Date Published: August 1, 2013
Publisher: Public Library of Science
Author(s): Aroa Relaño-Ginès, Audrey Gabelle, Claire Hamela, Maxime Belondrade, Danielle Casanova, Chantal Mourton-Gilles, Sylvain Lehmann, Carole Crozet, Neil A. Mabbott.
Prion diseases are irreversible progressive neurodegenerative diseases, leading to severe incapacity and death. They are characterized in the brain by prion amyloid deposits, vacuolisation, astrocytosis, neuronal degeneration, and by cognitive, behavioural and physical impairments. There is no treatment for these disorders and stem cell therapy therefore represents an interesting new approach. Gains could not only result from the cell transplantation, but also from the stimulation of endogenous neural stem cells (NSC) or by the combination of both approaches. However, the development of such strategies requires a detailed knowledge of the pathology, particularly concerning the status of the adult neurogenesis and endogenous NSC during the development of the disease. During the past decade, several studies have consistently shown that NSC reside in the adult mammalian central nervous system (CNS) and that adult neurogenesis occurs throughout the adulthood in the subventricular zone of the lateral ventricle or the Dentate Gyrus of the hippocampus. Adult NSC are believed to constitute a reservoir for neuronal replacement during normal cell turnover or after brain injury. However, the activation of this system does not fully compensate the neuronal loss that occurs during neurodegenerative diseases and could even contribute to the disease progression. We investigated here the status of these cells during the development of prion disorders. We were able to show that NSC accumulate and replicate prions. Importantly, this resulted in the alteration of their neuronal fate which then represents a new pathologic event that might underlie the rapid progression of the disease.
Prion diseases or transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative disorders, which include Creutzfeldt-Jakob disease in humans, scrapie in sheep and goats, and bovine spongiform encephalopathy in cattle. Their origin can be genetic, sporadic or infectious and there is currently no available treatment preventing the widespread neurodegeneration occurring in these disorders. TSEs are pathophysiologically characterized by the accumulation in the brain of a pathogenic abnormal isoform of a protein termed PrP scrapie (PrPSc) . According to the prion hypothesis, the infectious isoform PrPSc can trigger the autocatalytic conversion of the neuronal host-encoded PrPC into PrPSc through a poorly understood misfolding process , rendering the progression of the disease dependent upon PrP expression. Several studies have reported early, severe and selective loss of GABAergic interneurons in prion diseases , . These specific changes in neuronal subset may underlie some of the clinical symptoms in prions. The diagnosis of these diseases is difficult and often leaves only a short therapeutic window after the appearance of the first clinical signs . Although important efforts have been made to understand the physiopathogenesis of neurodegenerative disorders, Prion diseases are still incurable and new therapeutic approaches such as cell therapy need to be explored. As a matter of fact, the widespread existence of endogenous neural stem cells (NSC) in the adult brain ,  offers hope that these endogenous cells may be harnessed to repair cellular damages caused by brain injuries. During the past decade, several studies have consistently shown that (i) NSC reside in the adult mammalian CNS and that (ii) adult neurogenesis occurs throughout the adulthood in the subventricular zone (SVZ) of the lateral ventricle (LV) or the Dentate Gyrus (DG) of the hippocampus (H). Accumulating evidences have clearly shown that a large number of newborn neurons can be generated from adult NSC, and integrate into pre-existing neural circuits . Under physiological conditions, adult NSC follow a highly stereotypic differentiation path to generate neurons in the olfactory bulb and the DG. Adult neurogenesis is also highly sensitive to environmental cues, physiological stimuli and neuronal activity, suggesting that the tailored addition of new neurons might serve specific neuronal functions . Endogenous NSC may also provide a cellular reservoir for replacement of cell lost during normal cell turnover but also after brain injury , . In neurodegenerative affections, particularly those involving pathogenic protein misfolding, the field of adult neurogenesis only begins to be explored. The results are not always consistent between studies. For instance, hippocampal neurogenesis is increased in patients with AD , but it is decreased in some transgenic mouse models of AD . Following brain injuries, adult neurogenesis can be increased and is even accompanied by a migration of neural precursors towards the injured area , . However, the activation of this system does not fully compensate the neuronal loss that occurs during diseases and could even contribute to the disease progression .
The presence of PrPSc among lateral ventricle NSC and neuroblasts incited us to isolate NSC cells from the brain of prion infected mice. We then checked whether these cells were infected by prion or not and assess the impact this could have on their neuronal differentiation. Our results show for the first time that NSC present in prion infected mouse brain accumulate PrPSc, which leads to an alteration of their neuronal fate. Indeed, we isolated adult neural stem cells from neurogenic adult brain area (the dentate gyrus and the lateral wall of the lateral ventricles) of both prion infected and non infected mice. Importantly, we were able to keep these cells in culture for several subpassages while maintaining their prion replication. The neuronal differentiation of the infected cells was shown to be compromised since less neuroblasts and less newborn neurons were obtained when the cells were placed in a neuronal differentiation medium. This was also accompanied by an increase in the amount of astrocytes.
Five-week-old C57Bl/6J female mice were anesthetized via intraperitoneal route with 100 µg/g of ketamine (Imalgène, Merial, Lyon, France) and 5 µg/g of xylazine (Rompun, Bayer, Leverkusen, Germany). They were then intracranially inoculated with the ME7 prion strain (1%, 20 µl) and with 1% homogenate of healthy brain as control (mock). Mice were housed in an A3 facility. Transgenic mice expressing EGFP under beta-actin promoter kindly provided by Dr. M. Okabe were also used. KO-PrP mice were kindly provided by Dr C. Weissmann. All animal work has been conducted according to relevant national guidelines of the French Ethical Committee (decree 87–848) and European Community Directive 86/609/EEC regarding mice. Experiments were performed with the approval of the Regional Languedoc Roussillon Ethical Committee for Animal Experiments under the registration number CEEA-LR-1006. They were performed in the Biohazard prevention area (A3) (Biorad/Université MontpellierII).