Manipulating gene expression in DRG sensory neurones

Mammalian sensory neurones that respond to tissue damaging stimuli are known as nociceptors. Activation of these neurones can induce a sensation of pain. The aim of this thesis was to develop methods to manipulate gene expression in rodent sensory neurones, in order to understand more about the molecular mechanisms involved in pain pathways. Three approaches were compared: viral gene delivery, antisense gene knock-down, and transgenic knock-out mice. Firstly, novel recombinant Herpes simplex viruses were used to infect sensory ganglia, and the efficiency of gene transfer assessed using histochemical markers. Secondly, antisense oligonucleotides directed against genes involved in determining neuronal excitability were delivered intrathecally, and the effects on gene expression and behaviour were assessed. Finally, two transgenic mouse lines in which the bacterial recombinase Cre was expressed downstream of sensory neuron specific promoters were analysed. These mice can be used to delete genes flanked by lox-p sites in a tissue-specific manner. The pattern of expression of Cre recombinase was assessed using reporter mice that express beta-galactosidase down stream of lox-p flanked stop (poly-adenylation) sites. Experiments with modified Herpes simplex viruses expressing green fluorescent protein (GFP) showed that injection of virus into the sciatic nerve resulted in more infected DRG neurones than footpad injection. A maximum transduction level of 7.9 1.3 % of all neurones was achieved one month after sciatic delivery. The majority of transduced neurones (67 1%) were large-diameter neurones. This subpopulation is not generally responsible for the transmission of noxious stimuli. Only 3.4 0.8% of peripherin positive neurones (a population that includes the majority of nociceptors) were successfully transduced. This level of transduction was too low to be used effectively. Vectors expressing VP22 a protein that enhances cell-to-cell viral spread were made, but were unstable. The use of these HSV vectors is thus limited to experimental situations where infection of only a few cells is adequate (for example, examining the function of secreted molecules). Experiments with labelled antisense probes gave much higher rates of neuronal transduction in comparison to viral transduction methods ( 80%). Unlike HSV-vectors however, there was no preferential transduction of neuronal cells, and all cell types were susceptible to oligonucleotide uptake. Intrathecal administration of antisense oligonucleotides directed against the annexin light chain protein p11 resulted in lowered levels of expression of the sodium channel Nav1.8 that requires p11 for insertion into the plasma membrane. Behavioural experiments were carried out to investigate the changes in both normal sensory function, and the alterations in inflammatory-related hyperalgesia after the administration of p11 antisense molecules. It was shown that this approach could successfully modulate the subcellular location of Nav1.8 protein. The results point to a dramatic and specific role for BDNF in the processing of noxious stimuli. In summary, it is clear that a variety of methods can be used to manipulate gene expression in sensory neurones to examine mechanisms involved in nociception. In this thesis, successful experiments using antisense oligonucleotides and nociceptor-specific Cre-expressing mice were carried out. It is concluded that the most effective route to manipulating nociceptor gene expression is the use of transgenic mice, but that antisense strategies have some advantages in terms of simplicity and speed. The use of HSV vectors is limited at present due to the low nociceptor transduction efficiency that can be achieved by relatively non-invasive delivery methods necessitating further improvements in vector design before their full potential can be realised. (Abstract shortened by UMI.)

Mislocalization of neuronal tau in the absence of tangle pathology in phosphomutant tau knockin mice.

Hyperphosphorylation and fibrillar aggregation of the microtubule-associated protein tau are key features of Alzheimer's disease and other tauopathies. To investigate the involvement of tau phosphorylation in the pathological process, we generated a pair of complementary phosphomutant tau knockin mouse lines. One exclusively expresses phosphomimetic tau with 18 glutamate substitutions at serine and/or threonine residues in the proline-rich and first microtubule-binding domains to model hyperphosphorylation, whereas its phosphodefective counterpart has matched alanine substitutions. Consistent with expected effects of genuine phosphorylation, association of the phosphomimetic tau with microtubules and neuronal membranes is severely disrupted in vivo, whereas the phosphodefective mutations have more limited or no effect. Surprisingly, however, age-related mislocalization of tau is evident in both lines, although redistribution appears more widespread and more pronounced in the phosphomimetic tau knockin. Despite these changes, we found no biochemical or immunohistological evidence of pathological tau aggregation in mice of either line up to at least 2 years of age. These findings raise important questions about the role of tau phosphorylation in driving pathology in human tauopathies

Manipulating gene expression in sensory neurons of DRG

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Age-dependent axonal transport and locomotor changes and tau hypophosphorylation in a "P301L" tau knockin mouse.

Tauopathies are characterized by hyperphosphorylation of the microtubule-associated protein tau and its accumulation into fibrillar aggregates. Toxic effects of aggregated tau and/or dysfunction of soluble tau could both contribute to neural defects in these neurodegenerative diseases. We have generated a novel knockin mouse model of an inherited tauopathy, frontotemporal dementia with parkinsonism linked to tau mutations on chromosome 17 (FTDP-17T). We incorporated a single mutation, homologous to the common FTDP-17T P301L mutation, directly into the endogenous mouse gene, mimicking the human disease situation. These mice express P301L-equivalent mutant tau at normal physiological levels from the knockin allele. Importantly, in contrast to existing transgenic mouse models that overexpress human P301L mutant tau, no overt tau pathology developed during the normal lifespan of the knockin mice. In fact, overall phosphorylation of tau was reduced, perhaps due to reduced microtubule binding. However, homozygous knockin mice did display intriguing age-dependent changes in axonal transport of mitochondria, and increased spontaneous locomotor activity in old age. These could represent early consequences of the tau dysfunction that eventually precipitates pathogenesis in humans.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe