The impact of metallothionein-II on microglial response to tumor necrosis factor-alpha (TNFα) and downstream effects on neuronal regeneration.

BACKGROUND: The extracellular environment plays an important role in supporting the regeneration of axons after injury. Metallothionein-II (MTII) is a metal-binding protein known for its neuroprotective effect by directly stimulating the growth of axons after injury. Previous studies have shown that MTII also modulates the response of astrocytes and microglia after injury. However, a detailed analysis describing how MTII modulates the interaction between microglia and neurons is lacking. METHODS: We introduced fluorescently labelled MTII into the cortex at the time of needlestick injury to investigate the cellular uptake of MTII using immunohistochemistry with antibodies against cell-type-specific markers. The role of MTII in modulating the effect of microglia on axon outgrowth following an inflammatory response is further investigated using a co-culture model involving primary rodent microglia pre-treated with TNFα and primary rodent cortical neurons. The axon lengths were assessed 24 h after the plating of the neurons onto treated microglia. We also utilised siRNA to knockdown the expression of LRP1, which allows us to investigate the role of LRP1 receptors in the MTII-mediated effect of microglia on axon outgrowth. RESULTS: Fluorescently labelled MTII was found to be associated with neurons, astrocytes and microglia following injury in vivo. Microglia-neuron co-culture experiments demonstrated that exogenous MTII altered the response of microglia to TNFα. The neurons plated onto the TNFα-stimulated microglia pre-treated with MTII have shown a significantly longer axonal length compare to the TNFα-stimulated microglia without the MTII treatment. This suggested that MTII reduce cytokine-stimulated activation of microglia, which would ordinarily impair neurite outgrowth. This inhibitory effect of MTII on activated microglia was blocked by siRNA-mediated downregulation of LRP1 receptor expression in microglia, suggesting that MTII acts via the LRP1 receptor on microglia. CONCLUSIONS: This study demonstrates that exogenous MTII acts via the LRP1 receptor to alter the inflammatory response of microglia following TNFα stimulation, providing a more supportive environment for axon growth.

Computational and experimental investigation of Li-doped ionic liquid electrolytes: [pyr14][TFSI], [pyr13][FSI], and [EMIM][BF4].

We employ molecular dynamics (MD) simulation and experiment to investigate the structure, thermodynamics, and transport of N-methyl-N-butylpyrrolidinium bis(trifluoromethylsufonyl)imide ([pyr14][TFSI]), N-methyl-N-propylpyrrolidinium bis(fluorosufonyl)imide ([pyr13][FSI]), and 1-ethyl-3-methylimidazolium boron tetrafluoride ([EMIM][BF4]), as a function of Li-salt mole fraction (0.05 ≤ xLi(+) ≤ 0.33) and temperature (298 K ≤ T ≤ 393 K). Structurally, Li(+) is shown to be solvated by three anion neighbors in [pyr14][TFSI] and four anion neighbors in both [pyr13][FSI] and [EMIM][BF4], and at all levels of xLi(+) we find the presence of lithium aggregates. Pulsed field gradient spin-echo NMR measurements of diffusion and electrochemical impedance spectroscopy measurements of ionic conductivity are made for the neat ionic liquids as well as 0.5 molal solutions of Li-salt in the ionic liquids. Bulk ionic liquid properties (density, diffusion, viscosity, and ionic conductivity) are obtained with MD simulations and show excellent agreement with experiment. While the diffusion exhibits a systematic decrease with increasing xLi(+), the contribution of Li(+) to ionic conductivity increases until reaching a saturation doping level of xLi(+) = 0.10. Comparatively, the Li(+) conductivity of [pyr14][TFSI] is an order of magnitude lower than that of the other liquids, which range between 0.1 and 0.3 mS/cm. Our transport results also demonstrate the necessity of long MD simulation runs (∼200 ns) to converge transport properties at room temperature. The differences in Li(+) transport are reflected in the residence times of Li(+) with the anions (τ(Li/-)), which are revealed to be much larger for [pyr14][TFSI] (up to 100 ns at the highest doping levels) than in either [EMIM][BF4] or [pyr13][FSI]. Finally, to comment on the relative kinetics of Li(+) transport in each liquid, we find that while the net motion of Li(+) with its solvation shell (vehicular) significantly contributes to net diffusion in all liquids, the importance of transport through anion exchange increases at high xLi(+) and in liquids with large anions.

Hematopoietic stem cell and gene therapy corrects primary neuropathology and behavior in mucopolysaccharidosis IIIA mice.

Mucopolysaccharidosis IIIA (MPS IIIA or Sanfilippo disease) is a neurodegenerative disorder caused by a deficiency in the lysosomal enzyme sulfamidase (SGSH), catabolizing heparan sulfate (HS). Affected children present with severe behavioral abnormalities, sleep disturbances, and progressive neurodegeneration, leading to death in their second decade. MPS I, a similar neurodegenerative disease accumulating HS, is treated successfully with hematopoietic stem cell transplantation (HSCT) but this treatment is ineffectual for MPS IIIA. We compared HSCT in MPS IIIA mice using wild-type donor cells transduced ex vivo with lentiviral vector-expressing SGSH (LV-WT-HSCT) versus wild-type donor cell transplant (WT-HSCT) or lentiviral-SGSH transduced MPS IIIA cells (LV-IIIA-HSCT). LV-WT-HSCT results in 10% of normal brain enzyme activity, near normalization of brain HS and GM2 gangliosides, significant improvements in neuroinflammation and behavioral correction. Both WT-HSCT and LV-IIIA-HSCT mediated improvements in GM2 gangliosides and neuroinflammation but were less effective at reducing HS or in ameliorating abnormal HS sulfation and had no significant effect on behavior. This suggests that HS may have a more significant role in neuropathology than neuroinflammation or GM2 gangliosides. These data provide compelling evidence for the efficacy of gene therapy in conjunction with WT-HSCT for neurological correction of MPS IIIA where conventional transplant is ineffectual.

Systemic iridovirus from threespine stickleback Gasterosteus aculeatus represents a new megalocytivirus species (family Iridoviridae).

Megalocytiviruses have been associated with epizootics resulting in significant economic losses in public aquaria and food-fish and ornamental fish industries, as well as threatening wild fish stocks. The present report describes characteristics of the first megalocytivirus from a wild temperate North American fish, the threespine stickleback Gasterosteus aculeatus. Moribund and dead fish sampled after transfer to quarantine for an aquarium exhibit had amphophilic to basophilic intracytoplasmic inclusions (histopathology) and icosahedral virions (transmission electron microscopy) consistent with an iridovirus infection. Phylogenetic analyses of the major capsid, ATPase, and DNA polymerase genes confirmed the virus as the first known member of the genus Megalocytivirus (family Iridoviridae) from a gasterosteid fish. The unique biologic and genetic properties of this virus are sufficient to establish a new Megalocytivirus species to be formally known as the threespine stickleback iridovirus (TSIV). The threespine stickleback is widely distributed throughout the northern hemisphere in both freshwater and estuarine environments. The presence of megalocytiviruses with broad host specificity and detrimental economic and ecologic impacts among such a widely dispersed fish species indicates the need for sampling of other stickleback populations as well as other North American sympatric marine and freshwater ichthyofauna.

Metallothionein promotes regenerative axonal sprouting of dorsal root ganglion neurons after physical axotomy.

Prior studies have reported that metallothionein I/II (MT) promote regenerative axonal sprouting and neurite elongation of a variety of central nervous system neurons after injury. In this study, we evaluated whether MT is capable of modulating regenerative axon outgrowth of neurons from the peripheral nervous system. The effect of MT was firstly investigated in dorsal root ganglion (DRG) explants, where axons were scratch-injured in the presence or absence of exogenous MT. The application of MT led to a significant increase in regenerative sprouting of neurons 16 h after injury. We show that the pro-regenerative effect of MT involves an interaction with the low-density lipoprotein receptor megalin, which could be blocked using the competitive antagonist RAP. Pre-treatment with the mitogen-activated protein kinase (MAPK) inhibitor PD98059 also completely abrogated the effect of exogenous MT in promoting axonal outgrowth. Interestingly, we only observed megalin expression in neuronal soma and not axons in the DRG explants. To investigate this matter, an in vitro injury model was established using Campenot chambers, which allowed the application of MT selectively into either the axonal or cell body compartments after scratch injury was performed to axons. At 16 h after injury, regenerating axons were significantly longer only when exogenous MT was applied solely to the soma compartment, in accordance with the localized expression of megalin in neuronal cell bodies. This study provides a clear indication that MT promotes axonal regeneration of DRG neurons, via a megalin- and MAPK-dependent mechanism.

Tg2576 cortical neurons that express human Ab are susceptible to extracellular Aß-induced, K+ efflux dependent neurodegeneration.

BACKGROUND: One of the key pathological features of AD is the formation of insoluble amyloid plaques. The major constituent of these extracellular plaques is the beta-amyloid peptide (Aß), although Aß is also found to accumulate intraneuronally in AD. Due to the slowly progressive nature of the disease, it is likely that neurons are exposed to sublethal concentrations of both intracellular and extracellular Aß for extended periods of time. RESULTS: In this study, we report that daily exposure to a sublethal concentration of Aß(1-40) (1 µM) for six days induces substantial apoptosis of cortical neurons cultured from Tg2576 mice (which express substantial but sublethal levels of intracellular Aß). Notably, untreated Tg2576 neurons of similar age did not display any signs of apoptosis, indicating that the level of intracellular Aß present in these neurons was not the cause of toxicity. Furthermore, wildtype neurons did not become apoptotic under the same chronic Aß(1-40) treatment. We found that this apoptosis was linked to Tg2576 neurons being unable to maintain K(+) homeostasis following Aß treatment. Furthermore, blocking K(+) efflux protected Tg2576 neurons from Aß-induced neurotoxicity. Interestingly, chronic exposure to 1 µM Aß(1-40) caused the generation of axonal swellings in Tg2576 neurons that contained dense concentrations of hyperphosphorylated tau. These were not observed in wildtype neurons under the same treatment conditions. CONCLUSIONS: Our data suggest that when neurons are chronically exposed to sublethal levels of both intra- and extra-cellular Aß, this causes a K(+)-dependent neurodegeneration that has pathological characteristics similar to AD.

The native copper- and zinc-binding protein metallothionein blocks copper-mediated Abeta aggregation and toxicity in rat cortical neurons.

BACKGROUND: A major pathological hallmark of AD is the deposition of insoluble extracellular beta-amyloid (Abeta) plaques. There are compelling data suggesting that Abeta aggregation is catalysed by reaction with the metals zinc and copper. METHODOLOGY/PRINCIPAL FINDINGS: We now report that the major human-expressed metallothionein (MT) subtype, MT-2A, is capable of preventing the in vitro copper-mediated aggregation of Abeta1-40 and Abeta1-42. This action of MT-2A appears to involve a metal-swap between Zn7MT-2A and Cu(II)-Abeta, since neither Cu10MT-2A or carboxymethylated MT-2A blocked Cu(II)-Abeta aggregation. Furthermore, Zn7MT-2A blocked Cu(II)-Abeta induced changes in ionic homeostasis and subsequent neurotoxicity of cultured cortical neurons. CONCLUSIONS/SIGNIFICANCE: These results indicate that MTs of the type represented by MT-2A are capable of protecting against Abeta aggregation and toxicity. Given the recent interest in metal-chelation therapies for AD that remove metal from Abeta leaving a metal-free Abeta that can readily bind metals again, we believe that MT-2A might represent a different therapeutic approach as the metal exchange between MT and Abeta leaves the Abeta in a Zn-bound, relatively inert form.

Identification of c-myc coding region determinant RNA sequences and structures cleaved by an RNase1-like endoribonuclease.

The coding region of c-myc mRNA encompassing the coding region determinant (CRD) nucleotides (nts) 1705-1792 is critical in regulating c-myc mRNA stability. This is in part due to the susceptibility of c-myc CRD RNA to attack by an endoribonuclease. We have previously purified and characterized a mammalian endoribonuclease that cleaves c-myc CRD RNA in vitro. This enzyme is tentatively identified as a 35 kDa RNase1-like endonuclease. In an effort to understand the sequence and secondary structure requirements for RNA cleavage by this enzyme, we have determined the secondary structure of the c-myc CRD RNA nts 1705-1792 using RNase probing technique. The secondary structure of c-myc CRD RNA possesses five stems; two of which contain 4 base pairs (stems I and V) and three consisting of 3 base pairs (stems II, III, and IV). Endonucleolytic assays using the c-myc CRD and several c-myc CRD mutants as substrates led to the following conclusions: (i) the enzyme prefers to cleave in between the dinucleotides UA, CA, and UG in single-stranded regions; (ii) the enzyme is more specific towards UA dinucleotides. These properties further distinguish the enzyme from previously described mammalian endonuclease that cleaves c-myc mRNA in vitro.

Role of the forkhead protein FoxO1 in beta cell compensation to insulin resistance.

Diabetes is associated with defective beta cell function and altered beta cell mass. The mechanisms regulating beta cell mass and its adaptation to insulin resistance are unknown. It is unclear whether compensatory beta cell hyperplasia is achieved via proliferation of existing beta cells or neogenesis from progenitor cells embedded in duct epithelia. We have used transgenic mice expressing a mutant form of the forkhead-O1 transcription factor (FoxO1) in both pancreatic ductal and endocrine beta cells to assess the contribution of these 2 compartments to islet expansion. We show that the mutant FoxO1 transgene prevents beta cell replication in 2 models of beta cell hyperplasia, 1 due to peripheral insulin resistance (Insulin receptor transgenic knockouts) and 1 due to ectopic local expression of IGF2 (Elastase-IGF2 transgenics), without affecting insulin secretion. In contrast, we failed to detect a specific effect of the FoxO1 transgene on the number of periductal beta cells. We propose that beta cell compensation to insulin resistance is a proliferative response of existing beta cells to growth factor signaling and requires FoxO1 nuclear exclusion.

Deletion of long-range sequences at Sox10 compromises developmental expression in a mouse model of Waardenburg-Shah (WS4) syndrome.

The transcription factor SOX10 is mutated in the human neurocristopathy Waardenburg-Shah syndrome (WS4), which is characterized by enteric aganglionosis and pigmentation defects. SOX10 directly regulates genes expressed in neural crest lineages, including the enteric ganglia and melanocytes. Although some SOX10 target genes have been reported, the mechanisms by which SOX10 expression is regulated remain elusive. Here, we describe a transgene-insertion mutant mouse line (Hry) that displays partial enteric aganglionosis, a loss of melanocytes, and decreased Sox10 expression in homozygous embryos. Mutation analysis of Sox10 coding sequences was negative, suggesting that non-coding regulatory sequences are disrupted. To isolate the Hry molecular defect, Sox10 genomic sequences were collected from multiple species, comparative sequence analysis was performed and software was designed (ExactPlus) to identify identical sequences shared among species. Mutation analysis of conserved sequences revealed a 15.9 kb deletion located 47.3 kb upstream of Sox10 in Hry mice. ExactPlus revealed three clusters of highly conserved sequences within the deletion, one of which shows strong enhancer potential in cultured melanocytes. These studies: (i) present a novel hypomorphic Sox10 mutation that results in a WS4-like phenotype in mice; (ii) demonstrate that a 15.9 kb deletion underlies the observed phenotype and likely removes sequences essential for Sox10 expression; (iii) combine a novel in silico method for comparative sequence analysis with in vitro functional assays to identify candidate regulatory sequences deleted in this strain. These studies will direct further analyses of Sox10 regulation and provide candidate sequences for mutation detection in WS4 patients lacking a SOX10-coding mutation.

An enhancer element at the Igf2/H19 locus drives gene expression in both imprinted and non-imprinted tissues.

The insulin-like growth factor 2 (Igf2) gene encodes a potent growth factor that is expressed in multiple tissues during embryonic development. Expression at this locus is mediated by genomic imprinting. In the developing endodermal tissues, imprinting of Igf2 is mediated by the interaction of a set of enhancers downstream of the linked H19 gene with a differentially methylated domain (DMD) that lies approximately 2-4 kb upstream of H19 that has a boundary or insulator function in the hypomethylated state. In the remainder of tissues that express Igf2 and H19, the cis elements that drive their correct expression and imprinting are not well understood. In addition, enhancers driving expression of Igf2 in the choroid plexus and leptomeninges, tissues where the gene is thought not to be imprinted, have not been isolated. Here we show that biallelic (non-imprinted) expression within the choroid plexus is restricted to the epithelium, and we provide evidence that a conserved intergenic region functions as an enhancer for Igf2 both in tissues where the gene is imprinted, and where Igf2 is biallelically expressed. The presence of an enhancer for imprinted tissues in the intergenic region argues for the existence of imprinting controls distinct from the DMD, which may be provided by differential methylation at sites proximal to Igf2.

Fission of pancreatic islets during postnatal growth of the mouse.

A cell composition analysis was made of the pancreatic islets in postnatal H253 mice. This line has a lacZ insertion on the X chromosome so that in female hemizygotes 50% of cells should be positive for beta-galactosidase and 50% negative. Immediately after birth, the islets were of a heterogeneous cell composition. However, by 4 weeks some islets have become homogeneous. This suggests that islets progress towards monoclonality in a similar way to the intestinal crypts and stomach gastric glands. Pancreatic islets may therefore represent 'structural proliferative units' in the overall histological organization of the pancreas. Reduction of genetic heterogeneity might arise from cell turnover, fission of islets or both. Analysis of the cell composition of the X-inactivation mosaic mice also provides the first clear evidence for islet fission in pancreatic development. Irregularly shaped islets resembling dumb-bells, with a characteristic neck of alpha-cells, were observed with decreasing frequency with increasing age. Three-dimensional reconstruction confirmed their resemblance to conjoined islets. The cell composition analysis showed: (1) the relatedness of the two sides of a dumb-bell islet is significantly higher than between two non-dumb-bell islets and (2) the relatedness of two randomly selected islets decreases as the distance between them increases. This suggests that dumb-bell islets are in a state of fission rather than fusion, and that islet fission is a mode of islet production in the postnatal pancreas.

Disruption of the imprinted Grb10 gene leads to disproportionate overgrowth by an Igf2-independent mechanism.

To investigate the function of the Grb10 adapter protein, we have generated mice in which the Grb10 gene was disrupted by a gene-trap insertion. Our experiments confirm that Grb10 is subject to genomic imprinting with the majority of Grb10 expression arising from the maternally inherited allele. Consistent with this, disruption of the maternal allele results in overgrowth of both the embryo and placenta such that mutant mice are at birth approximately 30% larger than normal. This observation establishes that Grb10 is a potent growth inhibitor. In humans, GRB10 is located at chromosome 7p11.2-p12 and has been associated with Silver-Russell syndrome, in which approximately 10% of those affected inherit both copies of chromosome 7 from their mother. Our results indicate that changes in GRB10 dosage could, in at least some cases, account for the severe growth retardation that is characteristic of Silver-Russell syndrome. Because Grb10 is a signaling protein capable of interacting with tyrosine kinase receptors, we tested genetically whether Grb10 might act downstream of insulin-like growth factor 2, a paternally expressed growth-promoting gene. The result indicates that Grb10 action is essentially independent of insulin-like growth factor 2, providing evidence that imprinting acts on at least two major fetal growth axes in a manner consistent with parent-offspring conflict theory.

Structural-proliferative units and organ growth: effects of insulin-like growth factor 2 on the growth of colon and skin.

Many epithelial renewal tissues in vertebrates are organised into structural-proliferative units. We have examined the effect of IGF2 dose on the structure of structural-proliferative units in skin and colon. The mouse strains used were the Igf2 knockout, wild type and K:Igf2, a transgenic in which Igf2 is overexpressed under control of a keratin promoter. For both skin and colon, the histological organisation of structural-proliferative units was unaltered with increasing IGF2 dose, although there was a higher fraction of dividing cells in the proliferative compartment. In the colon an increase in IGF2 dose increases the overall area of the epithelium. This is due to an increase in the number of crypts with no change of cell size or of crypt area. Growth stimulation appears to be due to a reduction in the duration of crypt fission. The conclusion is that the IGF2 pathway can stimulate the multiplication of colonic crypts independently of stimulating increased cell proliferation. The results for the skin are consistent with this. An increase of IGF2 dose increases the proportion of dividing cells in the basal layer, the thickness of the epidermis and the total area of the epidermis. By comparison with Drosophila, these results show no effects on cell size, but do show the possibility of inducing disproportionate growth. These differences may represent properties of the SPU organisation that is characteristic of vertebrate tissues.