Stem cell based therapy for the treatment of prion diseases

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 19-06-2009Transmissible spongiform encephalopathies or prion diseases are invariably fatal neurodegenerative disorders affecting humans and both domestic and free-ranging animals {Aguzzi, 2004 #5}. Their central feature is the post-translational conversion of PrPc, a hostencoded cellular prion protein into an abnormal isoform PrPSc {Prusiner, 1982 #8}, {Collinge, 2001 #84}. This transition appears to involve only a conformational change rather than covalent modification and confers PrPSc detergent insolubility and partial resistance to proteolytic degradation. The neuropathological characteristics present are: vacuolization (spongiosis), neuronal death, astrocytic activation or gliosis, and PrPSc deposits {Prusiner, 1991#7}. Prion diseases are progressive, fatal, and to date there is neither early diagnostic nor effective treatment. Conventional drug therapeutic approaches remain several obstacles and limitations in vivo. For this reason, new and innovative therapies constitute an area of intense research interest. A major focus of research consists therefore in the identification of new molecules that could interfere with in vivo prion propagation. Promising therapeutic approaches to block the production of PrPC/PrPSc are based on PrP RNA interference, passive or active immunization, dominant negative inhibition of PrPSc formation, and inhibition of interactions between PrPSc and other cofactors. Other alternative strategies would consist in the development of a gene or/and cell therapy system. The combination of both of therapeutic strategies would target not only the formation of toxic aggregates, but also the brain destruction already incurred. Stem cell based therapy in prion disorders remains a poorly explored option and there is only one study reporting the use of stem cells for therapeutic purposes in prion diseases {Brown, 2001 #360}. Therefore, in this work, we have chosen to develop a cell replacement therapeutic approach, since therapeutic strategies using stem cell grafting have given rise to encouraging results in the treatment of Parkinson’s disease {Bjorklund, 2000 #354}, {Rodriguez-Gomez, 2007 #355}, Huntington's disease {Kendall, 1998 #356}, {Dunnett, 2004 #357}, {Bachoud-Levi, 1998 #358} and amyotrophic lateral sclerosis {Silani, 2004 #359}. Our aim is to replace the damaged neurons by the transplantation of stem cells from two different origins: embryonic and foetal derived from various mice. In a first part, I will describe these two cell types, their set up, and their common neural differentiation process {Lee SH, 2000 #246}, {Ray, 2006 #385}, {Pollard, 2008 #382}. In an attempt to analyse the fate of these cells in vivo, they will be graft in the brain of healthy mice. As we obtained some teratomas in the brain of the mice grafted with cells from embryonic origin, we carry out the final therapeutic approaches in prion infected mice with foetal NSC. The results obtained are encouraging and efforts and progress in stem cell therapy should continue given the potential they represent not only for the treatment of prion diseases but also for the other neurodegenerative disorders

Biomass measurements of single neurites in vitro using optical wavefront microscopy

International audienceQuantitative phase microscopies (QPMs) enable label-free, non-invasive observation of living cells in culture, for arbitrarily long periods of time. One of the main benefits of QPMs compared with fluorescence microscopy is the possibility to measure the dry mass of individual cells or organelles. While QPM dry mass measurements on neural cells have been reported this last decade, dry mass measurements on their neurites has been very little addressed. Because neurites are tenuous objects, they are difficult to precisely characterize and segment using most QPMs. In this article, we use cross-grating wavefront microscopy (CGM), a high-resolution wavefront imaging technique, to measure the dry mass of individual neurites of primary neurons in vitro. CGM is based on the simple association of a cross-grating positioned in front of a camera, and can detect wavefront distortions smaller than a hydrogen atom (∼0.1 nm). In this article, an algorithm for dry-mass measurement of neurites from CGM images is detailed and provided. With objects as small as neurites, we highlight the importance of dealing with the diffraction rings for proper image segmentation and accurate biomass measurements. The high precision of the measurements we obtain using CGM and this semi-manual algorithm enabled us to detect periodic oscillations of neurites never observed before, demonstrating the sufficient degree of accuracy of CGM to capture the cell dynamics at the single neurite level, with a typical precision of 2%, i.e., 0.08 pg in most cases, down to a few fg for the smallest objects

Cellules souches dentaires : mythe ou espoir en médecine neurorégénératrice ?

International audienceThe use of dental stem cells has raised many hopes in the development of new treatments for neurodegenerative diseases. According to current statistics, about 1 in 6 people in the world would be affected by a neurological disease. This number continues to increase as the world's population ages, making neurodegenerative diseases probably the one of the major challenges of public health in the 21st century. Neurodegenerative diseases are characterized mainly by a progressive loss of cognitive abilities and patient autonomy related to loss and degeneration of neurons in brain structures. Unfortunately, today, the only treatments available for this type of disease do only relieve the symptoms, they do not treat them, and few clinical trials have been truly convincing to date. Hence, hope lies for these diseases in the development of other therapeutic approaches. As such, dental stem cells could be a promising area of research because of their rapid growth, their great capacity for differentiation into different types of cells (among neuronal ones for some of them) and how easy they can be obtained, without raising ethical issues as for example for embryonic stem cells.L’utilisation des cellules souches dentaires a fait naître de nombreux espoirs dans le développement de nouveaux traitements destinés aux maladies neurodégénératives. Si l’on se réfère aux statistiques actuelles, environ 1 personne sur 6 dans le monde serait atteinte par une maladie neurologique. Ce nombre ne cesse d’augmenter au fur et à mesure que la population mondiale vieillit, faisant des maladies neurodégénératives probablement l’un des principaux défis de la santé publique du XXIe siècle. Les maladies neurodégénératives se caractérisent principalement par une perte progressive des facultés cognitives et de l’autonomie des patients liée à une perte et une dégénérescence des neurones des structures cérébrales. Malheureusement, force est de constater que les seuls traitements disponibles actuellement pour ce type de pathologies ne font que soulager les symptômes et non les traiter et que peu d’essais cliniques à ce jour ont été véritablement probants. L’espoir réside donc pour ces maladies neurodégénératives dans le développement de nouvelles approches thérapeutiques. Les cellules souches dentaires pourraient constituer une nouvelle voie de recherche en thérapie cellulaire, de par leur croissance rapide, leur grande capacité de différenciation en différents types cellulaires (y compris en cellules neuronales pour certaines) et la facilité avec laquelle elles peuvent être obtenues sans soulever de problèmes d’éthique comme par exemple pour les cellules souches embryonnaires

Elements modulating the prion species barrier and its passage consequences.

The specific characteristics of Transmissible Spongiform Encephalopathy (TSE) strains may be altered during passage across a species barrier. In this study we investigated the biochemical and biological characteristics of Bovine Spongiform Encephalopathy (BSE) after transmission in both natural host species (cattle, sheep, pigs and mice) and in transgenic mice overexpressing the corresponding cellular prion protein (PrPC) in comparison with other non-BSE related prions from the same species. After these passages, most features of the BSE agent remained unchanged. BSE-derived agents only showed slight modifications in the biochemical properties of the accumulated PrPSc, which were demonstrated to be reversible upon re-inoculation into transgenic mice expressing bovine-PrPC. Transmission experiments in transgenic mice expressing bovine, porcine or human-PrP revealed that all BSE-derived agents were transmitted with no or a weak transmission barrier. In contrast, a high species barrier was observed for the non-BSE related prions that harboured an identical PrP amino acid sequence, supporting the theory that the prion transmission barrier is modulated by strain properties (presumably conformation-dependent) rather than by PrP amino acid sequence differences between host and donor. As identical results were observed with prions propagated either in natural hosts or in transgenic mouse models, we postulate that the species barrier and its passage consequences are uniquely governed by the host PrPC sequence and not influenced by other host genetic factors. The results presented herein reinforce the idea that the BSE agent is highly promiscuous, infecting other species, maintaining its properties in the new species, and even increasing its capabilities to jump to other species including humans. These data are essential for the development of an accurate risk assessment for BSE

Electrophoretic profiles and antibody labelling of PrP^res in BSE-derived prions.

<p>PrP^res was detected by western blot using the mAbs Sha31 (A) and 12B2 (B) in the different BSE-derived prions propagated either in natural hosts or in transgenic mouse models over-expressing their corresponding PrP^C sequence: cattle-BSE (lanes 1 and 11), BoTg-BSE (lanes 2 and 12), sheep-BSE (lane 3), OvTg-BSE (lane 4), pig-BSE (lane 5), PoTg-BSE (lane 6), mouse-BSE (lane 7), MoTga20-BSE (lane 8), human-vCJD (lane 13), HuTg-BSE (lane 14). Sheep-scrapie (lane 9), mouse-RML (lane 10), human-sCJD (lane 15) and atypical cattle-BSE H (lane 16) were included as control non-BSE related prions. Panels A and B were loaded with the same quantities of PrP^res extracted from each sample. MW, molecular weight in kilodaltons.</p

PrP^res in BoPrP-Tg110 mice.

<p>Electrophoretic profiles and antibody labelling of PrP^res as detected by mAbs Sha31 (A) and 12B2 (B) in brain extracts from BoPrP-Tg110 mice inoculated with the different BSE-derived prions: cattle-BSE (lane 1), sheep-BSE (lane 2), OvTg-BSE (lane 3), pig-BSE (lane 4), PoTg-BSE (lane 5), mouse-BSE (lane 6), MoTga20-BSE (lane 7), human-vCJD (lane 8), HuTg-BSE (lane 9). Brain extracts from BoPrP-Tg110 mice inoculated with atypical cattle-BSE H (lane 10), sheep-scrapie (lane 11) and mouse-RML (lane 12) were included as a control non-BSE related prion propagated in the same mouse model. Panels A and B were loaded with the same quantities of PrP^res extracted from each sample. MW, molecular weight in kilodaltons.</p

Glycoform ratios of PrP^res detected by Western blotting.

<p>PrP^res was detected with the Sha31 monoclonal antibody in the different BSE-derived prions propagated either in the natural hosts (A) or in transgenic mouse models over-expressing their corresponding PrP^C sequence (B). Atypical cattle-BSE H, sheep-scrapie, mouse-RML and human-sCJD isolates (C) are included for comparison purposes. Values are the normalized means from at least six repeated runs. Arrows indicate the reading direction of the axis.</p