Cre/lox-assisted non-invasive in vivo tracking of specific cell populations by positron emission tomography.

Many pathophysiological processes are associated with proliferation, migration or death of distinct cell populations. Monitoring specific cell types and their progeny in a non-invasive, longitudinal and quantitative manner is still challenging. Here we show a novel cell-tracking system that combines Cre/lox-assisted cell fate mapping with a thymidine kinase (sr39tk) reporter gene for cell detection by positron emission tomography (PET). We generate Rosa26-mT/sr39tk PET reporter mice and induce sr39tk expression in platelets, T lymphocytes or cardiomyocytes. As proof of concept, we demonstrate that our mouse model permits longitudinal PET imaging and quantification of T-cell homing during inflammation and cardiomyocyte viability after myocardial infarction. Moreover, Rosa26-mT/sr39tk mice are useful for whole-body characterization of transgenic Cre mice and to detect previously unknown Cre activity. We anticipate that the Cre-switchable PET reporter mice will be broadly applicable for non-invasive long-term tracking of selected cell populations in vivo.Non-invasive cell tracking is a powerful method to visualize cells in vivo under physiological and pathophysiological conditions. Here Thunemann et al. generate a mouse model for in vivo tracking and quantification of specific cell types by combining a PET reporter gene with Cre-dependent activation that can be exploited for any cell population for which a Cre mouse line is available.

A combinatorial approach to identify calpain cleavage sites in the Machado-Joseph disease protein ataxin-3.

Ataxin-3, the disease protein in Machado-Joseph disease, is known to be proteolytically modified by various enzymes including two major families of proteases, caspases and calpains. This processing results in the generation of toxic fragments of the polyglutamine-expanded protein. Although various approaches were undertaken to identify cleavage sites within ataxin-3 and to evaluate the impact of fragments on the molecular pathogenesis of Machado-Joseph disease, calpain-mediated cleavage of the disease protein and the localization of cleavage sites remained unclear. Here, we report on the first precise localization of calpain cleavage sites in ataxin-3 and on the characterization of the resulting breakdown products. After confirming the occurrence of calpain-derived fragmentation of ataxin-3 in patient-derived cell lines and post-mortem brain tissue, we combined in silico prediction tools, western blot analysis, mass spectrometry, and peptide overlay assays to identify calpain cleavage sites. We found that ataxin-3 is primarily cleaved at two sites, namely at amino acid positions D208 and S256 and mutating amino acids at both cleavage sites to tryptophan nearly abolished ataxin-3 fragmentation. Furthermore, analysis of calpain cleavage-derived fragments showed distinct aggregation propensities and toxicities of C-terminal polyglutamine-containing breakdown products. Our data elucidate the important role of ataxin-3 proteolysis in the pathogenesis of Machado-Joseph disease and further emphasize the relevance of targeting this disease pathway as a treatment strategy in neurodegenerative disorders.

In vivo assessment of riluzole as a potential therapeutic drug for spinocerebellar ataxia type 3.

Spinocerebellar ataxia type 3 (SCA3) is an autosomal dominantly inherited neurodegenerative disorder for which no curative therapy is available. The cause of this disease is the expansion of a CAG repeat in the so-called ATXN3 gene leading to an expanded polyglutamine stretch in the ataxin-3 protein. Although the function of ataxin-3 has been defined as a deubiquitinating enzyme, the pathogenic pathway underlying SCA3 remains to be deciphered. Besides others, also the glutamatergic system seems to be altered in SCA3. The antiglutamatergic substance riluzole has thus been suggested as a potential therapeutic agent for SCA3. To assess whether riluzole is effective in the treatment of SCA3 in vivo, we used a phenotypically well-characterized conditional mouse model previously generated by us. Treatment with 10 mg/kg riluzole in the drinking water was started when mice showed impairment in rotarod performance. Post-symptomatic treatment with riluzole carried out for a period of 10 months led to reduction of the soluble ataxin-3 level and an increase in ataxin-3 positive accumulations, but did not improve motor deficits measured by rotarod. There was also no positive effect on home cage behavior or body weight. We even observed a pronounced reduction of calbindin expression in Purkinje cells in riluzole-treated mice. Thus, long-term treatment with riluzole was not able to alleviate disease symptoms observed in transgenic SCA3 mice and should be considered with caution in the treatment of human patients. Assessing riluzole as a potential treatment for spinocerebellar ataxia type 3 (SCA3) had no beneficial, but rather a worsening effect on our transgenic SCA3 mouse model. We hypothesize that: Riluzole treatment enhanced glutamate release in ATXN3-expressing cells leading to an increased Ca(2+) influx resulting in Purkinje cell damage shown by loss of calbindin expression.

The Basis CL cemented femoral stem: results after 8.9 years follow-up.

This prospective study was conducted to demonstrate that the matte-finish Basis CL cemented endoprosthetic stem delivers good qualitative results after 10 years. Between January and December 1999, 205 consecutive hips (201 patients; 74.5 ± 6.8 years at surgery) underwent primary total hip arthroplasty with the Basis CL and the same acetabular cup (RM Classic cup) at a single institution. Follow-up data at 10 years was available for 120 hips (average follow-up of 8.9 years, ±2.9). Mean Harris Hip Score improved from 39.5 ± 16.8 at baseline to 75.9 ± 16.7 at 10-year follow-up (p<0.001). Four hips required revision during the study: three for infection and one for pain. There were no cases of aseptic loosening, implant migration, or stem fracture. Cumulative survival at 10 years was 97.4% with the endpoint of revision for any reason. In conclusion, results with the matte-finish cemented Basis CL indicated that it was safe and effective after medium-term follow-up.