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京都大学 / 新制・課程博士 / 博士(医学) / 甲第25006号 / 医博第5040号 / 新制||医||1070(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 高橋 淳, 教授 荒川 芳輝, 教授 林 康紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Les précurseurs neuraux dérivés de cellules souches pluripotentes induites (iPS-NPCs) peuvent représenter la source cellulaire autologue idéale pour la thérapie cellulaire visant à promouvoir la remyélinisation et la neuroprotection des maladies de la myéline. Jusqu'à présent, le potentiel thérapeutique de ces cellules a été abordé dans des conditions néonatales. Cependant, l'efficacité de la réparation et de la sécurité de ces cellules dans le système nerveux central (SNC), une condition associée à une diminution de la plasticité cellulaire et effarouchement, reste à être bien traités. D'ailleurs, il reste à démontrer si le comportement de ces cellules ressemble à celle des NPCs du SNC. D'abord, j'ai comparé des iPS-NPCs de souris avec des cellules embryonnaires du SNC, in vitro et après greffe dans des modèles de démyélinisation de la moelle épinière de souris adulte. Nos données ont révélé la capacité de survie, intégration, migration et différenciation rapide des cellules greffées en oligodendrocytes matures. Les cellules greffées ont généré de la myéline compacte autour des axones, la restauration de n¿uds de Ranvier et la vitesse de conduction aussi efficacement que les précurseurs du SNC dérivés tandis supplantant cellules endogènes. Ensuite, pour valider la fonctionnalité des précurseurs gliaux humains dérivés des iPS-NPC, je les ai transplantés dans des modèles nouveau-nés et adultes de dys/démyélinisation. Mes données ont montré la migration généralisée, l'intégration et génération de oligodendrocytes fonctionnels, la formation de la myéline compacte tout en reconstruisant n¿uds de Ranvier dans chez les nouveau-nés et les adultes greffés. / Induced pluripotent stem cell-derived neural precursor cells (iPS-NPCs) may represent the ideal autologous cell source for cell-based therapy to promote remyelination and neuroprotection in myelin diseases and can serve as suitable tools to model myelin disorders or to test the potential of pharmacological compounds. So far the therapeutic potential of these cells was approached in neonatal conditions. However, the repair efficacy and safety of these cells in the demyelinated adult central nervous system (CNS), a condition associated with decreased cell plasticity and scaring, remains to be well addressed. Moreover, whether the therapeutic behavior of these pluripotent-derived cells resembles that of physiologically committed CNS-derived precursors remains elusive. First, I used mouse iPS-NPCs and compared them side-by-side to embryonic CNS-derived cells, in vitro and in vivo after engraftment in models of adult spinal cord demyelination. My data revealed the prominent capacity of survival, safe integration, migration and timely differentiation of the grafted cells into mature oligodendrocytes. Grafted cells generated compact myelin around host axons, restoring nodes of Ranvier and conduction velocity as efficiently as CNS-derived precursors while outcompeting endogenous cells. Second, to validate the functionality of human iPS-NPC-derived glial precursors, I transplanted them in newborn and adult models of dys/demyelination. My data showed widespread migration, integration and extensive generation of functional oligodendrocytes ensheathing host axons, forming compact myelin while reconstructing nodes of Ranvier both in newborn grafted and adult demyelination contexts.

Au cours des 10 dernières années, le laboratoire de JP Hugnot s’est concentré sur les différents pools de progéniteurs et de cellules souches trouvés dans la moelle épinière adulte, chez l’homme comme chez la souris. Ceci est important pour mener ce type de recherche car la moelle épinière est affectée par plusieurs maladies neurodégénératives telles que la sclérose latérale amyotrophique (SLA) et des lésions traumatiques pour lesquelles il n'existe pas de traitement curatif. Chez des animaux comme le poisson zèbre, la moelle épinière peut se régénérer après une lésion en raison de l'activation de progéniteurs / cellules souches endogènes. Ainsi, en recherchant la présence et les propriétés de telles cellules chez les mammifères, en particulier les humains, on pourrait exploiter ces cellules pour la régénération, y compris les neurones. Nous avons procédé au profilage de l'ARN pour comparer la niche de cellules souches humaine et de souris et la niche de cellules souches de souris de la moelle épinière lésée ou non lésée. Cette niche est particulièrement intéressante dans la mesure où, chez les anamniotes, les cellules de l'épendymoglie radiale situées dans cette région sont multipotentes et peuvent générer de nouveaux motoneurones après une lésion. et des cellules similaires, mais non identiques, sont présentes chez la souris. Chez les mammifères, après la lésion, ces cellules de niche prolifèrent et migrent activement pour générer principalement des cellules astrocytaires et peu d'oligodendrocytes qui participent à la cicatrice gliale et à la régénération en fournissant un facteur neurotrophique tel que le CNTF, le HGF et l'IGF-1. Cette niche contient au moins 5 types de cellules et un nouveau type de cellules dorsales exprimant les facteurs de transcription Msx1 et Id4 a été identifié. Ces résultats indiquent que la niche de la moelle épinière adulte chez la Souris et chez l'homme est une mosaïque de cellules ayant différentes origines développementales et conservant des niveaux élevés de gènes de développement neural. Les interactions gliales-neuronales qui soutiennent et maintiennent les neurones intacts peuvent influer sur les maladies neurodégénératives. L'une de ces cellules gliales est l'oligodendrocyte satellite ou cellules satellites périneuronales (PNC). Les PNC sont étroitement associés au soma de gros neurones et largement répandus dans la substance grise du cortex et de la moelle épinière. Cependant, les propriétés cellulaires et les rôles fonctionnels de ces oligodendrocytes non myélinisants n'ont pas encore été découverts. Dans cette étude, les cellules positives à la nestine-GFP sont associées à des neurones immunocolorés pour l'antigène nucléaire neuronal dans le cortex et la moelle épinière. Nous avons identifié les PNC comme étant des cellules positives pour la CNPase qui ne sont ni des cellules progénitrices d'oligodendrocytes (PDGFRa) ni des oligodendrocytes myélinisants (MBP). Ces données suggèrent que les PNC pourraient affecter la survie neuronale ainsi que le processus de myélinisation dans des conditions de démyélinisation. En outre, il pourrait être impliqué dans des maladies neurodégénératives telles que la sclérose en plaques et la sclérose latérale amyotrophique en raison de leur interaction avec les motoneurones. / Over the last 10 years, JP Hugnot’s lab has been focusing on the different pools of progenitors and stem cells found in the adult spinal cord both in human and mouse. This is important to conduct this kind of research as the spinal cord is affected by several neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and traumatic lesions for which there is no cure. In anamniotes such as Zebrafish, the spinal cord can regenerate after lesion due to endogenous progenitors/stem cells activation. So by investigating the presence and properties of such cells in mammals especially human, one could possibly harness those cells toward regeneration including neurons. We conducted RNA profiling to compare human vs mouse stem cell niche and lesioned vs non lesioned spinal cord mouse stem cell niche. This niche is particularly interesting as in anamniotes, radial ependymoglia cells located in this region are multipotent and can generate new motoneurons after lesion. And similar, albeit non identical, cells are present in mouse. In mammals, after lesion, these niche cells actively proliferate and migrate to generate mainly astrocytic cells and few oligodendrocytes which participate to the glial scar and regeneration by providing neurotrophic factor such as CNTF, HGF, and IGF-1. This niche contains at least 5 cell types and here a new dorsal cell type expressing Msx1 and Id4 transcription factors was identified. These results indicated that the adult spinal cord niche in mouse and human is a mosaic of cells with different developmental origin and maintaining high levels of neural developmental genes. Glial-neuronal interactions supporting and keeping neurons intact can be influence neurodegenerative diseases. One of these glial cells is the satellite oligodendrocyte or so called perineuronal satellite cells (PNCs). PNCs are tightly associated to the soma of large neurons and widely spread in the grey matter of the CNS both cortex and spinal cord. However the cellular properties and functional roles of these unmyelinating oligodendrocytes are not yet discovered. In this study, nestin-GFP positive cells are associated to neurons immunostained for neuronal nuclear antigen in both cortex and spinal cord. We identified PNCs as CNPase positive cells that are neither oligodendrocyte progenitor cells (PDGFRa) nor myelinating oligodendrocytes (MBP). These data suggest that PNCs might affect neuronal survival as well as the myelination process in demyelinating conditions. Also it could be implicated in neurodegenerative diseases such as multiple sclerosis and amyotrophic lateral sclerosis due to their interaction with motor neurons.

La production physiologique continue de cellules myélinisantes dans le système nerveux (SN) de mammifère offre de nouvelles perspectives thérapeutiques. Lors d’une atteinte de la myéline, une régénération endogène impliquant la génération d’oligodendrocytes s’engage. Ce processus repose sur la mobilisation de progéniteurs oligodendrocytaires parenchymateux et de progéniteurs de la zone sous-ventriculaire (SVZ). Cette réparation ne permet cependant pas une récupération fonctionnelle systématique. Nos travaux ont pour but d’identifier les facteurs qui contrôlent les différentes étapes de régénération. Ils révèlent une réexpression du CNTF et une surexpression des héparans sulfates protéoglycanes (HSPGs) suite à une démyélinisation du corps calleux. Ces changements de l’environnement péri-lésionnel régulent positivement le processus de remyélinisation. Nous avons montré un impact direct de l’expression post-lésionnelle du CNTF sur la mobilisation des deux sources cellulaires. Différents tests in vitro ont identifié le CNTF comme facteur chémoattractant pour ces cellules. Nos données montrent également que des modifications de sulfatation des héparans sulfates (HS) protéoglycanes contrôlées par la N-désacétylase-Sulfotransférase 1 des cellules du lignage oligodendrocytaire s’établissent en bordure de lésion et créent un microenvironnement favorable à la régénération. Divers test fonctionnels in vivo et in vitro révèlent le rôle clef des HSPGs dans la cinétique de démyélinisation et de remyélinisation en régulant la mobilisation des cellules du lignage oligodendrocytaire et l’activation microgliale. / In the mammal’s nervous system, the ongoing production of new myelinating cells on has open news therapeutic perspectives for demyelinating diseases. An endogenous regeneration process involving the generation of oligodendrocytes can occur following demyelination. This process relies on the mobilization of an endogenous reservoir of progenitor cells located in the adult brain: The parenchymal oligodendrocyte precursors and the subventricular zone derived neural progenitors. However, these endogenous repair attempts do not permit an efficient functional recovery. These failures are mainly due to mobilization, differentiation or to the generation of a hostile environment for the repair process. Our work is focusing on the identification of factors regulating those events. Our data show the reexpression of CNTF and overexpression of heparan sulphate proteoglycans (HSPGs) following a focal demyelination of the corpus callosum in adult mice. These environmental changes favor myelin repair. We show a direct impact of the post-lesional expression of CNTF on the mobilization of both cellular sources. Using various in vitro assays, we showed that CNTF is acting on the two cellular sources as a chemoattractant factor. Our data also show that sulfation modifications of HSPGs performed by the deacetylase-N-sulfotransferase 1 (Ndst1) on oligodendrocyte lineage cells occurred around the lesion and created a permissive microenvironment for the regenerative process. Various in vitro and in vivo functional assays demonstrated the key role of HSPGs in demyelination and remyelination dynamic by controlling mobilization of the oligodendrocyte lineage cells and microglial activation.

The structural integrity of the white matter is required for neuronal communication within the brain which is essential for normal cognitive function. Post-mortem and clinical imaging studies of elderly individuals have demonstrated that white matter integrity is weakened with increasing age which is proposed to underlie age-related cognitive decline. Whilst the exact mechanisms are unknown it is thought that modest age-related reductions in cerebral blood flow, termed chronic cerebral hypoperfusion, may contribute to white matter disruption and impaired cognition with ageing. Investigating the effects of white matter integrity in humans is limited as it is difficult to definitively ascertain a cause and effect relationship. Indeed, elderly individuals with cerebral hypoperfusion often have co-existing disease such as hypertension thus the effects of hypoperfusion in isolation cannot be determined. This has led to the development of a mouse model of chronic cerebral hypoperfusion which provides the opportunity to directly assess whether cerebral hypoperfusion results in disruption to white matter and cognitive impairment. This is achieved by applying small wire coils around both common carotid arteries of the mouse resulting in a global reduction in cerebral blood flow. Importantly the extent of blood flow reduction is dependent on the internal diameter of the coils meaning that differing severities of hypoperfusion can be studied. Previous studies using this model have demonstrated diffuse white matter pathology in white matter tracts including the corpus callosum, internal capsule and optic tract following 1 month of hypoperfusion which is accompanied by impaired spatial working memory. This thesis sought to test the hypothesis that chronic cerebral hypoperfusion would influence the structural integrity of nodal and paranodal domains of myelinated axons of the white matter and result in decreased numbers of oligodendroglial cells. It was additionally hypothesised that treatment with the anti-inflammatory and antioxidant drug dimethyl fumarate (DMF) would ameliorate structural and functional alterations to white matter following hypoperfusion. Aim 1 – To determine the impact of chronic cerebral hypoperfusion on the structural integrity of nodal and paranodal domains of myelinated axons The first aim of this thesis was to investigate the effects of chronic cerebral hypoperfusion on the structural integrity of nodal and paranodal domains of myelinated axons. This was addressed by examining key myelin and axonal proteins found at nodal, paranodal and internodal domains. This revealed significant alterations to the distribution of voltage-gated sodium (Nav1.6) channels at nodes of Ranvier which were differentially altered in response to increasing durations of chronic cerebral hypoperfusion. Specifically an increase in the Nav1.6+ domain length was observed in the corpus callosum following 3 days (p < 0.0001) and 1 month (p < 0.001) of chronic cerebral hypoperfusion but was not significantly different from sham controls following 6 weeks of hypoperfusion (p = 0.066). A significant decrease in Nav1.6 domain length was observed following 3 months of hypoperfusion (p = 0.003). Assessment of paranodal integrity was carried out by measuring nodal gap length and by ultrastructural analysis of paranodal domains. This revealed pronounced alterations to nodal gap length, loss of paranodal septate-like junctions and abnormal morphology of paranodal loops. Furthermore this study revealed a significant loss of myelin associated glycoprotein, a key protein involved in the maintenance of axon-glial integrity, as early as 3 days following the onset of hypoperfusion. A further aim of this study was to examine potential mechanisms underlying the observed alterations to nodal and paranodal domains following cerebral hypoperfusion. It was hypothesised that increased inflammation and accumulation of mitochondria at nodes of Ranvier would be observed following hypoperfusion. The extent of inflammation was assessed by counting numbers of microglia which revealed no significant difference between groups following 3 days of hypoperfusion (p = 0.425) but a significant increase in microglial number was observed following 1 month of hypoperfusion (p = 0.001). In addition, assessment of mitochondrial distribution along myelinated axons revealed decreased numbers of nodes containing mitochondria following 6 weeks of hypoperfusion (p = 0.03) with no difference between groups observed following 3 months (p = 0.742). Taken together the results from this study provide evidence that chronic cerebral hypoperfusion results in dynamic alterations in the localisation of Nav1.6 channels which are accompanied by disruption to paranodal domains and impaired axon-glial integrity. Furthermore microglial number does not appear to mediate nodal and paranodal disruption following 3 days but may contribute to ongoing pathology following 1 month of chronic cerebral hypoperfusion. Aim 2 – To determine the effects of chronic cerebral hypoperfusion on oligodendroglial populations. The second aim of this thesis was to determine the effect of chronic cerebral hypoperfusion on numbers of mature oligodendrocytes and oligodendrocyte precursor cells (OPCs). This revealed a significant decrease in numbers of both populations following 3 days of cerebral hypoperfusion however following 1 month numbers of OPCs were restored and a significant increase in mature oligodendrocyte number was observed. Assessment of OPC proliferation demonstrated low numbers of proliferating cells but revealed that a proportion of newly generated cells had differentiated into mature oligodendrocytes. To determine a potential mechanism involved in OPC differentiation following cerebral hypoperfusion the expression of the GPR17 receptor was examined which has recently been reported to mediate OPC differentiation in response to injury. The results demonstrated decreased expression of GPR17 following 3 days of hypoperfusion (p = 0.007) with no difference between groups observed following 1 month (p = 0.362) indicating that this receptor is not involved in differentiation of OPCs following hypoperfusion. Taken together the results from this study show that mature oligodendrocytes and OPCs are lost early in response to hypoperfusion but that these cells recover over time, highlighting the regenerative capacity of the white matter following cerebral hypoperfusion.Aim 3 – To investigate whether modulation of inflammation and oxidative stress could ameliorate alterations to white matter structure and function following severe chronic cerebral hypoperfusion The third and final aim of this thesis was to determine whether treatment with the anti-inflammatory and antioxidant drug DMF could ameliorate structural and functional alterations to white matter following severe chronic cerebral hypoperfusion. This was achieved by examining myelin and axonal integrity in addition to numbers of oligodendrocytes and OPCs following 7 days of severe chronic cerebral hypoperfusion. This revealed that myelin integrity was significantly decreased in vehicle-treated hypoperfused animals as compared to shams (p = 0.005). However no differences in myelin integrity were observed between sham and hypoperfused mice treated with DMF (p = 0.312). In contrast to the previous study, numbers of oligodendrocytes and OPCs were not altered following severe hypoperfusion however DMF treatment led to significantly increased numbers of oligodendrocytes in sham animals (p = 0.003). Assessment of white matter function using electrophysiology revealed that the conduction velocity of myelinated axons was significantly increased in DMF-treated hypoperfused animals as compared to those treated with vehicle (p = 0.04). Taken together the results of this study demonstrate that modulation of inflammation and oxidative stress may improve structural and functional white matter alterations following chronic cerebral hypoperfusion. Conclusions: The results presented in this thesis demonstrate that chronic cerebral hypoperfusion results in structural alterations to myelinated axons and to oligodendroglial populations within the white matter which are accompanied by impaired spatial working memory. Whilst previous studies using the model have reported that cerebral hypoperfusion results in diffuse white matter pathology, this study has highlighted the vulnerability of nodal and paranodal domains of myelinated axons as regions which are altered early in response to hypoperfusion. Furthermore, characterisation of oligodendroglial populations has revealed that these cells are replaced over time despite ongoing hypoperfusion which demonstrates the regenerative capacity of the white matter following cerebral hypoperfusion. Critically the results presented in this thesis demonstrate that treatment with DMF improved the function of myelinated axons in response to severe reductions in cerebral blood flow and thus may represent an appropriate therapeutic strategy for chronic cerebral hypoperfusion.

Aging is associated with a loss of cognitive function related to learning, memory, and executive function with varying severity. Although there is no age-related loss of neurons in healthy aging, myelin damage accumulates and is associated with cognitive decline. The brain’s resident macrophages, microglia, are responsible for clearing damaged myelin and promoting subsequent oligodendrocyte-mediated remyelination. To test the hypothesis that age-related dysfunction of microglial phagocytosis and oligodendrocyte remyelination capacity contributes to myelin pathology and cognitive impairment. To test this, rhesus monkeys from across the lifespan (7-30 years of age) were tested in three specific aims. 1) To characterize gene expression of myelin basic protein (MBP) in the brain and clearance of MBP to the cerebrospinal fluid (CSF) in relation to age-related myelin pathology. The density of myelinated axons visualized using label-free spectral confocal reflectance imaging did not correlate with age, but was significantly lower in aged animals with cognitive impairment. Next, MBP gene expression was measured using qPCR in the dorsal prefrontal cortex along with quantification of MBP protein levels in the CSF using ELISA. Age-dependent increases of MBP gene expression in the brain and MBP protein levels in the CSF were observed. Interestingly, MBP levels in the CSF were lower in animals with cognitive impairment. 2) To test the hypothesis that microglia would become increasingly primed for phagocytosis with age-related myelin pathology. The number of microglia immunostained with galectin-3, a marker for phagocytic activation, was quantified in the frontal white matter and increases in both aging and cognitive decline were detected. 3) To evaluate the hypothesis that lipofuscin, an age-related accumulation indicative of autophagic dysfunction, would accumulate and impair glial cells of the white matter in aged animals. Lipofuscin accumulation was increased with age in the frontal white matter and the size of lipofuscin clusters was associated with cognitive impairment. Lipofuscin was found primarily in microglia and oligodendrocytes, but not in astrocytes. These data suggest that lipofuscin burden in microglia and oligodendrocytes inhibits their homeostatic functions resulting in improper myelin clearance and turnover, leading to a devastating feed-forward cycle of myelin damage that contributes to age-related cognitive impairment.

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Myelin is an extensive cell membrane produced by oligodendrocytes to ensheath neuronal axons in the central nervous system with the primary goal of maximizing the efficiency of electrochemical impulse transmission. During brain development, oligodendrocytes differentiate into myelin forming cells in a tightly regulated process which makes them vulnerable to multiple insults. Previous results from the laboratory showed that the timing of oligodendrocyte differentiation and rat brain myelination were altered by perinatal exposure to buprenorphine and methadone, opioid analogues used for treating pregnant addicts. The mechanism by which these opioids exerted their effects involved two opioid receptors, the μ-opioid receptor (MOR) and the nociceptin/orphanin FQ receptor (NOR). However, the role of these receptors and their endogenous ligands in controlling the timing of myelination under normal physiological conditions of brain development is not known. In this dissertation, we found that the endogenous MOR ligand endomorphin-1 (EM-1) acts as a strong promoter of rat pre-oligodendrocyte differentiation, but surprisingly, this effect is observed only in cells isolated from female pups. Interestingly, the stimulatory action of EM-1 was abolished upon co-incubation with the endogenous NOR ligand, nociceptin. Moreover, injections of NOR antagonist to 9-day-old female and male rat pups accelerated rat brain myelination in female rat pups with no significant changes in their male counterparts. Interestingly, the lack of major sex-dependent differences in developmental brain levels of EM-1 and nociceptin and the presence of the two receptors MOR and NOR in male and female oligodendrocytes suggested that the observed sex-specific responses may be highly dependent on critical intrinsic sex-dependent differences within these cells. Although nociceptin alone did not exert observable effects on pre-oligodendrocyte maturation, it increased the number of cells expressing Ki-67, a cell proliferation indicator, in oligodendrocyte progenitor cultures. These results suggest that nociceptin may be playing a stage specific role in oligodendrocyte development during brain maturation. The finding of critical functions of EM-1 and nociceptin in the developing female oligodendrocytes and brain myelination highlights the need for considering sexual dimorphism in the design of safer and more effective therapeutic approaches for treating opioid abuse, pain, and demyelinating disease as multiple sclerosis.

In embryonic stem cells (ESCs) neural differentiation is elicited in the absence of extrinsic signaling in minimal conditions. This ‘default mechanism’ in ESCs produces neural stem cells termed primitive neural stem cells, which can subsequently yield FGF2-dependent definitive neural stem cells (dNSCs). We hypothesized that dNSCs have properties similar to neural stem/progenitor cells derived from the adult brain (aNPCs). The neural differentiation profile of the cell-types was characterized in vitro and in vivo following transplantation into the Shiverer mouse. The dNSCs produced a differentiation profile similar to that of aNPCs and both cell-types produced oligodendrocytes. This is the first demonstration of the in vivo differentiation of neural stem cells, derived from ESCs through the default mechanism, into the oligodendrocyte lineage. We conclude that dNSCs are a similar cell population to aNPCs. The default mechanism is a promising approach to generate neural stem cells and their progeny from pluripotent cell populations.