Cellular and molecular properties of the neuroprotective Wldˢ gene

The aim of this thesis is to advance our understanding of the mechanisms controlling nerve degeneration, by examining the molecular interactions of the Wldˢ gene and its chimeric protein product. Wallerian degeneration is normally complete within twenty-four to forty-eight hours of axotomy, but in the mouse mutant C57B1/Wldˢ the process is delayed by up to three weeks. The Wldˢ mutation comprises an 85Kb tandem triplication of genomic region containing the complete sequence for Rbp7 (retinoid binding protein 7), Nmnatl (nicotinamide mononucleotide adenyltransferase) and the N-70 amino acids of Ube4b (a ubiquitin ligase). At the boundaries of this in frame tandem triplication, a chimeric "fusion" gene is formed comprising the N-70 Ube4b linked to Nmnat. The protein product localises to nuclei. Transgenic expression of the chimeric gene has previously been shown to be sufficient to reproduce the Wldˢ nerve-degeneration phenotype in both mice and rats.The main objectives in my research were; 1. To develop a rapid and effective method for determining the copy number of Wldˢ alleles in mutant and transgenic Wldˢ mouse lines. 2. To map the regional distribution and sub cellular localisation of the Wldˢ chimeric protein product within the nervous system. 3. To test the hypothesis that the expression of the constituent components of the Wldˢ chimeric gene is sufficient to alter mRNA levels of other genes that may themselves be downstream effectors of the Wldˢ neuroprotective phenotype.1. A rapid and cost effective method for genotyping was developed using quantitative real time PCR on genomic DNA from the spontaneous Wldˢ mutant mouse and transgenic lines. This method allows the determination of Wldˢ copy number (specifically a region of the N10-Ube4b portion of the Wldˢ gene) for genotyping and calculating insertion number in transgenic lines.2. A highly specific antibody generated against the Wld-18 peptide confirms that the Wlcf protein product is localised to neuronal nuclei. Mapping of the Wldˢ protein product in the CNS showed that the neuronal distribution is not uniform either in the spontaneous mutant or the transgenic models. The intranuclear distribution varied between and within cell types; some neurones showed intense, particulate staining, others showed fine speckling, while others had large nuclear inclusions that varied in shape. Cerebellar granule cells in the Wldˢ mouse have a consistent expression pattern, with about 90% of cells containing at least one large nuclear inclusion of Wldˢ protein. The expression of Wldˢ - containing inclusions in these cells increased with postnatal age in vivo and also in vitro in primary cultures and cell lines. The inclusions persist, evidently without detriment to the animals as they mature. Coimmunostaining and/or co-transfection studies in the HEK293 cell lines suggested that the Wldˢ protein co-localises with certain transcription factors including HDAC5; and members of the SMRT family; and a member of the ubiquitin proteasome system, VCP.3. Microarray analysis and quantitative real time PCR of Wldˢ compared with wildtype C57B16 mouse cerebellar mRNA indicated differences in expression levels for at least 11 genes outside the Wldˢ locus. Some of these genes were down-regulated, but others were up-regulated. The greatest differences were strong down-regulation of Pttgl and strong up-regulation of a homologue of Edrl. Transfection of human (HEK293) cells with a Wldˢ-eGFP construct mimicked changes shown in Wldˢ mouse mRNA levels. The sub-cellular distribution of Wldˢ protein is dependent on both the Nmnat-1 and Ube4b regions of the protein and both are required to target Wldˢ protein to discrete intranuclear foci. The induced changes in Pttgl mRNA levels, but not the Edrl-like transcript, were partly mimicked by transfecting HEK293 cells with the Nmnat-1 component of the chimeric gene, or by exogenous administration of NAD at a concentration of ImM. This effect was blocked by sirtenol, an inhibitor of the nuclear transcription factor Sirtl. Transfection with the N10-Ube4b component of the Wldˢ gene partly mimicked up-regulation of the Edrllike transcript but had no effect on expression levels of Pttg-1 mRNA. Wldˢ-positive cerebellar granule cells show a protective phenotype, but Pttgl-KO peripheral nerves do not.Together, these studies suggest that the Wldˢ gene product functions as a bidirectional regulator of gene expression, driving up and down the transcription of several specific genes via more than one intra-nuclear signalling pathway. The summed effects of these variations in mRNA levels may be required for full expression of the Wldˢ neuroprotective phenotype. Identification of the genes and proteins ultimately responsible may open up new possibilities for understanding and treatment of the consequences of injury to the nervous system, whether induced by trauma or by neurodegenerative disease

Synaptic Protection in the Brain of WldS Mice Occurs Independently of Age but Is Sensitive to Gene-Dose

Disruption of synaptic connectivity is a significant early event in many neurodegenerative conditions affecting the aging CNS, including Alzheimer's disease and Parkinson's disease. Therapeutic approaches that protect synapses from degeneration in the aging brain offer the potential to slow or halt the progression of such conditions. A range of animal models expressing the slow Wallerian Degeneration (Wld(S)) gene show robust neuroprotection of synapses and axons from a wide variety of traumatic and genetic neurodegenerative stimuli in both the central and peripheral nervous systems, raising that possibility that Wld(S) may be useful as a neuroprotective agent in diseases with synaptic pathology. However, previous studies of neuromuscular junctions revealed significant negative effects of increasing age and positive effects of gene-dose on Wld(S)-mediated synaptic protection in the peripheral nervous system, raising doubts as to whether Wld(S) is capable of directly conferring synapse protection in the aging brain.We examined the influence of age and gene-dose on synaptic protection in the brain of mice expressing the Wld(S) gene using an established cortical lesion model to induce synaptic degeneration in the striatum. Synaptic protection was found to be sensitive to Wld(S) gene-dose, with heterozygous Wld(S) mice showing approximately half the level of protection observed in homozygous Wld(S) mice. Increasing age had no influence on levels of synaptic protection. In contrast to previous findings in the periphery, synapses in the brain of old Wld(S) mice were just as strongly protected as those in young mice.Our study demonstrates that Wld(S)-mediated synaptic protection in the CNS occurs independently of age, but is sensitive to gene dose. This suggests that the Wld(S) gene, and in particular its downstream endogenous effector pathways, may be potentially useful therapeutic agents for conferring synaptic protection in the aging brain

Applying modern Omic technologies to the Neuronal Ceroid Lipofuscinoses

The Neuronal Ceroid Lipofuscinoses are a group of severe and progressive neurodegenerative disorders, which generally present during childhood. With new treatments emerging on the horizon, there is a growing need to understand the specific disease mechanisms as well as identify prospective biomarkers for use to stratify patients and monitor treatment. The use of Omics technologies to NCLs has the potential to address this need. We discuss the recent use and outcomes of Omics to various forms of NCL including identification of interactomes, affected biological pathways and potential biomarker candidates. We also identify common pathways affected in NCL across the reviewed studies

Modified cell cycle status in a mouse model of altered neuronal vulnerability (slow Wallerian degeneration; Wlds)

Profiling of gene expression changes in mice harbouring the neurodegenerative Wlds mutation shows a strong correlation between changes in cell cycle pathways and altered vulnerability of terminally differentiated neurons

Microarray profiling emphasizes transcriptomic differences between hippocampal in vivo tissue and in vitro cultures" for publication in Brain Communications.

From Crossref journal articles via Jisc Publications RouterHistory: epub 2021-07-07, issued 2021-07-07Article version: AMPublication status: PublishedAbstract Primary hippocampal cell cultures are routinely used as an experimentally accessible model platform for the hippocampus and brain tissue in general. Containing multiple cell types including neurons, astrocytes and microglia in a state that can be readily analysed optically, biochemically and electrophysiologically, such cultures have been used in many in vitro studies. To what extent the in vivo environment is recapitulated in primary cultures in an on-going question. Here we compare the transcriptomic profiles of primary hippocampal cell cultures and intact hippocampal tissue. In addition, by comparing profiles from wild type and the PrP 101LL transgenic model of prion disease, we also demonstrate that gene conservation is predominantly conserved across genetically altered lines

Systemic restoration of UBA1 ameliorates disease in spinal muscular atrophy

Acknowledgments Blood biochemistry analysis and serum analysis were performed by the Easter Bush Pathology Department, University of Edinburgh. Animal husbandry was performed by Centre for Integrative Physiology bio-research restructure technical staff, University of Edinburgh. Assistance with intravenous injections was provided by Ian Coldicott (University of Sheffield) and Hannah Shorrock (University of Edinburgh). Human blood cDNA was a gift to GH from Kathy Evans, University of Edinburgh. Imaging was performed at the IMPACT imaging facility, University of Edinburgh, with technical assistance from Anisha Kubasik-Thayil. The authors would also like to thank Lyndsay Murray for technical discussions relating to qRT-PCR analysis. This work was supported by funding from the SMA Trust and the Anatomical Society (via grants to THG); the Euan MacDonald Centre for Motor Neurone Disease Research (via grants to THG and SHP); the Wellcome Trust (via grants to EJNG and THG); Muscular Dystrophy UK (via grants to THG and CGB); a Elphinstone Scholarship from the University of Aberdeen (to SHP); and The French Muscular Dystrophy Association (via grants to CM and JC).Peer reviewedPublisher PD

An optimized comparative proteomic approach as a tool in neurodegenerative disease research.

Recent advances in proteomic technologies now allow unparalleled assessment of the molecular composition of a wide range of sample types. However, the application of such technologies and techniques should not be undertaken lightly. Here, we describe why the design of a proteomics experiment itself is only the first step in yielding high-quality, translatable results. Indeed, the effectiveness and/or impact of the majority of contemporary proteomics screens are hindered not by commonly considered technical limitations such as low proteome coverage but rather by insufficient analyses. Proteomic experimentation requires a careful methodological selection to account for variables from sample collection, through to database searches for peptide identification to standardised post-mass spectrometry options directed analysis workflow, which should be adjusted for each study, from determining when and how to filter proteomic data to choosing holistic versus trend-wise analyses for biologically relevant patterns. Finally, we highlight and discuss the difficulties inherent in the modelling and study of the majority of progressive neurodegenerative conditions. We provide evidence (in the context of neurodegenerative research) for the benefit of undertaking a comparative approach through the application of the above considerations in the alignment of publicly available pre-existing data sets to identify potential novel regulators of neuronal stability