Threshold for epileptiform activity is elevated in prion knockout mice.

Prion protein (PrP) is abundant in the nervous system, but its role remains uncertain. Prion diseases depend on an aggregation of the protein that is likely to interfere with its normal function. Loss of function does not in itself cause neurodegeneration, but whether it contributes to the clinical features of the disease remains an open question. Patients with classical Creutzfeldt-Jakob disease (CJD) have a higher than expected incidence of epilepsy. To study the mechanisms by which loss of PrP function may underlie changes in vulnerability to epilepsy in disease, we used several acute epilepsy models: we applied a variety of convulsant treatments (zero-magnesium, bicuculline, and pentylenetetrazol) to slices in vitro from PrP knockout (Prnp0/0) and control mice. In all three epilepsy models, we found that longer delays and/or higher concentrations of convulsants were necessary to generate spontaneous epileptiform activity in Prnp0/0 mice. These results together indicate an increased seizure threshold in Prnp0/0 mice, suggesting that loss of PrP function cannot explain a predisposition to seizures initiation in CJD.

Review: contribution of transgenic models to understanding human prion disease.

Transgenic mice expressing human prion protein in the absence of endogenous mouse prion protein faithfully replicate human prions. These models reproduce all of the key features of human disease, including long clinically silent incubation periods prior to fatal neurodegeneration with neuropathological phenotypes that mirror human prion strain diversity. Critical contributions to our understanding of human prion disease pathogenesis and aetiology have only been possible through the use of transgenic mice. These models have provided the basis for the conformational selection model of prion transmission barriers and have causally linked bovine spongiform encephalopathy with variant Creutzfeldt-Jakob disease. In the future these models will be essential for evaluating newly identified potentially zoonotic prion strains, for validating effective methods of prion decontamination and for developing effective therapeutic treatments for human prion disease.

Prion infectivity in variant Creutzfeldt-Jakob disease rectum.

BACKGROUND: Disease-related prion protein (PrP(Sc)) is readily detectable in lymphoreticular tissues in variant Creutzfeldt-Jakob disease (vCJD), but not in other forms of human prion disease. This distinctive pathogenesis, with the unknown population prevalence of asymptomatic vCJD infection, has led to significant concerns that secondary transmission of vCJD prions will occur through a wide range of surgical procedures. To date PrP(Sc):prion infectivity ratios have not been determined in vCJD, and it is unknown whether vCJD prions are similar to experimental rodent prions, where PrP(Sc) concentration typically reflects infectious prion titre. AIM: To investigate prion infectivity in vCJD tissue containing barely detectable levels of PrP(Sc). METHODS: Transgenic mice expressing only human PrP (Tg(HuPrP129M(+/+)Prnp(o/o))-35 and Tg(HuPrP129M(+/+)Prnp(o/o))-45 mice) were inoculated with brain or rectal tissue from a previously characterised patient with vCJD. These tissues contain the maximum and minimum levels of detectable PrP(Sc) that have been observed in vCJD. RESULTS: Efficient transmission of prion infection was observed in transgenic mice inoculated with vCJD rectal tissue containing PrP(Sc) at a concentration of 10(4.7)-fold lower than that in vCJD brain. CONCLUSIONS: These data confirm the potential risks for secondary transmission of vCJD prions via gastrointestinal procedures and support the use of PrP(Sc) as a quantitative marker of prion infectivity in vCJD tissues.

Rskalpha-actin/hIGF-1 transgenic mice with increased IGF-I in skeletal muscle and blood: impact on regeneration, denervation and muscular dystrophy.

Human IGF-I was over-expressed in skeletal muscles of C57/BL6xCBA mice under the control of the rat skeletal alpha-actin gene promoter. RT-PCR verified expression of the transgene in skeletal muscle but not in the liver of 1- and 21-day old heterozygote transgenic mice. The concentration of endogenous mouse IGF-I, measured by an immunoassay which does not detect human IGF-I, was not significantly different between transgenic mice and wild-type littermates (9.5 +/- 0.8 and 13.3 +/- 1.9 ng/g in muscle; 158.3 +/- 18.6 and 132.9 +/- 33.1 ng/ml in plasma, respectively). In contrast, quantitation with antibodies to human IGF-I showed an increase in IGF-I of about 100 ng/ml in plasma and 150 ng/g in muscle of transgenic mice at 6 months of age. Transgenic males, compared to their age matched wild-type littermates, had a significantly higher body weight (38.6 +/- 0.53 g vs. 35.8 +/- 0.64 g at 6 months of age; P < 0.001), dry fat-free carcass mass (5.51 +/- 0.085 vs. 5.08 +/- 0.092 g; P < 0.001) and myofibrillar protein mass (1.62 +/- 0.045 vs. 1.49 +/- 0.048 g; P < 0.05), although the fractional content of fat in the carcass was lower (167 +/- 7.0 vs. 197 +/- 7.7 g/kg wet weight) in transgenic animals. There was no evidence of muscle hypertrophy and no change in the proportion of slow type I myofibres in the limb muscles of Rskalpha-actin/hIGF-I transgenic mice at 3 or 6 months of age. Phenotypic changes in Rskalpha-actin/hIGF-I mice are likely to be due to systemic as well as autocrine/paracrine effects of overproduction of IGF-I due to expression of the human IGF-I transgene. The effect of muscle specific over-expression of Rskalpha-actin/hIGF-I transgene was tested on: (i) muscle regeneration in auto-transplanted whole muscle grafts; (ii) myofibre atrophy following sciatic nerve transection; and (iii) sarolemmal damage and myofibre necrosis in dystrophic mdx muscle. No beneficial effect of muscle specific over-expression of Rskalpha-actin/hIGF-I transgene was seen in these three experimental models.

Expression pattern of a mini human PrP gene promoter in transgenic mice.

The prion protein is central to the pathogenesis of prion diseases, although its exact function remains unclear. Although transgenic mice have been widely utilised in prion research, their PrP expression patterns have not been characterised in detail. We have studied the developmental temporal and spatial expression of a 214-bp mini human PrP promoter in transgenic mice. Transgene expression is first detected at embryonic day 12.5, a day earlier than previously reported for endogenous mouse gene by in situ hybridization. The general expression pattern closely mirrors that of the endogenous mouse PrP gene, such that this small and clearly defined transgene cassette can replace the need to use large cosmid based vectors for transgenetic modeling of human and animal prion disease.

Post-natal knockout of prion protein alters hippocampal CA1 properties, but does not result in neurodegeneration.

Prion protein (PrP) plays a crucial role in prion disease, but its physiological function remains unclear. Mice with gene deletions restricted to the coding region of PrP have only minor phenotypic deficits, but are resistant to prion disease. We generated double transgenic mice using the Cre-loxP system to examine the effects of PrP depletion on neuronal survival and function in adult brain. Cre-mediated ablation of PrP in neurons occurred after 9 weeks. We found that the mice remained healthy without evidence of neurodegeneration or other histopathological changes for up to 15 months post-knockout. However, on neurophysiological evaluation, they showed significant reduction of afterhyperpolarization potentials (AHPs) in hippocampal CA1 cells, suggesting a direct role for PrP in the modulation of neuronal excitability. These data provide new insights into PrP function. Furthermore, they show that acute depletion of PrP does not affect neuronal survival in this model, ruling out loss of PrP function as a pathogenic mechanism in prion disease and validating therapeutic approaches targeting PrP.

Isolation and functional characterisation of the promoter region of the human prion protein gene.

The human prion protein gene (PRNP) encodes a 33-35 kDa cell surface protein that is highly expressed in the central nervous system and is vital to the pathogenesis of prion diseases. We have characterised the promoter region of PRNP as a first step towards defining the mechanisms regulating its expression. Sequence analysis of a 2.7 kb genomic DNA fragment containing exon I and the 5'-flanking region of PRNP, revealed a number of putative transcriptional factor binding sites, including Sp1, Ap-1, Ap-2 and a CCAAT box. Transient transfection assays in tissue culture cells with constructs consisting of the wild-type and deletion mutants of the PRNP 2.7 kb genomic fragment driving a luciferase reporter gene, demonstrate an active promoter within a 273 bp region (-148 to +125, relative to the cap site).

Expression of human full-length and minidystrophin in transgenic mdx mice: implications for gene therapy of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disorder with a high spontaneous mutation rate and no effective treatment, hence development of genetic based therapies is an important goal. We report that expression of a recombinant human minidystrophin cDNA, compatible with current viral vectors, can significantly reduce the myopathic phenotype in transgenic mdx mice, even when expressed at only 20-30% of endogenous dystrophin levels at the sarcolemma. To the extent that data obtained in mouse studies are applicable to DMD, the virtual elimination of morphological and biochemical abnormalities in the mdx mouse supports the use of this cDNA in somatic gene therapy protocols for DMD.

Tissue specific expression of an alpha-skeletal actin-lacZ fusion gene during development in transgenic mice.

Transgenic mice carrying a chimaeric transgene containing 730 bp of the 5'-flanking sequences and the entire first intron of the rat alpha-skeletal actin gene fused to the lacZ reporter gene have been produced by microinjection. The lacZ reporter gene was used to verify the suitability of using the rat alpha-actin promoter elements to target expression of genes of agricultural and therapeutic value exclusively to skeletal and heart muscle cells and fibres of transgenic mice. Expression of the transgene indicates a tightly regulated developmental and muscle specific control of the rat alpha-skeletal actin gene, making it a useful promoter for gene targeting to muscle tissues. The cells destined to form muscle tissues in these transgenic mice are readily visualized in intact embryos by staining for beta-galactosidase activity, making them a suitable animal model for studying the origin and development of skeletal and cardiac muscle tissues.

Analysis of lines of mice selected for fat content. 3. Flux through the de novo lipid synthesis pathway.

The flux through the de novo fatty acid synthesis pathway was estimated in lines of mice which differed substantially in fat content following 26 generations of selection at 10 weeks of age. Previous estimates of lipogenic enzyme activities had indicated an increase in the capacity for lipogenesis in the Fat compared to the Lean line. Therefore the in vivo flux in lipogenesis was measured in both liver and gonadal fat pad (GFP) tissues of males at 5 and 10 weeks of age, using the rate of incorporation of 3H from 3H2O and 14C from acetate and citrate into total lipids. At both ages and in both tissues the Fat line had a higher flux, about 20% increase in the liver and up to three-fold increase (range 1.2- to 3.4-fold) in the GFP. We conclude that direct selection for fatness in mice has resulted in metabolic changes in the rate of de novo fatty acid synthesis, and that the changes are largely detectable before 10 weeks, the age of selection.

Analysis of lines of mice selected for fat content. 1. Correlated responses in the activities of NADPH-generating enzymes.

Estimates of the activities (Vmax) of four enzymes that generate the coenzyme NADPH, an absolute requirement for tissue fatty-acid synthesis, and of the concentration of NADP plus NADPH were made in lines of mice differing in fat content. These lines had been selected from the same base population for 20 generations, and 3 high, 3 low replicates and 1 unselected control were used. Analyses were performed on liver and gonadal fat pad (GFP) of males at 5 and 10 weeks of age. In both the liver and the GFP, measurable activities of the four enzymes: glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), isocitrate dehydrogenase (IDH) and malic enzyme (ME) expressed per mg soluble protein were, with minor exceptions, higher in the Fat (F) than in the Lean (L) lines at both ages; the highest ratio being 2.2 for ME in the GFP. The relationships between these measurable activities (Vmax) and in vivo lipogenesis are not however known. When expressed per gram tissue, the ratios for F to L in the GFP were less than 1 in most cases, presumably because of the very different adipocyte numbers and/or sizes between the lines. There were no significant differences between the lines in the concentration of NADP plus NADPH per gram tissue in liver or GFP, suggesting that F lines converted NADP to NADPH faster than L lines. It is predicted that selection on the enzyme activities would be less efficient than direct selection at changing fat content.