Neuroprotective effects of granulocyte-colony stimulating factor in a novel transgenic mouse model of SCA17

Spinocerebellar ataxia type 17 (SCA17) is an autosomal dominant inherited disorder characterized by degeneration of spinocerebellar tracts and selected brainstem neurons owing to the expansion of a CAG repeat of the human TATA-binding protein (hTBP) gene. To gain insight into the pathogenesis of this hTBP mutation, we generated transgenic mice with the mutant hTBP gene driven by the Purkinje specific protein (Pcp2/L7) gene promoter. Mice with the expanded hTBP allele developed ataxia within 2-5 months. Behavioral analysis of L7-hTBP transgenic mice showed reduced fall latency in a rotarod assay. Purkinje cell degeneration was identified by immunostaining of calbindin and IP3R1. Reactive gliosis and neuroinflammation occurred in the transgenic cerebellum, accompanied by up-regulation of GFAP and Iba1. The L7-hTBP transgenic mice were thus confirmed to recapitulate the SCA17 phenotype and were used as a disease model to explore the potential of granulocyte-colony stimulating factor in SCA17 treatment. Our results suggest that granulocyte-colony stimulating factor has a neuroprotective effect in these transgenic mice, ameliorating their neurological and behavioral deficits. These data indicate that the expression of the mutant hTBP in Purkinje cells is sufficient to produce cell degeneration and an ataxia phenotype, and constitutes a good model for better analysis of the neurodegeneration in SCA17.     

Mutation of a conserved active site residue converts tyrosyl-DNA phosphodiesterase I into a DNA topoisomerase I-dependent poison

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) catalyzes the resolution of 3' and 5' phospho-DNA adducts. A defective mutant, associated with the recessive neurodegenerative disease SCAN1, accumulates Tdp1-DNA complexes in vitro. To assess the conservation of enzyme architecture, a 2.0 A crystal structure of yeast Tdp1 was determined that is very similar to human Tdp1. Poorly conserved regions of primary structure are peripheral to an essentially identical catalytic core. Enzyme mechanism was also conserved, because the yeast SCAN1 mutant (H(432)R) enhanced cell sensitivity to the DNA topoisomerase I (Top1) poison camptothecin. A more severe Top1-dependent lethality of Tdp1H(432)N was drug-independent, coinciding with increased covalent Top1-DNA and Tdp1-DNA complex formation in vivo. However, both H(432) mutants were recessive to wild-type Tdp1. Thus, yeast H(432) acts in the general acid/base catalytic mechanism of Tdp1 to resolve 3' phosphotyrosyl and 3' phosphoamide linkages. However, the distinct pattern of mutant Tdp1 activity evident in yeast cells, suggests a more severe defect in Tdp1H(432)N-catalyzed resolution of 3' phospho-adducts.     

Some Observations upon Biochemical Causes of Ataxia and a New Disease Entity Ubiquinone, CoQ10 Deficiency

Some hereditary ataxias are treatable and the insight required for this has come from an in depth knowledge of the phenotypes and clinical biochemistry of the conditions. This has required both fundamental and translational clinical research. Prof John Blass was fortunate to begin his career at what we can now recognise as a golden era for such studies and he worked upon two important conditions; Refsum’s disease and Friedreich’s ataxia. More recently the mitochondrial encephalomyopathies have been described and similar investigative work has been undertaken upon them. Ubiquinone, CoQ₁₀, deficiency is the most recently recognised encephalomyopathy and is itself treatable. Though rare, it is becoming increasingly recognised and patients are benefiting from the same scholarly approach to its’ investigation as was afforded Refsums’ disease and Friedreich’s ataxia. A dedication to Professor John P. Blass.     

FGF14在神经退行疾病中的研究进展 *

成纤维细胞生长因子14(FGF14)是成纤维细胞生长因子家族中一员,主要在发育中及成熟的神经系统中表达。研究发现,FGF14在退行性神经系统中的脊髓小脑共济失调27型中发挥着重要的作用,FGF14的功能与各种离子通道关系密切,以钠离子通道为主,活性被神经元的兴奋性调节。根据FGF14的特点,归纳总结了FGF14目前的研究进展,为FGF14的基础和工程化研究提供理论依据。     

Chromatin Regulation of DNA Damage Repair and Genome Integrity in the Central Nervous System

With the continued extension of lifespan, aging and age-related diseases have become a major medical challenge to our society. Aging is accompanied by changes in multiple systems. Among these, the aging process in the central nervous system is critically important but very poorly understood. Neurons, as post-mitotic cells, are devoid of replicative associated aging processes, such as senescence and telomere shortening. However, because of the inability to self-replenish, neurons have to withstand challenge from numerous stressors over their lifetime. Many of these stressors can lead to damage of the neurons' DNA. When the accumulation of DNA damage exceeds a neuron's capacity for repair, or when there are deficiencies in DNA repair machinery, genome instability can manifest. The increased mutation load associated with genome instability can lead to neuronal dysfunction and ultimately to neuron degeneration. In this review, we first briefly introduce the sources and types of DNA damage and the relevant repair pathways in the nervous system (summarized in Fig. 1). We then discuss the chromatin regulation of these processes and summarize our understanding of the contribution of genomic instability to neurodegenerative diseases.     

SCA14 mutation V138E leads to partly unfolded PKCγ associated with an exposed C‐terminus,altered kinetics,phosphorylation and enhanced insolubilization

The protein kinase C γ (PKCγ) undergoes multistep activation and participates in various cellular processes in Purkinje cells. Perturbations in its phosphorylation state, conformation or localization can disrupt kinase signalling, such as in spinocerebellar ataxia type 14 (SCA14) that is caused by missense mutations in PRKCG encoding for PKCγ. We previously showed that SCA14 mutations enhance PKCγ membrane translocation upon stimulation owing to an altered protein conformation. As the faster translocation did not result in an increased function, we examined how SCA14 mutations induce this altered conformation of PKCγ and what the consequences of this conformational change are on PKCγ life cycle. Here, we show that SCA14‐related PKCγ‐V138E exhibits an exposed C‐terminus as shown by fluorescence resonance energy transfer‐fluorescence lifetime imaging microscopy in living cells, indicative of its partial unfolding. This conformational change was associated with faster phorbol 12‐myristate 13‐acetate‐induced translocation and accumulation of fully phosphorylated PKCγ in the insoluble fraction, which could be rescued by coexpressing PDK1 kinase that normally triggers PKCγ autophosphorylation. We propose that the SCA14 mutation V138E causes unfolding of the C1B domain and exposure of the C‐terminus of the PKCγ‐V138E molecule, resulting in a decrease of functional kinase in the soluble fraction.

     

Regulation of sheep α-TTP by dietary vitamin E and preparation of monoclonal antibody for sheep α-TTP

α-Tocopherol transfer protein (α-TTP) is a cytosolic protein that plays an important role in regulating concentrations of plasma α-tocopherol (the most bio-active form of vitamin E). Despite the central roles that α-TTP plays in maintaining vitamin E adequacy, we have only recently proved the existence of the α-TTP gene in sheep and, for the first time, cloned its full-length cDNA. However, the study of sheep α-TTP is still in its infancy. In the present study, thirty-five local male lambs of Tan sheep with similar initial body weight were randomly divided into five groups and fed with diets supplemented with 0 (control group), 20, 100, 200, 2000 IU·sheep^− 1·d^− 1 vitamin E for 120 days. At the end of the experiment, the plasma and liver vitamin E contents were analyzed first and then α-TTP mRNA and protein expression levels were determined by quantitative real-time PCR (qRT-PCR) and Western-blot analysis, respectively. In addition, as no sheep α-TTP antibody was available, a specific monoclonal antibody (McAb) against the ovine α-TTP protein was prepared. The effect of vitamin E supplementation was confirmed by the significant changes in the concentrations of vitamin E in the plasma and liver. As shown by qRT-PCR and Western-blot analysis, dietary vitamin E does not affect sheep α-TTP gene expression, except for high levels of vitamin E supplementation, which significantly increased expression at the protein level. Importantly, the specific sheep anti-α-TTP McAb we generated could provide optimal recognition in ELISA, Western-blot and immunohistochemistry assays, which will be a powerful tool in future studies of the biological functions of sheep α-TTP.