Characterisation of the pathogenic effects of the in vivo expression of an ALS-linked mutation in D-amino acid oxidase: Phenotype and loss of spinal cord motor neurons

Amyotrophic lateral sclerosis (ALS) is the most common adult-onset neuromuscular disorder characterised by selective loss of motor neurons leading to fatal paralysis. Current therapeutic approaches are limited in their effectiveness. Substantial advances in understanding ALS disease mechanisms has come from the identification of pathogenic mutations in dominantly inherited familial ALS (FALS). We previously reported a coding mutation in D-amino acid oxidase (DAOR199W) associated with FALS. DAO metabolises D-serine, an essential co-agonist at the N-Methyl-D-aspartic acid glutamate receptor subtype (NMDAR). Using primary motor neuron cultures or motor neuron cell lines we demonstrated that expression of DAOR199W, promoted the formation of ubiquitinated protein aggregates, activated autophagy and increased apoptosis. The aim of this study was to characterise the effects of DAOR199W in vivo, using transgenic mice overexpressing DAOR199W. Marked abnormal motor features, e.g. kyphosis, were evident in mice expressing DAOR199W, which were associated with a significant loss (19%) of lumbar spinal cord motor neurons, analysed at 14 months. When separated by gender, this effect was greater in females (26%; p< 0.0132). In addition, we crossed the DAOR199W transgenic mouse line with the SOD1G93A mouse model of ALS to determine whether the effects of SOD1G93A were potentiated in the double transgenic line (DAOR199W/SOD1G93A). Although overall survival was not affected, onset of neurological signs was significantly earlier in female double transgenic animals than their female SOD1G93A littermates (125 days vs 131 days, P = 0.0239). In summary, some significant in vivo effects of DAOR199W on motor neuron function (i.e. kyphosis and loss of motor neurons) were detected which were most marked in females and could contribute to the earlier onset of neurological signs in double transgenic females compared to SOD1G93A littermates, highlighting the importance of recognizing gender effects present in animal models of ALS

Defective axonal transport in motor neuron disease

Several recent studies have highlighted the role of axonal transport in the pathogenesis of motor neuron diseases. Mutations in genes that control microtubule regulation and dynamics have been shown to cause motor neuron degeneration in mice and in a form of human motor neuron disease. In addition, mutations in the molecular motors dynein and kinesins and several proteins associated with the membranes of intracellular vesicles that undergo transport cause motor neuron degeneration in humans and mice. Paradoxically, evidence from studies on the legs at odd angles (Loa) mouse and a transgenic mouse model for human motor neuron disease suggest that partial limitation of the function of dynein may in fact lead to improved axonal transport in the transgenic mouse, leading to delayed disease onset and increased life span

Cancer vaccines: uses of HLA transgenic mice compared to genetically modified mice

Many tumor antigens have been identified that can be targeted by the immune system. Animal models that have been genetically modified to express human HLA molecules instead of their own MHC antigens have shown to be valuable in the discovery of peptides derived from tumor antigens many of which have since been used in clinical trials with varying degrees of success. Although these models are not perfect, they nonetheless allow transplantable tumor models to be developed to evaluate novel vaccination strategies that can then be applied in humans. In addition animals that have been genetically modified to “spontaneously” generate tumors that will grow within their correct environment are of greater value for studying angiogenesis, metastasis and the relationship between the immune system and tumor in a physiological setting. In this review, mice genetically modified to express HLA genes or to spontaneously develop tumors are discussed, highlighting their advantages and limitations as preclinical models for cancer immunotherapy

In vivo evidence for NMDA receptor mediated excitotoxicity in a murine genetic model of Huntington Disease

N-methyl-D-aspartate receptor (NMDAR) mediated excitotoxicity is implicated as a proximate cause of neurodegeneration in Huntington Disease (HD). However, this hypothesis has not been tested rigorously in vivo. NMDAR NR2B-subunits are the predominant NR2 subunit expressed by the striatal medium spiny neurons that degenerate in HD. To test this hypothesis, we crossed a well validated murine genetic model of HD (Hdh(CAG)150) with a transgenic line overexpressing NMDAR NR2B-subunits. In the resulting double mutant line, we show exacerbation of selective striatal neuron degeneration. These results provide the first direct in vivo evidence of NR2B-NMDAR mediated excitotoxicity in the context of HD. Our results are consistent with prior suggestions that direct and/or indirect interactions of mutant huntingtin with NMDARs are a proximate cause of neurodegeneration in HD

Plant Cellular and Molecular Biotechnology: Following Mariotti's Steps

This review is dedicated to the memory of Prof. Domenico Mariotti, who significantly contributed to establishing the Italian research community in Agricultural Genetics and carried out the first experiments of Agrobacterium-mediated plant genetic transformation and regeneration in Italy during the 1980s. Following his scientific interests as guiding principles, this review summarizes the recent advances obtained in plant biotechnology and fundamental research aiming to: (i) Exploit in vitro plant cell and tissue cultures to induce genetic variability and to produce useful metabolites; (ii) gain new insights into the biochemical function of Agrobacterium rhizogenes rol genes and their application to metabolite production, fruit tree transformation, and reverse genetics; (iii) improve genetic transformation in legume species, most of them recalcitrant to regeneration; (iv) untangle the potential of KNOTTED1-like homeobox (KNOX) transcription factors in plant morphogenesis as key regulators of hormonal homeostasis; and (v) elucidate the molecular mechanisms of the transition from juvenility to the adult phase in Prunus tree species

Transgenic Overexpression of LARGE Induces alpha-Dystroglycan Hyperglycosylation in Skeletal and Cardiac Muscle

Background: LARGE is one of seven putative or demonstrated glycosyltransferase enzymes defective in a common group of muscular dystrophies with reduced glycosylation of alpha-dystroglycan. Overexpression of LARGE induces hyperglycosylation of alpha-dystroglycan in both wild type and in cells from dystroglycanopathy patients, irrespective of their primary gene defect, restoring functional glycosylation. Viral delivery of LARGE to skeletal muscle in animal models of dystroglycanopathy has identical effects in vivo, suggesting that the restoration of functional glycosylation could have therapeutic applications in these disorders. Pharmacological strategies to upregulate Large expression are also being explored.Methodology/Principal Findings: In order to asses the safety and efficacy of long term LARGE over-expression in vivo, we have generated four mouse lines expressing a human LARGE transgene. On observation, LARGE transgenic mice were indistinguishable from the wild type littermates. Tissue analysis from young mice of all four lines showed a variable pattern of transgene expression: highest in skeletal and cardiac muscles, and lower in brain, kidney and liver. Transgene expression in striated muscles correlated with alpha-dystroglycan hyperglycosylation, as determined by immunoreactivity to antibody IIH6 and increased laminin binding on an overlay assay. Other components of the dystroglycan complex and extracellular matrix ligands were normally expressed, and general muscle histology was indistinguishable from wild type controls. Further detailed muscle physiological analysis demonstrated a loss of force in response to eccentric exercise in the older, but not in the younger mice, suggesting this deficit developed over time. However this remained a subclinical feature as no pathology was observed in older mice in any muscles including the diaphragm, which is sensitive to mechanical load-induced damage.Conclusions/Significance: This work shows that potential therapies in the dystroglycanopathies based on LARGE upregulation and alpha-dystroglycan hyperglycosylation in muscle should be safe

Neuropathological investigations of three murine models of Huntington’s disease

Huntington’s disease (HD) is a purely genetic neurodegenerative disorder affecting approximately 1 in 10,000 people. It is most commonly associated with excessive involuntary movement, or chorea, combined with varying degrees of other motor, psychiatric and cognitive disturbances. Identification of the mutation in the HD gene prompted the generation of several transgenic mouse models. HD is but one of a family of at least 9 triplet repeat disorders, all of which exhibit protein aggregation by a similar mechanism. The understanding of one disease is therefore of importance to the understanding of them all. This thesis aims to be a comprehensive comparative study of three very different mouse models of HD elucidating the pathological changes that precede and accompany the disease process. The work described in this thesis presents a detailed account of a longitudinal study of the pathological changes that occur within the brains of founder generations of mice transgenic for exon 1 of the HD gene, containing a highly expanded CAG repeat, the R6 lines. I have determined the intracellular sites for deposition and accumulation of the mutant protein huntingtin (htt), within both the neurons and glia of the central nervous system. The progressive accumulation of additional proteins within these aggregates has been described. The temporal evolution and spatial distribution of the neuronal intranuclear inclusion (NII) was determined using both immunohistochemical and morphometric analyses. The cellular consequences resulting from the aggregation of mutant htt were also investigated. I have conducted a detailed morphometric analysis of neurones within the cerebral cortex, striatum and cerebellum throughout the period of protein deposition, until the eventual degeneration of these cells. The dendritic and somal changes resulting from the cellular disruption associated with these NII are also described. In a further series of experiments I have investigated the changes that occur in a novel model of HD, namely the conditional, doxycycline inducible double transgenic mouse, HD94 model. It was interesting to find that the same construct when differently manipulated in two mouse lines can produce such contrasting symptoms and pathology. This was highlighted by the comparison of immunohistochemical and morphometric analyses between the HD94 and the R6 lines, where the pattern of mutant protein deposition was found to vary significantly. Lastly I have studied a more genetically accurate murine model of HD, the HD80 ‘knock-in model’. These mice develop a pathology broadly similar to that of the R6 lines but markedly different to that of the HD94, and over a much longer time frame This detailed comparative analysis of the molecular and cellular pathology of three transgenic mouse models of HD provides new insights identifying novel and unique neuropathology and suggests new approaches for therapeutic treatments for this disease