>13</Superscript>C-decoupling during acquisition in <Superscript>13</Superscript>C-detected experiments with proteins and oligonucleotides

Significant resolution improvement in ¹³C,¹³C-TOCSY spectra of uniformly deuterated and ¹³C, ¹⁵N-labeled protein and ¹³C,¹⁵N-labeled RNA samples is achieved by introduction of multiple-band-selective ¹³C-homodecoupling applied simultaneously with ¹H- or ²H- and ¹⁵N-decoupling at all stages of multidimensional experiments including signal acquisition period. The application of single, double or triple band-selective ¹³C-decoupling in 2D-[¹³C,¹³C]-TOCSY experiments during acquisition strongly simplifies the homonuclear splitting pattern. The technical aspects of complex multiple-band homonuclear decoupling and hardware requirements are discussed. The use of this technique (i) facilitates the resonance assignment process as it reduces signal overlap in homonuclear ¹³C-spectra and (ii) possibly improves the signal-to-noise ratio through multiplet collapse. It can be applied in any ¹³C-detected experiment.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N, <Superscript>13</Superscript>C resonance assignment of human osteopontin

Osteopontin (OPN) is a 33.7 kDa intrinsically disordered protein and a member of the SIBLING family of proteins. OPN is bearing a signal peptide for secretion into the extracellular space, where it exerts its main physiological function, the control of calcium biomineralization. It is often involved in tumorigenic processes influencing proliferation, migration and survival, as well as the adhesive properties of cancer cells via CD44 and integrin signaling pathways. Here we report the nearly complete NMR chemical shift assignment of recombinant human osteopontin.     

NMR <Superscript>1</Superscript>H,<Superscript>13</Superscript>C, <Superscript>15</Superscript>N backbone and <Superscript>13</Superscript>C side chain resonance assignment of the G12C mutant of human K-Ras bound to GDP

K-Ras is a key driver of oncogenesis, accounting for approximately 80% of Ras-driven human cancers. The small GTPase cycles between an inactive, GDP-bound and an active, GTP-bound state, regulated by guanine nucleotide exchange factors and GTPase activating proteins, respectively. Activated K-Ras regulates cell proliferation, differentiation and survival by signaling through several effector pathways, including Raf-MAPK. Oncogenic mutations that impair the GTPase activity of K-Ras result in a hyperactivated state, leading to uncontrolled cellular proliferation and tumorogenesis. A cysteine mutation at glycine 12 is commonly found in K-Ras associated cancers, and has become a recent focus for therapeutic intervention. We report here ¹H^{N, 15}N, and ¹³C resonance assignments for the 19.3 kDa (aa 1–169) human K-Ras protein harboring an oncogenic G12C mutation in the GDP-bound form (K-RAS^G12C-GDP), using heteronuclear, multidimensional NMR spectroscopy. Backbone ¹H–¹⁵N correlations have been assigned for all non-proline residues, except for the first methionine residue.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N resonance assignment of human guanylate kinase

Human guanylate kinase (hGMPK) is a critical enzyme that, in addition to phosphorylating its physiological substrate (d)GMP, catalyzes the second phosphorylation step in the conversion of anti-viral and anti-cancer nucleoside analogs to their corresponding active nucleoside analog triphosphates. Until now, a high-resolution structure of hGMPK is unavailable and thus, we studied free hGMPK by NMR and assigned the chemical shift resonances of backbone and side chain ¹H, ¹³C, and ¹⁵N nuclei as a first step towards the enzyme’s structural and mechanistic analysis with atomic resolution.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N resonance assignments of a C-terminal domain of human CHD1

Chromatin remodelling proteins are an essential family of eukaryotic proteins. They harness the energy from ATP hydrolysis and apply it to alter chromatin structure in order to regulate all aspects of genome biology. Chromodomain helicase DNA-binding protein 1 (CHD1) is one such remodelling protein that has specialised nucleosome organising abilities and is conserved across eukaryotes. CHD1 possesses a pair of tandem chromodomains that directly precede the core catalytic Snf2 helicase-like domain, and a C-terminal SANT-SLIDE DNA-binding domain. We have identified an additional conserved domain in the C-terminal region of CHD1. Here, we report the backbone and side chain resonance assignments for this domain from human CHD1 at pH 6.5 and 25 °C (BMRB No. 25638).     

Backbone <Superscript>1</Superscript>H, <Superscript>15</Superscript>N,and <Superscript>13</Superscript>C resonance assignments of the Tom1 VHS domain

Efficient trafficking of ubiquitinated receptors (cargo) to endosomes requires the recruitment of adaptor proteins that exhibit ubiquitin-binding domains for recognition and transport. Tom1 is an adaptor protein that not only associates with ubiquitinated cargo but also represents a phosphoinositide effector during specific bacterial infections. This phosphoinositide-binding property is associated with its N-terminal Vps27, Hrs, STAM (VHS) domain. Despite its biological relevance, there are no resonance assignments of Tom1 VHS available that can fully characterize its molecular interactions. Here, we report the nearly complete ¹H, ¹⁵N, and ¹³C backbone resonance assignments of the VHS domain of human Tom1.     

Automated amino acid side-chain NMR assignment of proteins using <Superscript>13</Superscript>C- and <Superscript>15</Superscript>N-resolved 3D [<Superscript>1</Superscript>H,<Superscript>1</Superscript>H]-NOESY

ASCAN is a new algorithm for automatic sequence-specific NMR assignment of amino acid side-chains in proteins, which uses as input the primary structure of the protein, chemical shift lists of (1)H(N), (15)N, (13)C(alpha), (13)C(beta) and possibly (1)H(alpha) from the previous polypeptide backbone assignment, and one or several 3D (13)C- or (15)N-resolved [(1)H,(1)H]-NOESY spectra. ASCAN has also been laid out for the use of TOCSY-type data sets as supplementary input. The program assigns new resonances based on comparison of the NMR signals expected from the chemical structure with the experimentally observed NOESY peak patterns. The core parts of the algorithm are a procedure for generating expected peak positions, which is based on variable combinations of assigned and unassigned resonances that arise for the different amino acid types during the assignment procedure, and a corresponding set of acceptance criteria for assignments based on the NMR experiments used. Expected patterns of NOESY cross peaks involving unassigned resonances are generated using the list of previously assigned resonances, and tentative chemical shift values for the unassigned signals taken from the BMRB statistics for globular proteins. Use of this approach with the 101-amino acid residue protein FimD(25-125) resulted in 84% of the hydrogen atoms and their covalently bound heavy atoms being assigned with a correctness rate of 90%. Use of these side-chain assignments as input for automated NOE assignment and structure calculation with the ATNOS/CANDID/DYANA program suite yielded structure bundles of comparable quality, in terms of precision and accuracy of the atomic coordinates, as those of a reference structure determined with interactive assignment procedures. A rationale for the high quality of the ASCAN-based structure determination results from an analysis of the distribution of the assigned side chains, which revealed near-complete assignments in the core of the protein, with most of the incompletely assigned residues located at or near the protein surface.     

Near-complete <Superscript>1</Superscript>H, <Superscript>13</Superscript>C, <Superscript>15</Superscript>N resonance assignments of dimethylsulfoxide-denatured TGFBIp FAS1-4 A546T

The transforming growth factor beta induced protein (TGFBIp) is a major protein component of the human cornea. Mutations occurring in TGFBIp may cause corneal dystrophies, which ultimately lead to loss of vision. The majority of the disease-causing mutations are located in the C-terminal domain of TGFBIp, referred as the fourth fascilin-1 (FAS1-4) domain. In the present study the FAS1-4 Ala546Thr, a mutation that causes lattice corneal dystrophy, was investigated in dimethylsulfoxide using liquid-state NMR spectroscopy, to enable H/D exchange strategies for identification of the core formed in mature fibrils. Isotope-labeled fibrillated FAS1-4 A546T was dissolved in a ternary mixture 95/4/1 v/v/v% dimethylsulfoxide/water/trifluoroacetic acid, to obtain and assign a reference 2D ¹H–¹⁵N HSQC spectrum for the H/D exchange analysis. Here, we report the near-complete assignments of backbone and aliphatic side chain ¹H, ¹³C and ¹⁵N resonances for unfolded FAS1-4 A546T at 25 °C.     

Modulating P1 Adenosine Receptors in Disease Progression of SOD1G93A Mutant Mice

Amyotrophic lateral sclerosis (ALS) is a fatal progressing neurodegenerative disease; to date, despite the intense research effort, only two therapeutic options, with very limited effects, are available. The purinergic system has been indicated as a possible new therapeutic target for ALS, but the results are often contradictory and generally confused. The present study was designed to determine whether P1 adenosine receptor ligands affected disease progression in a transgenic model of ALS. SOD1^G93A mice were chronically treated, from presymptomatic stage, with a selective adenosine A_2A receptor agonist (CGS21680), antagonist (KW6002) or the A₁ receptor antagonist DPCPX. Body weight, motor performance and survival time were evaluated. The results showed that neither the stimulation nor the blockade of adenosine A_2A receptors modified the progressive loss of motor skills or survival of mSOD1^G93A mice. Conversely, blockade of adenosine A₁ receptors from the presymptomatic stage significantly attenuated motor disease progression and induced a non-significant increase of median survival in ALS mice. Our data confirm that the modulation of adenosine receptors can elicit very different (and even opposite) effects during the progression of ALS course, thus strengthens the importance of further studies to elucidated their real therapeutic potential in this pathology.

     

Dynamic Changes of CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript> Regulatory T Cells in NOD.H-2<Superscript>h4</Superscript> Mice with Iodine-Induced Autoimmune Thyroiditis

Iodine is an essential trace element for thyroid hormone synthesis and metabolism, either low or high intake may lead to thyroid disease, but the pathogenetic mechanisms by which iodine interacts with the thyroid autoimmune are poorly understood. We investigated the dynamic changes of CD4⁺CD25⁺ regulatory T cells in NOD.H-2^h4 mice with iodine-induced autoimmune thyroiditis (AIT), and explore potential immune mechanism of AIT induced by iodine. NOD.H-2^h4 mice were randomly divided into two groups, and received plain water or water containing 0.005% sodium iodide. Eight weeks after iodine provision, the incidences of thyroiditis, relative weights of thyroids, and serum thyroglobulin antibody titers in the iodine-supplied groups were significantly increased compared to the control groups (p < 0.05). The AIT mice had fewer CD4⁺CD25⁺Foxp3⁺ T cells and reduced Foxp3 mRNA expression in splenocytes compared with the controls (p < 0.01), and maintained relatively low levels during the development of thyroiditis. The changes described above aggravated gradually with the extension of iodine treatment. These data suggest that CD4⁺CD25⁺ regulatory T cells may be involved in the pathogenesis and development of AIT induced by iodine.     

Production of hemo- and immunoregulatory cytokines by erythroblast antigen<Superscript>+</Superscript> and glycophorin A<Superscript>+</Superscript> cells from human bone marrow

Background  

Erythroid nuclear cells (ENC) of the bone marrow (BM) have not previously been considered as important producers of wide spectrum of haemo- and immunoregulatory cytokines. The aim of the current work was to confirm the production of the main hemo- and immunoregulatory cytokines in human ENC from BM.