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Cell-based therapy against prion diseases
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ScienceDirect Cell-based therapy against prion diseases Aroa Relan˜o-Gine´s1,2, Sylvain Lehmann1,2,3 and Carole Crozet1 Despite multiple efforts to find treatments, prion diseases are still incurable. The currently available therapeutic strategies are mostly based on compounds to inhibit pathological PrP (PrPSc) accumulation, and cellular PrP (PrPC) conversion into PrPSc. However, they cannot reverse the pathological changes already present in the brain. Cell-based therapeutic strategies could promote the repair of the pre-existing brain damage. The few available data come mostly from preclinical studies using neural stem cells, bone marrow-derived microglia and mesenchymal stem cells, as cell sources. Moreover, the benefits of cell-based therapeutic strategies could be linked not only to the replacement of damaged cells, but also to the secretion of trophic factors by the grafted cells that might modulate inflammation, cell death, or endogenous neurogenesis. Addresses 1 Institute for Regenerative Medicine and Biotherapies (IRMB), Neural Stem Cell, MSC and Neurodegenerative Diseases - U1183 INSERM (Institut National de la Sante´ et de la Recherche Me´dicale), 80 rue Augustin Fliche, 34295 Montpellier, France 2 Universite´ de Montpellier, 163 rue Auguste Broussonet, 34090 Montpellier, France 3 Centre Hospitalo-Universitaire de Montpellier, 191 Av. du Doyen Gaston Giraud, 34295 Montpellier, France Corresponding author: Crozet, Carole ([email protected])
Current Opinion in Pharmacology 2019, 44:8–14 This review comes from a themed issue on Neurosciences – prion disease Edited by Roberto Chiesa and Ina Vorberg
https://doi.org/10.1016/j.coph.2018.11.001 1471-4892/ã 2019 Elsevier Ltd. All rights reserved.
Introduction Transmissible spongiform encephalopathies (TSE), also called prion diseases, are degenerative disorders of the central nervous system (CNS) leading to severe incapacity and death [1]. They affect both humans (mainly, Creutzfeldt-Jakob disease (CJD)) and animals (sheep scrapie, and bovine spongiform encephalopathies (BSE)). Human prion diseases exist in three main forms [2,3]: sporadic (80–90% of all CJD cases; 1.5 cases/million people/year), familial (5–15%, mutations in the PRNP gene encoding the cellular prion protein PrPC), and Current Opinion in Pharmacology 2019, 44:8–14
acquired (<5%, iatrogenic contamination during transplantation, blood transfusion, therapy with growth hormones, or dietary exposure to BSE prions in the case of the variant CJD in relatively young patients). TSE are characterized by a long incubation period, followed by the appearance of clinical symptoms leading to the fatal issue. TSE can be transmitted by intracerebral injection to primates and rodents that constitute very relevant experimental models. Neuronal vacuolization and loss, astrocyte proliferation and cerebral accumulation of pathological prion proteins (PrPSc) are typical TSE features. PrPSc results from the conformational conversion of the hostencoded cellular PrP protein (PrPC) that is mainly expressed in the CNS [1,4]. TSE are incurable diseases, mainly because of their late diagnosis when brain lesions are already severe, and the therapeutic window is limited. Moreover, the currently available therapeutic strategies only inhibit PrPSc accumulation and PrPC conversion into PrPSc, but cannot reverse the pre-existing brain lesions. Therefore, it is important to develop alternative strategies to promote the functional recovery of the damaged areas through cell replacement. Regenerative medicine has emerged as a promising option for treating neurologic diseases by using different cell types [5,6]: neural stem cells (NSC) derived from pluripotent stem cells, embryonic stem (ES) cells or induced pluripotent stem cells (iPSC); fetal neural stem cells (fNSC); neuronal precursor cells (NPC); mesenchymal stem cells (MSC); and even microglial cells. Stem cells (NSC and fNSC) are defined by their capacity to self-renew and to differentiate into neurons, astrocytes or oligodendrocytes. NPC are neuronal progenitors that are already more committed towards the neuronal lineage than NSC. They can be obtained from NSC in culture, and could be used in graft approaches. MSC are multipotent adult stem cells of mesodermal origin. They can differentiate into mesenchymal lineages [7], and also into non-mesodermal cell types in vitro, including neuronal and glial lineages [8– 10]. MSC and MSC-like cells can have different origins (bone marrow (BM), fat tissue, nasal mucosae, dental pulp . . . [11]). MSC, mostly derived from BM, have been extensively used for stem cell therapy approaches in several neurodegenerative diseases [10]. It is thought that they restore injured tissues through the secretion of various trophic factors [12], extracellular vesicle dispersion [13], and mitochondrial transfer [14]. They stimulate angiogenesis [15], the proliferation and differentiation of endogenous NSC [16], and integrate into tissues [17]. The great advantage of stem cells is that an almost unlimited amount of cells can be derived from one or few cells. However, the development of cell therapeutic approaches is still challenging. Moreover, outcome www.sciencedirect.com
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interpretation is complex because it must take into account not only the direct effect of cell replacement, but also the possible interactions between grafted cells and host tissue (e.g. secretion of trophic factors, cytokine, prion propagation in the grafted cells) [18,19]. For instance, it has been suggested that grafted NSC, which express glial-derived neurotrophic factor (GDNF), might upregulate brain-derived neurotrophic factor (BDNF) to support the survival of the existing neurons after ischemia [20]. They could also decrease the post-injury immune cell recruitment and pro-inflammatory cytokine expression in the brain. Moreover, endogenous NSC and adult neurogenesis also could be stimulated using stem cellbased therapeutic strategies. Indeed, it is now well admitted that in rodents, adult neurogenesis (i.e. the production of new neurons in the adult brain) occurs primarily in two areas: the dentate gyrus (DG) and the subventricular zone (SVZ) of the lateral ventricles (LV) [21]. Adult neurogenesis in human hippocampus is currently debated. However, a recent paper showed that proliferating NSC and immature neuronal cells are present throughout human aging, while the number of non-dividing quiescent stem cells, which are considered a NSC reservoir, decreases [22]. The equilibrium between endogenous NSC activity and quiescence is regulated by neurons located in remote brain areas that adapt neuron production to the physiological and pathological constraints. In rodents, adult neurogenesis can be increased by 30-fold after brain injury or during disease. This can be accompanied by migration of neural precursors towards the lesions [23,24]. In patients with Alzheimer’s or Huntington’s disease, the proliferation of neural precursors is stimulated and new neurons are generated [25,26]. These findings could help to better understand the disease and could be used to develop strategies to mobilize endogenous NSC to repair diseased or injured brain [22,27]. This review will discuss why adult NSC could be the basis for therapeutic interventions in prion disorders. It will also describe the available cell-based graft approaches for prion diseases.
Adult neural stem cells could be the basis for therapeutic interventions in prion diseases Adult neurogenesis has been poorly studied in prion diseases compared with other neurodegenerative diseases. In healthy mice, PrPC positively regulates endogenous neurogenesis in the SVZ and DG [28], and promotes survival and axon targeting of olfactory sensory neurons during adult neurogenesis [29,30]. In TSE, the situation might be more complex than in physiological conditions for two possible reasons: first, during neuronal differentiation, PrPC expression increases, and this PrPC could be converted into PrPSc, thus further promoting prion propagation in the brain, second, PrPC promotes www.sciencedirect.com
neural precursor proliferation during embryo and adult neurogenesis [28], and seems to be involved in neuronal survival [30]. Therefore, its conversion into PrPSc could inhibit neurogenesis and/or contribute to neurodegeneration [31]. Moreover, the self-healing capacity of adult brain is not enough to significantly improve the affected neuronal network because only a small proportion of new cells will integrate and survive. Immunohistochemistry analyses of brain tissues from prion-infected mice showed PrPSc accumulation within and in the vicinity of endogenous NSC in neurogenic areas (DG and lateral wall of the left LV) [32,33]. An increase of NSC proliferation was also reported in DG and/or LV of scrapie infected mice [34,35]. We also showed that NSC isolated from prioninfected mice accumulate and replicate PrPSc [32,33] and that these cells proliferate more rapidly and display more apoptosis (unpublished data). Moreover, neuron production from endogenous NSC is effective during the first stages of prion disease, but decreases during disease progression [35]. Indeed, adult neurogenesis seems to be first stimulated to produce new neurons to replace the lost/damaged cells. However, these newborn neurons cannot achieve neuronal maturation and most of them will die [32,33,35] (and unpublished data). Interestingly, analysis of prion propagation in mice lacking the NEIL3 enzyme (a DNA glycosidase involved in DNA repair) and in which adult neurogenesis is altered indicated that in these mice, the clinical phase is shortened, but not the survival time. This suggests that although adult neurogenesis cannot prevent the fatal outcome of prion diseases, it might delay the appearance of clinical signs [36]. Nevertheless, the precise mechanisms by which NEIL3 contributes to neuroprotection in prion diseases are not known. The mechanisms behind the impairment of adult neurogenesis in prion diseases are not known. Nevertheless, it could be interesting to determine whether inhibition of PrPSc propagation in NSC and the derived neurons might preserve the targeted cells, and possibly slow down disease progression. Moreover, inhibition of PrPSc accumulation/propagation could allow maintaining PrPC neuroprotective and pro-neurogenic functions. This could be achieved through gene engineering using viral vectors that express well characterized anti-prion molecules, such as anti-PrP single-chain variable fragment (scFv) antibodies, dominant negative PrP mutants, anti-PrP oligonucleotides for RNA interference [37–41]. It should be important to assess whether this type of approach might limit not only prion propagation/accumulation, but also maintain and preserve the neurogenesis niche to enable additional cell-based strategies.
Cell-graft-based therapeutic approaches Different cell types have been used for preclinical studies on cell-based therapies in prion infected mice: fNSC, NPC, MSC, and microglia. Current Opinion in Pharmacology 2019, 44:8–14
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Neural stem cells and neuronal precursor cells Despite their huge potential, no pre-clinical study in the prion field using NSC derived from pluripotent stem cells (ES or iPSC) has been published yet. The first study (Figure 1) on the use of NSC in prion diseases evaluated the effect of transplanting fNSC derived from prionresistant knockout (koPrP) mouse embryos in the hippocampus of asymptomatic scrapie-infected (C57Bl6/VM) wild-type (wt) mice (at 150 days post-inoculation, dpi) [42]. This was not enough to modify the incubation and survival times compared with control mice (only PBS) [42]. Interestingly, the number of functional neurons in the pyramidal cell layer was increased (by about 1.5 times) at day 21 post-graft and at the endpoint (250 dpi) compared with control mice [42]. Although small foci of transplanted cells were detected in the brain of grafted mice, it was not investigated whether these cells accumulated PrPSc and whether the higher neuron number resulted from the replacement of damaged cells, secretion of trophic factors that stimulate neuron survival, and/or reduction of inflammatory signaling. More recently, using fNSC from wild type mice that express wtPrP, from prion-resistant transgenic mice that express porcine PrP (poPrP), and from koPrP mice [43,44], we discovered that compared with PBS, NSC graft could significantly
prolong the incubation (by 20%) and survival times (by 13%) in wt mice infected with RML prions [45]. This effect occurred only when fNSC were grafted before the appearance of clinical signs, suggesting the existence of a specific time window for successful treatment. Moreover, we observed the positive effects on incubation and survival times with all fNSC types, independently of the background of the mice from which they were derived (wtPrP, koPrP, and poPrP). This was surprising because prions can infect and replicate only in wt NSC ex vivo, but not in poPrP NSC and koPrP NSC [43,44]. Because of technical constraints, we could not assess the presence of the grafted cells. Therefore, we could not determine whether prion replication occurred in the grafted fNSC. Importantly, the longer incubation time was correlated with a reduction of the number of astrocytes in areas close to the graft. This suggests that cell transplantation on its own might play a key role in the inflammatory process probably through secretion of trophic factors by fNSC. This hypothesis remains to be tested. Recently, Frid et al. described an autologous and heterologous fNPC-based therapy in transgenic mice (TgCJD mice) that mimic the human E200K PrP mutation (responsible for a genetic CJD) and show neurological deterioration during aging [46]. Newborn TgCJD mice
Figure 1
NSC koPrP-f NSC PoPrP-f SC wtPrP-fN
etal koPrP-f NSC
NPC TgCJD-f C P wtPrP-fN
Future directions
Stimulation of endogenous neurogenesis
Relaňo-Ginés et al., 2011
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Increase of the number of neurons, integration and survival of the grafted cells
Increase of incubation and survival time Decrease of atroglosis when cells are grafted before the appearance of clinical signs
Delay in disease progression in TgCJD model of genetic CJD
l cell
hMSC
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Slight increase of the survival period
Increase of the survival period in Chandler-infected mice Migration of hMSC towards brain lesions in mice infected with both strains
Shan et al., 2017 Increase of the survival period Migration and activation of microglia
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Pre-clinical studies using cell-based therapeutic approaches and future research directions.
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were grafted with fNPC from the brain of TgCJD or wt mouse embryos and were followed for 10 months. The graft of both fNPC types significantly delayed disease progression compared with non-transplanted mice. Similarly to our finding, both fNPC types (from TgCJD and wt mice) could prolong the disease incubation time (by 35%). Moreover, the number of endogenous NSC was increased in the brain of grafted animals, suggesting a proneurogenic effect of grafted cells [46]. The authors did [45,47] not assess PrPSc accumulation in the grafted cells. However, they showed the absence of PrPSc accumulation in endogenous NSC in TgCJD mice and the lack of prion transmission when TgCJD NPC were grafted in the brain of wt mice [46].
Microglial cells Microglial cells are considered the innate immune cells of the brain, and contribute to tissue homeostasis and synaptic plasticity regulation [48]. Their proliferation and activation are increased in pathological conditions, and they participate in the development and maintenance of the neuro-inflammatory response. Microglia can also trigger neurotoxic pathways, thus promoting neurodegeneration. They can also play a neuroprotective role, and seem necessary for controlling inflammatory, repair and regenerative processes [49]. On the basis of the dual role of microglia [50,51], several groups tried to better understand the involvement of these cells in disease development. For instance, to study microglia turnover in prion diseases, the RML prion strain was inoculated in the brain of mice that were transplanted with green fluorescent protein (GFP)-expressing bone marrow (BM) cells after irradiation [52]. Fifty percent of all brain resident microglia was replaced by GFP-positive bone marrow derivedmicroglial cells before the clinical manifestation of the disease. Although scrapie pathogenesis was not improved, prions did not replicate in GFP-positive microglial cells. Therefore, these cells could be used for the delivery of anti-prion molecules. This was tested by Fujita et al. [37] (Figure 1) by grafting microglial cells (murine Ra2 microglial cell line) that express anti-prion scFv antibodies in the brain of wt mice at week 7 (asymptomatic phase) after infection with the 22 L scrapie strain. The survival of these mice was significantly improved compared with animals grafted with GFP-expressing cells [37,53]. Moreover, in 22 L-infected mice, survival time increase was higher after Ra2 microglia graft than after direct infusion of anti-PrP antibodies [54], suggesting that the effect could be attributed to the migration of microglia to regions further away from the injection site. Conversely, survival time was not changed in mice grafted during the clinical phase (week 13 post-infection), and in mice inoculated with the Chandler scrapie strain. The authors did not assess whether the different results obtained with the 22 L and Chandler scrapie strains were due to their different tropism, and thus different behavior following microglia injection, or to the fact that the Chandler strain www.sciencedirect.com
infectious titer was 10-fold higher than that of the 22 L strain.
Mesenchymal stem cells In prion disorders, MSC therapeutic potential (Figure 1) was first studied using immortalized human MSC (hMSC) that express the LacZ gene [55]. In this pilot study to assess hMSC migration in brain, hMSC were transplanted on one side of the hippocampus or thalamus of wt mice infected with the Obihiro or Chandler scrapie strains. In these mice, hMSC were still present at week 3 after transplantation, not only in the injection side but also in the contralateral side. Interestingly, PrPSc accumulation, astrocytosis and spongiosis were more severe in mice inoculated with the Obihiro strain, and hMSC migration was positively correlated with the lesion severity. In contrast, only few hMSC were detected in noninfected mice. This finding suggests that in infected mice, the grafted hMSC are maintained and are attracted to lesions in different brain areas. Conversely, in noninfected mice, these cells seem not to be necessary, and most of them are probably eliminated. In addition, the authors found that the secretion/expression level of various trophic factors (e.g. BDNF, NT3, VEGF, NGF, and CNTF) [55,56] by migrating hMSC was higher in prioninfected mice than in mock-infected mice. As MSC can differentiate into neural lineages in specific situations, the authors also asked whether the grafted hMSC had given rise to neurons and astrocytes at week 3 post-graft. They found that some LacZ-positive cells expressed GFAP (astrocyte marker) or MAP2 (neuronal marker), suggesting that grafted cells can integrate and differentiate into neural lineages. Finally, the mean survival rate of Chandler-infected mice grafted with hMSC at 90dpi was slightly but significantly increased (by 5.3%) compared with non-grafted mice. Another recent study evaluated the therapeutic effect of the autologous transplantation of BM-MSCs in prion mouse models [57]. Transplantation of MSCs isolated from the mouse femur and tibia in Chandler straininfected mice at the beginning of the clinical phase (120 dpi) slightly (>5%) but significantly prolonged the survival rate, and promoted microglial cell activation, as indicated the increased expression of the Aif1 gene that encodes the microglial IBA-1 protein. However, no effect on PrPSc accumulation was reported.
Conclusion Regenerative medicine is expected to have tremendous medical, scientific and social impacts. It might offer the possibility to treat and cure diseases that are currently without adequate treatment. In prion diseases, only few preclinical trials have evaluated regenerative medicine strategies, and this paucity of studies probably reflects the complexity of such diseases. Moreover, several key questions need to be addressed when studying regenerative Current Opinion in Pharmacology 2019, 44:8–14
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medicine strategies for prion diseases, such as the optimal cell source, the ethical constraints of using fetal or embryonic NSC, the best delivery system, and how to ensure the long-term safety and the donor cell response. In addition, it is clear that cell therapies alone may not be enough to repair the brain lesions, mainly because of the current impossibility of early diagnosis of prion diseases. Therefore, it should be important to develop multimodal approaches that combine pharmacological drugs, immunomodulation to decrease brain inflammation, stimulation of endogenous neurogenesis, and gene, cell, or multiple cell therapies [58]. For instance, the double graft of NSC and MSC in a rat model of Huntington’s disease led to long-term behavioral benefits and increased the survival of the transplanted NSC. This finding suggests that MSC can favor a more suitable environment for NSC survival [13,16,59,60]. Finally, to promote therapeutic innovation in cell-based therapy for prion diseases, future research needs to address the effect of the grafted cells on the surrounding tissues and to improve the quality and safety of the cells to be grafted.
Conflict of interest statement Nothing declared.
Acknowledgments We thank the CJD Foundation for the financial support (2009) of the adult neurogenesis study and the INSERM (Institution) for current funding.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Prusiner SB: Prions. Proc Natl Acad Sci U S A 1998, 95: 13363-13383.
2.
Parchi P, de Boni L, Saverioni D, Cohen ML, Ferrer I, Gambetti P, Gelpi E, Giaccone G, Hauw JJ, Hoftberger R, Ironside JW et al.: Consensus classification of human prion disease histotypes allows reliable identification of molecular subtypes: an interrater study among surveillance centres in europe and USA. Acta Neuropathol 2012, 124:517-529.
3.
Will RG, Ironside JW, Hornlimann B, Zeidler M: Creutzfeldt-jakob disease. Lancet 1996, 347:65-66.
4.
Das AS, Zou WQ: Prions: beyond a single protein. Clini Microbiol Rev 2016, 29:633-658.
5.
Song CG, Zhang YZ, Wu HN, Cao XL, Guo CJ, Li YQ, Zheng MH, Han H: Stem cells: a promising candidate to treat neurological disorders. Neural Regen Res 2018, 13:1294-1304.
6.
Slack JMW: What is a stem cell? Wiley Interdiscip Rev Dev Biol 2018:e323.
7.
Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR: Multilineage potential of adult human mesenchymal stem cells. Science 1999, 284:143-147.
8.
Sanchez-Ramos JR: Neural cells derived from adult bone marrow and umbilical cord blood. J Neurosci Res 2002, 69:880-893.
Current Opinion in Pharmacology 2019, 44:8–14
9.
Tasnim N, Thakur V, Chattopadhyay M, Joddar B: The efficacy of graphene foams for culturing mesenchymal stem cells and their differentiation into dopaminergic neurons. Stem Cells Int 2018, 2018 3410168.
10. Volkman R, Offen D: Concise review: mesenchymal stem cells in neurodegenerative diseases. Stem Cells 2017, 35:1867-1880. 11. Hass R, Kasper C, Bohm S, Jacobs R: Different populations and sources of human mesenchymal stem cells (msc): a comparison of adult and neonatal tissue-derived msc. Cell Commun Signal 2011, 9. 12. Chopp M, Li Y: Treatment of neural injury with marrow stromal cells. Lancet Neurol 2002, 1:92-100. 13. de Godoy MA, Saraiva LM, de Carvalho LRP, Vasconcelos-DosSantos A, Beiral HJV, Ramos AB, Silva LRP, Leal RB, Monteiro VHS, Braga CV, de Araujo-Silva CA et al.: Mesenchymal stem cells and cell-derived extracellular vesicles protect hippocampal neurons from oxidative stress and synapse damage induced by amyloid-beta oligomers. J Biol Chem 2018, 293:1957-1975. 14. Rodriguez AM, Nakhle J, Griessinger E, Vignais ML: Intercellular mitochondria trafficking highlighting the dual role of mesenchymal stem cells as both sensors and rescuers of tissue injury. Cell Cycle 2018, 17:712-721. 15. Hamano K, Li TS, Kobayashi T, Kobayashi S, Matsuzaki M, Esato K: Angiogenesis induced by the implantation of selfbone marrow cells: a new material for therapeutic angiogenesis. Cell Transplant 2000, 9:439-443. 16. Munoz JR, Stoutenger BR, Robinson AP, Spees JL, Prockop DJ: Human stem/progenitor cells from bone marrow promote neurogenesis of endogenous neural stem cells in the hippocampus of mice. Proc Natl Acad Sci U S A 2005, 102:18171-18176. 17. Alvarez-Dolado M, Pardal R, Garcia-Verdugo JM, Fike JR, Lee HO, Pfeffer K, Lois C, Morrison SJ, Alvarez-Buylla A: Fusion of bonemarrow-derived cells with purkinje neurons, cardiomyocytes and hepatocytes. Nature 2003, 425:968-973. 18. Aguzzi A, Klein MA, Musahl C, Raeber AJ, Blattler T, Hegyi I, Frigg R, Brandner S: Use of brain grafts to study the pathogenesis of prion diseases. Essays Biochem 1998, 33:133-147. 19. Kordower JH, Chu Y, Hauser RA, Freeman TB, Olanow CW: Lewy body-like pathology in long-term embryonic nigral transplants in Parkinson’s disease. Nat Med 2008, 14:504-506. 20. Chen B, Gao XQ, Yang CX, Tan SK, Sun ZL, Yan NH, Pang YG, Yuan M, Chen GJ, Xu GT, Zhang K et al.: Neuroprotective effect of grafting gdnf gene-modified neural stem cells on cerebral ischemia in rats. Brain Res 2009, 1284:1-11. 21. Alvarez-Buylla A, Garcia-Verdugo JM: Neurogenesis in adult subventricular zone. J Neurosci 2002, 22:629-634. 22. Boldrini M, Fulmore CA, Tartt AN, Simeon LR, Pavlova I, Poposka V, Rosoklija GB, Stankov A, Arango V, Dwork AJ, Hen R et al.: Human hippocampal neurogenesis persists throughout aging. Cell Stem Cell 2018, 22:589-599 e585. Adult neurogenesis in human hippocampus is currently debated. In this paper, the authors show that proliferating NSC and immature neuronal cells (young neurons) are present throughout human aging, while the number of non-dividing quiescent (NSC reservoir) stem cells decreases. 23. Arvidsson A, Collin T, Kirik D, Kokaia Z, Lindvall O: Neuronal replacement from endogenous precursors in the adult brain after stroke. Nat Med 2002, 8:963-970. 24. Jin K, Sun Y, Xie L, Peel A, Mao XO, Batteur S, Greenberg DA: Directed migration of neuronal precursors into the ischemic cerebral cortex and striatum. Mol Cell Neurosci 2003, 24:171-189. 25. Jin K, Peel AL, Mao XO, Xie L, Cottrell BA, Henshall DC, Greenberg DA: Increased hippocampal neurogenesis in Alzheimer’s disease. Proc Natl Acad Sci U S A 2004, 101: 343-347.
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26. Curtis MA, Penney EB, Pearson AG, van Roon-Mom WM, Butterworth NJ, Dragunow M, Connor B, Faull RL: Increased cell proliferation and neurogenesis in the adult human Huntington’s disease brain. Proc Natl Acad Sci U S A 2003, 100:9023-9027. 27. Lim DA, Huang YC, Alvarez-Buylla A: The adult neural stem cell niche: Lessons for future neural cell replacement strategies. Neurosurg Clin North Am 2007, 18:81-92 ix. 28. Steele AD, Emsley JG, Ozdinler PH, Lindquist S, Macklis JD: Prion protein (prpc) positively regulates neural precursor proliferation during developmental and adult mammalian neurogenesis. Proc Natl Acad Sci U S A 2006, 103:3416-3421. 29. Parrie LE, Crowell JAE, Telling GC, Bessen RA: The cellular prion protein promotes olfactory sensory neuron survival and axon targeting during adult neurogenesis. Dev Biol 2018, 438:23-32. 30. Roucou X, Gains M, LeBlanc AC: Neuroprotective functions of prion protein. J Neurosci Res 2004, 75:153-161. 31. Westergard L, Christensen HM, Harris DA: The cellular prion protein (prp(c)): Its physiological function and role in disease. Biochim Biophys Acta 2007, 1772:629-644. 32. Relano-Gines A, Gabelle A, Hamela C, Belondrade M, Casanova D, Mourton-Gilles C, Lehmann S, Crozet C: Prion replication occurs in endogenous adult neural stem cells and alters their neuronal fate: Involvement of endogenous neural stem cells in prion diseases. PLoS Pathog 2013, 9:e1003485. 33. Relano-Gines A, Lehmann S, Crozet C: Prion diseases and adult neurogenesis: How do prions counteract the brain’s endogenous repair machinery? Prion 2014, 8:240-246. 34. Na YJ, Jin JK, Lee YJ, Choi EK, Carp RI, Kim YS: Increased neurogenesis in brains of scrapie-infected mice. Neurosci Lett 2009, 449:66-70. 35. Gomez-Nicola D, Suzzi S, Vargas-Caballero M, Fransen NL, AlMalki H, Cebrian-Silla A, Garcia-Verdugo JM, Riecken K, Fehse B, Perry VH: Temporal dynamics of hippocampal neurogenesis in chronic neurodegeneration. Brain 2014, 137:2312-2328. 36. Jalland CM, Scheffler K, Benestad SL, Moldal T, Ersdal C, Gunnes G, Suganthan R, Bjoras M, Tranulis MA: Neil3 induced neurogenesis protects against prion disease during the clinical phase. Sci Rep 2016, 6 37844. The authors used Neil3-deficient mice that lack the major pathway for oxidative DNA damage repair and in which neurogenesis is compromised. Following prion infection, the clinical stage of prion disease was shorter in Neil3-deficient mice than in wild type controls, and this was associated with reduced astrogliosis in the hippocampus and striatum. The expression of specific NSC and young neuron markers also was decreased in infected Neil3-deficient mice. These data strongly support the hypothesis that NEIL3-mediated neurogenesis induction protects against prion disease during the clinical phase. 37. Fujita K, Yamaguchi Y, Mori T, Muramatsu N, Miyamoto T, Yano M, Miyata H, Ootsuyama A, Sawada M, Matsuda H, Kaji R et al.: Effects of a brain-engraftable microglial cell line expressing anti-prion scfv antibodies on survival times of mice infected with scrapie prions. Cell Mol Neurobiol 2011, 31:999-1008. 38. Toupet K, Compan V, Crozet C, Mourton-Gilles C, MestreFrances N, Ibos F, Corbeau P, Verdier JM, Perrier V: Effective gene therapy in a mouse model of prion diseases. PLoS One 2008, 3:e2773. 39. Crozet C, Lin YL, Mettling C, Mourton-Gilles C, Corbeau P, Lehmann S, Perrier V: Inhibition of prpsc formation by lentiviral gene transfer of prp containing dominant negative mutations. J Cell Sci 2004, 117:5591-5597. 40. White MD, Mallucci GR: Rnai for the treatment of prion disease: A window for intervention in neurodegeneration? CNS Neurol Disorders Drug Targets 2009, 8:342-352. 41. Lehmann S, Relano-Gines A, Resina S, Brillaud E, Casanova D, Vincent C, Hamela C, Poupeau S, Laffont M, Gabelle A, Delaby C et al.: Systemic delivery of sirna down regulates brain prion protein and ameliorates neuropathology in prion disorder. PLoS One 2014, 9:e88797. www.sciencedirect.com
42. Brown K, Brown J, Ritchie DL, Sales J, Fraser R: Fetal cell grafts provide long-term protection against scrapie induced neuronal loss. Neuroreport 2001, 12:77-82. 43. Bueler H, Aguzzi A, Sailer A, Greiner RA, Autenried P, Aguet M, Weissmann C: Mice devoid of prp are resistant to scrapie. Cell 1993, 73:1339-1347. 44. Wells GA, Hawkins SA, Austin AR, Ryder SJ, Done SH, Green RB, Dexter I, Dawson M, Kimberlin RH: Studies of the transmissibility of the agent of bovine spongiform encephalopathy to pigs. J Gen Virol 2003, 84:1021-1031. 45. Relano-Gines A, Lehmann S, Bencsik A, Herva ME, Torres JM, Crozet CA: Stem cell therapy extends incubation and survival time in prion-infected mice in a time window-dependant manner. J Infect Dis 2011, 204:1038-1045. 46. Frid K, Binyamin O, Fainstein N, Keller G, Ben-Hur T, Gabizon R: Autologous neural progenitor cell transplantation into newborn mice modeling for e200k genetic prion disease delays disease progression. Neurobiol Aging 2018, 65:192-200. The authors used a transgenic mouse model that harbors the equivalent of the human E200K mutation in the gene encoding PrP and responsible for genetic CJD to assess the effect of NPC therapy. During aging, these mice display chronic progressive neurodegeneration that is associated with PrPSc deposition. Grafting NPC derived from fetal brain of the same transgenic mice and of wild type mice delayed this neurological deterioration. Importantly, wild type PrP and mutant PrP could not be converted into PrPSc in NPC exposed to prions. 47. Herva ME, Relano-Gines A, Villa A, Torres JM: Prion infection of differentiated neurospheres. J Neurosci Methods 2010, 188:270-275. 48. Kreutzberg GW: Microglia: A sensor for pathological events in the cns. Trends Neurosci 1996, 19:312-318. 49. Aguzzi A, Zhu C: Microglia in prion diseases. J Clin Invest 2017, 127:3230-3239. 50. Zhu C, Herrmann US, Falsig J, Abakumova I, Nuvolone M, Schwarz P, Frauenknecht K, Rushing EJ, Aguzzi A: A neuroprotective role for microglia in prion diseases. J Exp Med 2016, 213:1047-1059. Using a cerebellar organotypic slice culture model and a mouse model in which PrP is overexpressed, the authors showed that ablation of microglial cells results in a drastic aggravation of prion neurotoxicity and in an increase of PrPSc deposits. They obtained similar results also in a transgenic mouse model in which microglia is deficient. 51. Williams A, Lucassen PJ, Ritchie D, Bruce M: Prp deposition, microglial activation, and neuronal apoptosis in murine scrapie. Exp Neurol 1997, 144:433-438. 52. Priller J, Prinz M, Heikenwalder M, Zeller N, Schwarz P, Heppner FL, Aguzzi A: Early and rapid engraftment of bone marrow-derived microglia in scrapie. J Neurosci 2006, 26:11753-11762. 53. Donofrio G, Heppner FL, Polymenidou M, Musahl C, Aguzzi A: Paracrine inhibition of prion propagation by anti-prp singlechain fv miniantibodies. J Virol 2005, 79:8330-8338. 54. Song CH, Furuoka H, Kim CL, Ogino M, Suzuki A, Hasebe R, Horiuchi M: Effect of intraventricular infusion of anti-prion protein monoclonal antibodies on disease progression in prion-infected mice. J Gen Virol 2008, 89:1533-1544. 55. Song CH, Honmou O, Ohsawa N, Nakamura K, Hamada H, Furuoka H, Hasebe R, Horiuchi M: Effect of transplantation of bone marrow-derived mesenchymal stem cells on mice infected with prions. J Virol 2009, 83:5918-5927. 56. Li Y, Chen J, Chen XG, Wang L, Gautam SC, Xu YX, Katakowski M, Zhang LJ, Lu M, Janakiraman N, Chopp M: Human marrow stromal cell therapy for stroke in rat: neurotrophins and functional recovery. Neurology 2002, 59:514-523. 57. Shan Z, Hirai Y, Nakayama M, Hayashi R, Yamasaki T, Hasebe R, Song CH, Horiuchi M: Therapeutic effect of autologous compact bone-derived mesenchymal stem cell transplantation on prion disease. J Gen Virol 2017, 98: 2615-2627. This article reports the first graft of autologous MSC isolated from femur and tibia in Chandler prion-infected mice, 120 days post-inoculation. This Current Opinion in Pharmacology 2019, 44:8–14
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led to an increase of the incubation period and of survival, albeit marginal. However, the accumulation of pathological PrPSc was not inhibited. 58. Relano-Gines A, Gabelle A, Lehmann S, Milhavet O, Crozet C: Gene and cell therapy for prion diseases. Infect Disord Drug Targets 2009, 9:58-68. 59. Teixeira FG, Carvalho MM, Neves-Carvalho A, Panchalingam KM, Behie LA, Pinto L, Sousa N, Salgado AJ: Secretome of mesenchymal progenitors from the umbilical cord acts as
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modulator of neural/glial proliferation and differentiation. Stem Cell Rev 2015, 11:288-297. 60. Rossignol J, Fink K, Davis K, Clerc S, Crane A, Matchynski J, Lowrance S, Bombard M, Dekorver N, Lescaudron L, Dunbar GL: Transplants of adult mesenchymal and neural stem cells provide neuroprotection and behavioral sparing in a transgenic rat model of Huntington’s disease. Stem Cells 2014, 32:500-509.
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ScienceDirect Cell-based therapy against prion diseases Aroa Relan˜o-Gine´s1,2, Sylvain Lehmann1,2,3 and Carole Crozet1 Despite multiple efforts to find treatments, prion diseases are still incurable. The currently available therapeutic strategies are mostly based on compounds to inhibit pathological PrP (PrPSc) accumulation, and cellular PrP (PrPC) conversion into PrPSc. However, they cannot reverse the pathological changes already present in the brain. Cell-based therapeutic strategies could promote the repair of the pre-existing brain damage. The few available data come mostly from preclinical studies using neural stem cells, bone marrow-derived microglia and mesenchymal stem cells, as cell sources. Moreover, the benefits of cell-based therapeutic strategies could be linked not only to the replacement of damaged cells, but also to the secretion of trophic factors by the grafted cells that might modulate inflammation, cell death, or endogenous neurogenesis. Addresses 1 Institute for Regenerative Medicine and Biotherapies (IRMB), Neural Stem Cell, MSC and Neurodegenerative Diseases - U1183 INSERM (Institut National de la Sante´ et de la Recherche Me´dicale), 80 rue Augustin Fliche, 34295 Montpellier, France 2 Universite´ de Montpellier, 163 rue Auguste Broussonet, 34090 Montpellier, France 3 Centre Hospitalo-Universitaire de Montpellier, 191 Av. du Doyen Gaston Giraud, 34295 Montpellier, France Corresponding author: Crozet, Carole ([email protected])
Current Opinion in Pharmacology 2019, 44:8–14 This review comes from a themed issue on Neurosciences – prion disease Edited by Roberto Chiesa and Ina Vorberg
https://doi.org/10.1016/j.coph.2018.11.001 1471-4892/ã 2019 Elsevier Ltd. All rights reserved.
Introduction Transmissible spongiform encephalopathies (TSE), also called prion diseases, are degenerative disorders of the central nervous system (CNS) leading to severe incapacity and death [1]. They affect both humans (mainly, Creutzfeldt-Jakob disease (CJD)) and animals (sheep scrapie, and bovine spongiform encephalopathies (BSE)). Human prion diseases exist in three main forms [2,3]: sporadic (80–90% of all CJD cases; 1.5 cases/million people/year), familial (5–15%, mutations in the PRNP gene encoding the cellular prion protein PrPC), and Current Opinion in Pharmacology 2019, 44:8–14
acquired (<5%, iatrogenic contamination during transplantation, blood transfusion, therapy with growth hormones, or dietary exposure to BSE prions in the case of the variant CJD in relatively young patients). TSE are characterized by a long incubation period, followed by the appearance of clinical symptoms leading to the fatal issue. TSE can be transmitted by intracerebral injection to primates and rodents that constitute very relevant experimental models. Neuronal vacuolization and loss, astrocyte proliferation and cerebral accumulation of pathological prion proteins (PrPSc) are typical TSE features. PrPSc results from the conformational conversion of the hostencoded cellular PrP protein (PrPC) that is mainly expressed in the CNS [1,4]. TSE are incurable diseases, mainly because of their late diagnosis when brain lesions are already severe, and the therapeutic window is limited. Moreover, the currently available therapeutic strategies only inhibit PrPSc accumulation and PrPC conversion into PrPSc, but cannot reverse the pre-existing brain lesions. Therefore, it is important to develop alternative strategies to promote the functional recovery of the damaged areas through cell replacement. Regenerative medicine has emerged as a promising option for treating neurologic diseases by using different cell types [5,6]: neural stem cells (NSC) derived from pluripotent stem cells, embryonic stem (ES) cells or induced pluripotent stem cells (iPSC); fetal neural stem cells (fNSC); neuronal precursor cells (NPC); mesenchymal stem cells (MSC); and even microglial cells. Stem cells (NSC and fNSC) are defined by their capacity to self-renew and to differentiate into neurons, astrocytes or oligodendrocytes. NPC are neuronal progenitors that are already more committed towards the neuronal lineage than NSC. They can be obtained from NSC in culture, and could be used in graft approaches. MSC are multipotent adult stem cells of mesodermal origin. They can differentiate into mesenchymal lineages [7], and also into non-mesodermal cell types in vitro, including neuronal and glial lineages [8– 10]. MSC and MSC-like cells can have different origins (bone marrow (BM), fat tissue, nasal mucosae, dental pulp . . . [11]). MSC, mostly derived from BM, have been extensively used for stem cell therapy approaches in several neurodegenerative diseases [10]. It is thought that they restore injured tissues through the secretion of various trophic factors [12], extracellular vesicle dispersion [13], and mitochondrial transfer [14]. They stimulate angiogenesis [15], the proliferation and differentiation of endogenous NSC [16], and integrate into tissues [17]. The great advantage of stem cells is that an almost unlimited amount of cells can be derived from one or few cells. However, the development of cell therapeutic approaches is still challenging. Moreover, outcome www.sciencedirect.com
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interpretation is complex because it must take into account not only the direct effect of cell replacement, but also the possible interactions between grafted cells and host tissue (e.g. secretion of trophic factors, cytokine, prion propagation in the grafted cells) [18,19]. For instance, it has been suggested that grafted NSC, which express glial-derived neurotrophic factor (GDNF), might upregulate brain-derived neurotrophic factor (BDNF) to support the survival of the existing neurons after ischemia [20]. They could also decrease the post-injury immune cell recruitment and pro-inflammatory cytokine expression in the brain. Moreover, endogenous NSC and adult neurogenesis also could be stimulated using stem cellbased therapeutic strategies. Indeed, it is now well admitted that in rodents, adult neurogenesis (i.e. the production of new neurons in the adult brain) occurs primarily in two areas: the dentate gyrus (DG) and the subventricular zone (SVZ) of the lateral ventricles (LV) [21]. Adult neurogenesis in human hippocampus is currently debated. However, a recent paper showed that proliferating NSC and immature neuronal cells are present throughout human aging, while the number of non-dividing quiescent stem cells, which are considered a NSC reservoir, decreases [22]. The equilibrium between endogenous NSC activity and quiescence is regulated by neurons located in remote brain areas that adapt neuron production to the physiological and pathological constraints. In rodents, adult neurogenesis can be increased by 30-fold after brain injury or during disease. This can be accompanied by migration of neural precursors towards the lesions [23,24]. In patients with Alzheimer’s or Huntington’s disease, the proliferation of neural precursors is stimulated and new neurons are generated [25,26]. These findings could help to better understand the disease and could be used to develop strategies to mobilize endogenous NSC to repair diseased or injured brain [22,27]. This review will discuss why adult NSC could be the basis for therapeutic interventions in prion disorders. It will also describe the available cell-based graft approaches for prion diseases.
Adult neural stem cells could be the basis for therapeutic interventions in prion diseases Adult neurogenesis has been poorly studied in prion diseases compared with other neurodegenerative diseases. In healthy mice, PrPC positively regulates endogenous neurogenesis in the SVZ and DG [28], and promotes survival and axon targeting of olfactory sensory neurons during adult neurogenesis [29,30]. In TSE, the situation might be more complex than in physiological conditions for two possible reasons: first, during neuronal differentiation, PrPC expression increases, and this PrPC could be converted into PrPSc, thus further promoting prion propagation in the brain, second, PrPC promotes www.sciencedirect.com
neural precursor proliferation during embryo and adult neurogenesis [28], and seems to be involved in neuronal survival [30]. Therefore, its conversion into PrPSc could inhibit neurogenesis and/or contribute to neurodegeneration [31]. Moreover, the self-healing capacity of adult brain is not enough to significantly improve the affected neuronal network because only a small proportion of new cells will integrate and survive. Immunohistochemistry analyses of brain tissues from prion-infected mice showed PrPSc accumulation within and in the vicinity of endogenous NSC in neurogenic areas (DG and lateral wall of the left LV) [32,33]. An increase of NSC proliferation was also reported in DG and/or LV of scrapie infected mice [34,35]. We also showed that NSC isolated from prioninfected mice accumulate and replicate PrPSc [32,33] and that these cells proliferate more rapidly and display more apoptosis (unpublished data). Moreover, neuron production from endogenous NSC is effective during the first stages of prion disease, but decreases during disease progression [35]. Indeed, adult neurogenesis seems to be first stimulated to produce new neurons to replace the lost/damaged cells. However, these newborn neurons cannot achieve neuronal maturation and most of them will die [32,33,35] (and unpublished data). Interestingly, analysis of prion propagation in mice lacking the NEIL3 enzyme (a DNA glycosidase involved in DNA repair) and in which adult neurogenesis is altered indicated that in these mice, the clinical phase is shortened, but not the survival time. This suggests that although adult neurogenesis cannot prevent the fatal outcome of prion diseases, it might delay the appearance of clinical signs [36]. Nevertheless, the precise mechanisms by which NEIL3 contributes to neuroprotection in prion diseases are not known. The mechanisms behind the impairment of adult neurogenesis in prion diseases are not known. Nevertheless, it could be interesting to determine whether inhibition of PrPSc propagation in NSC and the derived neurons might preserve the targeted cells, and possibly slow down disease progression. Moreover, inhibition of PrPSc accumulation/propagation could allow maintaining PrPC neuroprotective and pro-neurogenic functions. This could be achieved through gene engineering using viral vectors that express well characterized anti-prion molecules, such as anti-PrP single-chain variable fragment (scFv) antibodies, dominant negative PrP mutants, anti-PrP oligonucleotides for RNA interference [37–41]. It should be important to assess whether this type of approach might limit not only prion propagation/accumulation, but also maintain and preserve the neurogenesis niche to enable additional cell-based strategies.
Cell-graft-based therapeutic approaches Different cell types have been used for preclinical studies on cell-based therapies in prion infected mice: fNSC, NPC, MSC, and microglia. Current Opinion in Pharmacology 2019, 44:8–14
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Neural stem cells and neuronal precursor cells Despite their huge potential, no pre-clinical study in the prion field using NSC derived from pluripotent stem cells (ES or iPSC) has been published yet. The first study (Figure 1) on the use of NSC in prion diseases evaluated the effect of transplanting fNSC derived from prionresistant knockout (koPrP) mouse embryos in the hippocampus of asymptomatic scrapie-infected (C57Bl6/VM) wild-type (wt) mice (at 150 days post-inoculation, dpi) [42]. This was not enough to modify the incubation and survival times compared with control mice (only PBS) [42]. Interestingly, the number of functional neurons in the pyramidal cell layer was increased (by about 1.5 times) at day 21 post-graft and at the endpoint (250 dpi) compared with control mice [42]. Although small foci of transplanted cells were detected in the brain of grafted mice, it was not investigated whether these cells accumulated PrPSc and whether the higher neuron number resulted from the replacement of damaged cells, secretion of trophic factors that stimulate neuron survival, and/or reduction of inflammatory signaling. More recently, using fNSC from wild type mice that express wtPrP, from prion-resistant transgenic mice that express porcine PrP (poPrP), and from koPrP mice [43,44], we discovered that compared with PBS, NSC graft could significantly
prolong the incubation (by 20%) and survival times (by 13%) in wt mice infected with RML prions [45]. This effect occurred only when fNSC were grafted before the appearance of clinical signs, suggesting the existence of a specific time window for successful treatment. Moreover, we observed the positive effects on incubation and survival times with all fNSC types, independently of the background of the mice from which they were derived (wtPrP, koPrP, and poPrP). This was surprising because prions can infect and replicate only in wt NSC ex vivo, but not in poPrP NSC and koPrP NSC [43,44]. Because of technical constraints, we could not assess the presence of the grafted cells. Therefore, we could not determine whether prion replication occurred in the grafted fNSC. Importantly, the longer incubation time was correlated with a reduction of the number of astrocytes in areas close to the graft. This suggests that cell transplantation on its own might play a key role in the inflammatory process probably through secretion of trophic factors by fNSC. This hypothesis remains to be tested. Recently, Frid et al. described an autologous and heterologous fNPC-based therapy in transgenic mice (TgCJD mice) that mimic the human E200K PrP mutation (responsible for a genetic CJD) and show neurological deterioration during aging [46]. Newborn TgCJD mice
Figure 1
NSC koPrP-f NSC PoPrP-f SC wtPrP-fN
etal koPrP-f NSC
NPC TgCJD-f C P wtPrP-fN
Future directions
Stimulation of endogenous neurogenesis
Relaňo-Ginés et al., 2011
Frid et al., 2018
Increase of the number of neurons, integration and survival of the grafted cells
Increase of incubation and survival time Decrease of atroglosis when cells are grafted before the appearance of clinical signs
Delay in disease progression in TgCJD model of genetic CJD
l cell
hMSC
Brown, K.L et al., 2000
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Graft of cells expressing dominant negative PrP mutants, amtiPrP RNAi
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Fujita et al., 2011
Song et al., 2009
Slight increase of the survival period
Increase of the survival period in Chandler-infected mice Migration of hMSC towards brain lesions in mice infected with both strains
Shan et al., 2017 Increase of the survival period Migration and activation of microglia
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Pre-clinical studies using cell-based therapeutic approaches and future research directions.
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Cell therapy in prion diseases Relan˜o-Gine´s, Lehmann and Crozet 11
were grafted with fNPC from the brain of TgCJD or wt mouse embryos and were followed for 10 months. The graft of both fNPC types significantly delayed disease progression compared with non-transplanted mice. Similarly to our finding, both fNPC types (from TgCJD and wt mice) could prolong the disease incubation time (by 35%). Moreover, the number of endogenous NSC was increased in the brain of grafted animals, suggesting a proneurogenic effect of grafted cells [46]. The authors did [45,47] not assess PrPSc accumulation in the grafted cells. However, they showed the absence of PrPSc accumulation in endogenous NSC in TgCJD mice and the lack of prion transmission when TgCJD NPC were grafted in the brain of wt mice [46].
Microglial cells Microglial cells are considered the innate immune cells of the brain, and contribute to tissue homeostasis and synaptic plasticity regulation [48]. Their proliferation and activation are increased in pathological conditions, and they participate in the development and maintenance of the neuro-inflammatory response. Microglia can also trigger neurotoxic pathways, thus promoting neurodegeneration. They can also play a neuroprotective role, and seem necessary for controlling inflammatory, repair and regenerative processes [49]. On the basis of the dual role of microglia [50,51], several groups tried to better understand the involvement of these cells in disease development. For instance, to study microglia turnover in prion diseases, the RML prion strain was inoculated in the brain of mice that were transplanted with green fluorescent protein (GFP)-expressing bone marrow (BM) cells after irradiation [52]. Fifty percent of all brain resident microglia was replaced by GFP-positive bone marrow derivedmicroglial cells before the clinical manifestation of the disease. Although scrapie pathogenesis was not improved, prions did not replicate in GFP-positive microglial cells. Therefore, these cells could be used for the delivery of anti-prion molecules. This was tested by Fujita et al. [37] (Figure 1) by grafting microglial cells (murine Ra2 microglial cell line) that express anti-prion scFv antibodies in the brain of wt mice at week 7 (asymptomatic phase) after infection with the 22 L scrapie strain. The survival of these mice was significantly improved compared with animals grafted with GFP-expressing cells [37,53]. Moreover, in 22 L-infected mice, survival time increase was higher after Ra2 microglia graft than after direct infusion of anti-PrP antibodies [54], suggesting that the effect could be attributed to the migration of microglia to regions further away from the injection site. Conversely, survival time was not changed in mice grafted during the clinical phase (week 13 post-infection), and in mice inoculated with the Chandler scrapie strain. The authors did not assess whether the different results obtained with the 22 L and Chandler scrapie strains were due to their different tropism, and thus different behavior following microglia injection, or to the fact that the Chandler strain www.sciencedirect.com
infectious titer was 10-fold higher than that of the 22 L strain.
Mesenchymal stem cells In prion disorders, MSC therapeutic potential (Figure 1) was first studied using immortalized human MSC (hMSC) that express the LacZ gene [55]. In this pilot study to assess hMSC migration in brain, hMSC were transplanted on one side of the hippocampus or thalamus of wt mice infected with the Obihiro or Chandler scrapie strains. In these mice, hMSC were still present at week 3 after transplantation, not only in the injection side but also in the contralateral side. Interestingly, PrPSc accumulation, astrocytosis and spongiosis were more severe in mice inoculated with the Obihiro strain, and hMSC migration was positively correlated with the lesion severity. In contrast, only few hMSC were detected in noninfected mice. This finding suggests that in infected mice, the grafted hMSC are maintained and are attracted to lesions in different brain areas. Conversely, in noninfected mice, these cells seem not to be necessary, and most of them are probably eliminated. In addition, the authors found that the secretion/expression level of various trophic factors (e.g. BDNF, NT3, VEGF, NGF, and CNTF) [55,56] by migrating hMSC was higher in prioninfected mice than in mock-infected mice. As MSC can differentiate into neural lineages in specific situations, the authors also asked whether the grafted hMSC had given rise to neurons and astrocytes at week 3 post-graft. They found that some LacZ-positive cells expressed GFAP (astrocyte marker) or MAP2 (neuronal marker), suggesting that grafted cells can integrate and differentiate into neural lineages. Finally, the mean survival rate of Chandler-infected mice grafted with hMSC at 90dpi was slightly but significantly increased (by 5.3%) compared with non-grafted mice. Another recent study evaluated the therapeutic effect of the autologous transplantation of BM-MSCs in prion mouse models [57]. Transplantation of MSCs isolated from the mouse femur and tibia in Chandler straininfected mice at the beginning of the clinical phase (120 dpi) slightly (>5%) but significantly prolonged the survival rate, and promoted microglial cell activation, as indicated the increased expression of the Aif1 gene that encodes the microglial IBA-1 protein. However, no effect on PrPSc accumulation was reported.
Conclusion Regenerative medicine is expected to have tremendous medical, scientific and social impacts. It might offer the possibility to treat and cure diseases that are currently without adequate treatment. In prion diseases, only few preclinical trials have evaluated regenerative medicine strategies, and this paucity of studies probably reflects the complexity of such diseases. Moreover, several key questions need to be addressed when studying regenerative Current Opinion in Pharmacology 2019, 44:8–14
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medicine strategies for prion diseases, such as the optimal cell source, the ethical constraints of using fetal or embryonic NSC, the best delivery system, and how to ensure the long-term safety and the donor cell response. In addition, it is clear that cell therapies alone may not be enough to repair the brain lesions, mainly because of the current impossibility of early diagnosis of prion diseases. Therefore, it should be important to develop multimodal approaches that combine pharmacological drugs, immunomodulation to decrease brain inflammation, stimulation of endogenous neurogenesis, and gene, cell, or multiple cell therapies [58]. For instance, the double graft of NSC and MSC in a rat model of Huntington’s disease led to long-term behavioral benefits and increased the survival of the transplanted NSC. This finding suggests that MSC can favor a more suitable environment for NSC survival [13,16,59,60]. Finally, to promote therapeutic innovation in cell-based therapy for prion diseases, future research needs to address the effect of the grafted cells on the surrounding tissues and to improve the quality and safety of the cells to be grafted.
Conflict of interest statement Nothing declared.
Acknowledgments We thank the CJD Foundation for the financial support (2009) of the adult neurogenesis study and the INSERM (Institution) for current funding.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Prusiner SB: Prions. Proc Natl Acad Sci U S A 1998, 95: 13363-13383.
2.
Parchi P, de Boni L, Saverioni D, Cohen ML, Ferrer I, Gambetti P, Gelpi E, Giaccone G, Hauw JJ, Hoftberger R, Ironside JW et al.: Consensus classification of human prion disease histotypes allows reliable identification of molecular subtypes: an interrater study among surveillance centres in europe and USA. Acta Neuropathol 2012, 124:517-529.
3.
Will RG, Ironside JW, Hornlimann B, Zeidler M: Creutzfeldt-jakob disease. Lancet 1996, 347:65-66.
4.
Das AS, Zou WQ: Prions: beyond a single protein. Clini Microbiol Rev 2016, 29:633-658.
5.
Song CG, Zhang YZ, Wu HN, Cao XL, Guo CJ, Li YQ, Zheng MH, Han H: Stem cells: a promising candidate to treat neurological disorders. Neural Regen Res 2018, 13:1294-1304.
6.
Slack JMW: What is a stem cell? Wiley Interdiscip Rev Dev Biol 2018:e323.
7.
Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR: Multilineage potential of adult human mesenchymal stem cells. Science 1999, 284:143-147.
8.
Sanchez-Ramos JR: Neural cells derived from adult bone marrow and umbilical cord blood. J Neurosci Res 2002, 69:880-893.
Current Opinion in Pharmacology 2019, 44:8–14
9.
Tasnim N, Thakur V, Chattopadhyay M, Joddar B: The efficacy of graphene foams for culturing mesenchymal stem cells and their differentiation into dopaminergic neurons. Stem Cells Int 2018, 2018 3410168.
10. Volkman R, Offen D: Concise review: mesenchymal stem cells in neurodegenerative diseases. Stem Cells 2017, 35:1867-1880. 11. Hass R, Kasper C, Bohm S, Jacobs R: Different populations and sources of human mesenchymal stem cells (msc): a comparison of adult and neonatal tissue-derived msc. Cell Commun Signal 2011, 9. 12. Chopp M, Li Y: Treatment of neural injury with marrow stromal cells. Lancet Neurol 2002, 1:92-100. 13. de Godoy MA, Saraiva LM, de Carvalho LRP, Vasconcelos-DosSantos A, Beiral HJV, Ramos AB, Silva LRP, Leal RB, Monteiro VHS, Braga CV, de Araujo-Silva CA et al.: Mesenchymal stem cells and cell-derived extracellular vesicles protect hippocampal neurons from oxidative stress and synapse damage induced by amyloid-beta oligomers. J Biol Chem 2018, 293:1957-1975. 14. Rodriguez AM, Nakhle J, Griessinger E, Vignais ML: Intercellular mitochondria trafficking highlighting the dual role of mesenchymal stem cells as both sensors and rescuers of tissue injury. Cell Cycle 2018, 17:712-721. 15. Hamano K, Li TS, Kobayashi T, Kobayashi S, Matsuzaki M, Esato K: Angiogenesis induced by the implantation of selfbone marrow cells: a new material for therapeutic angiogenesis. Cell Transplant 2000, 9:439-443. 16. Munoz JR, Stoutenger BR, Robinson AP, Spees JL, Prockop DJ: Human stem/progenitor cells from bone marrow promote neurogenesis of endogenous neural stem cells in the hippocampus of mice. Proc Natl Acad Sci U S A 2005, 102:18171-18176. 17. Alvarez-Dolado M, Pardal R, Garcia-Verdugo JM, Fike JR, Lee HO, Pfeffer K, Lois C, Morrison SJ, Alvarez-Buylla A: Fusion of bonemarrow-derived cells with purkinje neurons, cardiomyocytes and hepatocytes. Nature 2003, 425:968-973. 18. Aguzzi A, Klein MA, Musahl C, Raeber AJ, Blattler T, Hegyi I, Frigg R, Brandner S: Use of brain grafts to study the pathogenesis of prion diseases. Essays Biochem 1998, 33:133-147. 19. Kordower JH, Chu Y, Hauser RA, Freeman TB, Olanow CW: Lewy body-like pathology in long-term embryonic nigral transplants in Parkinson’s disease. Nat Med 2008, 14:504-506. 20. Chen B, Gao XQ, Yang CX, Tan SK, Sun ZL, Yan NH, Pang YG, Yuan M, Chen GJ, Xu GT, Zhang K et al.: Neuroprotective effect of grafting gdnf gene-modified neural stem cells on cerebral ischemia in rats. Brain Res 2009, 1284:1-11. 21. Alvarez-Buylla A, Garcia-Verdugo JM: Neurogenesis in adult subventricular zone. J Neurosci 2002, 22:629-634. 22. Boldrini M, Fulmore CA, Tartt AN, Simeon LR, Pavlova I, Poposka V, Rosoklija GB, Stankov A, Arango V, Dwork AJ, Hen R et al.: Human hippocampal neurogenesis persists throughout aging. Cell Stem Cell 2018, 22:589-599 e585. Adult neurogenesis in human hippocampus is currently debated. In this paper, the authors show that proliferating NSC and immature neuronal cells (young neurons) are present throughout human aging, while the number of non-dividing quiescent (NSC reservoir) stem cells decreases. 23. Arvidsson A, Collin T, Kirik D, Kokaia Z, Lindvall O: Neuronal replacement from endogenous precursors in the adult brain after stroke. Nat Med 2002, 8:963-970. 24. Jin K, Sun Y, Xie L, Peel A, Mao XO, Batteur S, Greenberg DA: Directed migration of neuronal precursors into the ischemic cerebral cortex and striatum. Mol Cell Neurosci 2003, 24:171-189. 25. Jin K, Peel AL, Mao XO, Xie L, Cottrell BA, Henshall DC, Greenberg DA: Increased hippocampal neurogenesis in Alzheimer’s disease. Proc Natl Acad Sci U S A 2004, 101: 343-347.
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Cell therapy in prion diseases Relan˜o-Gine´s, Lehmann and Crozet 13
26. Curtis MA, Penney EB, Pearson AG, van Roon-Mom WM, Butterworth NJ, Dragunow M, Connor B, Faull RL: Increased cell proliferation and neurogenesis in the adult human Huntington’s disease brain. Proc Natl Acad Sci U S A 2003, 100:9023-9027. 27. Lim DA, Huang YC, Alvarez-Buylla A: The adult neural stem cell niche: Lessons for future neural cell replacement strategies. Neurosurg Clin North Am 2007, 18:81-92 ix. 28. Steele AD, Emsley JG, Ozdinler PH, Lindquist S, Macklis JD: Prion protein (prpc) positively regulates neural precursor proliferation during developmental and adult mammalian neurogenesis. Proc Natl Acad Sci U S A 2006, 103:3416-3421. 29. Parrie LE, Crowell JAE, Telling GC, Bessen RA: The cellular prion protein promotes olfactory sensory neuron survival and axon targeting during adult neurogenesis. Dev Biol 2018, 438:23-32. 30. Roucou X, Gains M, LeBlanc AC: Neuroprotective functions of prion protein. J Neurosci Res 2004, 75:153-161. 31. Westergard L, Christensen HM, Harris DA: The cellular prion protein (prp(c)): Its physiological function and role in disease. Biochim Biophys Acta 2007, 1772:629-644. 32. Relano-Gines A, Gabelle A, Hamela C, Belondrade M, Casanova D, Mourton-Gilles C, Lehmann S, Crozet C: Prion replication occurs in endogenous adult neural stem cells and alters their neuronal fate: Involvement of endogenous neural stem cells in prion diseases. PLoS Pathog 2013, 9:e1003485. 33. Relano-Gines A, Lehmann S, Crozet C: Prion diseases and adult neurogenesis: How do prions counteract the brain’s endogenous repair machinery? Prion 2014, 8:240-246. 34. Na YJ, Jin JK, Lee YJ, Choi EK, Carp RI, Kim YS: Increased neurogenesis in brains of scrapie-infected mice. Neurosci Lett 2009, 449:66-70. 35. Gomez-Nicola D, Suzzi S, Vargas-Caballero M, Fransen NL, AlMalki H, Cebrian-Silla A, Garcia-Verdugo JM, Riecken K, Fehse B, Perry VH: Temporal dynamics of hippocampal neurogenesis in chronic neurodegeneration. Brain 2014, 137:2312-2328. 36. Jalland CM, Scheffler K, Benestad SL, Moldal T, Ersdal C, Gunnes G, Suganthan R, Bjoras M, Tranulis MA: Neil3 induced neurogenesis protects against prion disease during the clinical phase. Sci Rep 2016, 6 37844. The authors used Neil3-deficient mice that lack the major pathway for oxidative DNA damage repair and in which neurogenesis is compromised. Following prion infection, the clinical stage of prion disease was shorter in Neil3-deficient mice than in wild type controls, and this was associated with reduced astrogliosis in the hippocampus and striatum. The expression of specific NSC and young neuron markers also was decreased in infected Neil3-deficient mice. These data strongly support the hypothesis that NEIL3-mediated neurogenesis induction protects against prion disease during the clinical phase. 37. Fujita K, Yamaguchi Y, Mori T, Muramatsu N, Miyamoto T, Yano M, Miyata H, Ootsuyama A, Sawada M, Matsuda H, Kaji R et al.: Effects of a brain-engraftable microglial cell line expressing anti-prion scfv antibodies on survival times of mice infected with scrapie prions. Cell Mol Neurobiol 2011, 31:999-1008. 38. Toupet K, Compan V, Crozet C, Mourton-Gilles C, MestreFrances N, Ibos F, Corbeau P, Verdier JM, Perrier V: Effective gene therapy in a mouse model of prion diseases. PLoS One 2008, 3:e2773. 39. Crozet C, Lin YL, Mettling C, Mourton-Gilles C, Corbeau P, Lehmann S, Perrier V: Inhibition of prpsc formation by lentiviral gene transfer of prp containing dominant negative mutations. J Cell Sci 2004, 117:5591-5597. 40. White MD, Mallucci GR: Rnai for the treatment of prion disease: A window for intervention in neurodegeneration? CNS Neurol Disorders Drug Targets 2009, 8:342-352. 41. Lehmann S, Relano-Gines A, Resina S, Brillaud E, Casanova D, Vincent C, Hamela C, Poupeau S, Laffont M, Gabelle A, Delaby C et al.: Systemic delivery of sirna down regulates brain prion protein and ameliorates neuropathology in prion disorder. PLoS One 2014, 9:e88797. www.sciencedirect.com
42. Brown K, Brown J, Ritchie DL, Sales J, Fraser R: Fetal cell grafts provide long-term protection against scrapie induced neuronal loss. Neuroreport 2001, 12:77-82. 43. Bueler H, Aguzzi A, Sailer A, Greiner RA, Autenried P, Aguet M, Weissmann C: Mice devoid of prp are resistant to scrapie. Cell 1993, 73:1339-1347. 44. Wells GA, Hawkins SA, Austin AR, Ryder SJ, Done SH, Green RB, Dexter I, Dawson M, Kimberlin RH: Studies of the transmissibility of the agent of bovine spongiform encephalopathy to pigs. J Gen Virol 2003, 84:1021-1031. 45. Relano-Gines A, Lehmann S, Bencsik A, Herva ME, Torres JM, Crozet CA: Stem cell therapy extends incubation and survival time in prion-infected mice in a time window-dependant manner. J Infect Dis 2011, 204:1038-1045. 46. Frid K, Binyamin O, Fainstein N, Keller G, Ben-Hur T, Gabizon R: Autologous neural progenitor cell transplantation into newborn mice modeling for e200k genetic prion disease delays disease progression. Neurobiol Aging 2018, 65:192-200. The authors used a transgenic mouse model that harbors the equivalent of the human E200K mutation in the gene encoding PrP and responsible for genetic CJD to assess the effect of NPC therapy. During aging, these mice display chronic progressive neurodegeneration that is associated with PrPSc deposition. Grafting NPC derived from fetal brain of the same transgenic mice and of wild type mice delayed this neurological deterioration. Importantly, wild type PrP and mutant PrP could not be converted into PrPSc in NPC exposed to prions. 47. Herva ME, Relano-Gines A, Villa A, Torres JM: Prion infection of differentiated neurospheres. J Neurosci Methods 2010, 188:270-275. 48. Kreutzberg GW: Microglia: A sensor for pathological events in the cns. Trends Neurosci 1996, 19:312-318. 49. Aguzzi A, Zhu C: Microglia in prion diseases. J Clin Invest 2017, 127:3230-3239. 50. Zhu C, Herrmann US, Falsig J, Abakumova I, Nuvolone M, Schwarz P, Frauenknecht K, Rushing EJ, Aguzzi A: A neuroprotective role for microglia in prion diseases. J Exp Med 2016, 213:1047-1059. Using a cerebellar organotypic slice culture model and a mouse model in which PrP is overexpressed, the authors showed that ablation of microglial cells results in a drastic aggravation of prion neurotoxicity and in an increase of PrPSc deposits. They obtained similar results also in a transgenic mouse model in which microglia is deficient. 51. Williams A, Lucassen PJ, Ritchie D, Bruce M: Prp deposition, microglial activation, and neuronal apoptosis in murine scrapie. Exp Neurol 1997, 144:433-438. 52. Priller J, Prinz M, Heikenwalder M, Zeller N, Schwarz P, Heppner FL, Aguzzi A: Early and rapid engraftment of bone marrow-derived microglia in scrapie. J Neurosci 2006, 26:11753-11762. 53. Donofrio G, Heppner FL, Polymenidou M, Musahl C, Aguzzi A: Paracrine inhibition of prion propagation by anti-prp singlechain fv miniantibodies. J Virol 2005, 79:8330-8338. 54. Song CH, Furuoka H, Kim CL, Ogino M, Suzuki A, Hasebe R, Horiuchi M: Effect of intraventricular infusion of anti-prion protein monoclonal antibodies on disease progression in prion-infected mice. J Gen Virol 2008, 89:1533-1544. 55. Song CH, Honmou O, Ohsawa N, Nakamura K, Hamada H, Furuoka H, Hasebe R, Horiuchi M: Effect of transplantation of bone marrow-derived mesenchymal stem cells on mice infected with prions. J Virol 2009, 83:5918-5927. 56. Li Y, Chen J, Chen XG, Wang L, Gautam SC, Xu YX, Katakowski M, Zhang LJ, Lu M, Janakiraman N, Chopp M: Human marrow stromal cell therapy for stroke in rat: neurotrophins and functional recovery. Neurology 2002, 59:514-523. 57. Shan Z, Hirai Y, Nakayama M, Hayashi R, Yamasaki T, Hasebe R, Song CH, Horiuchi M: Therapeutic effect of autologous compact bone-derived mesenchymal stem cell transplantation on prion disease. J Gen Virol 2017, 98: 2615-2627. This article reports the first graft of autologous MSC isolated from femur and tibia in Chandler prion-infected mice, 120 days post-inoculation. This Current Opinion in Pharmacology 2019, 44:8–14
14 Neurosciences – prion disease
led to an increase of the incubation period and of survival, albeit marginal. However, the accumulation of pathological PrPSc was not inhibited. 58. Relano-Gines A, Gabelle A, Lehmann S, Milhavet O, Crozet C: Gene and cell therapy for prion diseases. Infect Disord Drug Targets 2009, 9:58-68. 59. Teixeira FG, Carvalho MM, Neves-Carvalho A, Panchalingam KM, Behie LA, Pinto L, Sousa N, Salgado AJ: Secretome of mesenchymal progenitors from the umbilical cord acts as
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modulator of neural/glial proliferation and differentiation. Stem Cell Rev 2015, 11:288-297. 60. Rossignol J, Fink K, Davis K, Clerc S, Crane A, Matchynski J, Lowrance S, Bombard M, Dekorver N, Lescaudron L, Dunbar GL: Transplants of adult mesenchymal and neural stem cells provide neuroprotection and behavioral sparing in a transgenic rat model of Huntington’s disease. Stem Cells 2014, 32:500-509.
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