Université Libre de Bruxelles Faculté de Médecine
Neural Precursor Cells: Interaction with Blood‐brain barrier and Neuroprotective effect
in an animal model of Cerebellar degeneration
Thèse présenté en vue de l’obtention du grade académique de Docteur en Sciences Biomédicales
Satyan Chintawar
Promoteur : Professeur Massimo Pandolfo
2009
Neural Precursor Cells: Interaction with Blood‐brain barrier and Neuroprotective effect
in an animal model of Cerebellar degeneration
Dedicated to
Millions of people Suffering from
Neurodegenerative disorders
And
Dear Aai‐Baba (my parents)
ACM – astrocyte conditioned media ACSF ‐ artificial cerebrospinal fluid ANOVA – analysis of variance aNPCs – adult neural precursor cells ATP – adenosine triphosphate AXN1 – ataxin‐1
BBB – blood‐brain barrier
BBB‐ECs – blood‐brain barrier endothelial cells BDA – biotynalated dextran‐amine
BDNF – brain‐derived neurotrophic factor bFGF – beta fibroblast growth factor BrdU – bromodeoxyuridine
Calb – calbindin CBX – carbenoxolone
CCL2 – chemokine (C‐C motif) ligand 2 CE – coefficient of error
CFSE – carboxyfluorescein diacetate succinimidyl ester CNS – central nervous system
CV – coefficient of variation Cx26 – connexin26
Cx43 – connexin43 Cxs – connexins Cy3 – cyanine3
DAPI – 4',6‐diamidino‐2‐phenylindole DCN – deep cerebellar nuclei
ECs – endothelial cells
EGF – epidermal growth factor FBS – fetal bovine serum FCS – fetal calf serum GalC – galactocerebroside
GBSS – Gey’s balanced salt solution GCL – granule cell layer
GDNF – glial cell line‐derived neurotrophic factor GFAP – glial fibrillary acidic protein
GFP – green fluorescent protein GJI – gap junction inhibitor GL – granular layer
hfNPCs – human fetal neural precursor cells hNSCs – human neural stem cells
HPAECs – human pulmonary artery endothelial cells HSCs ‐ hematopoietic stem cells
i.p. – intraperitoneal IL‐8 – interleukin‐8
ISNPCs – induced self‐renewing neural progenitor/stem cells LIF – leukemia inhibitory factor
LY – lucifer yellow
MAP‐2 – microtubule associated protein‐2 MCP‐1 – monocyte chemoattractant protein‐1 ML – molecular layer
mNSCs – mouse neural stem cells NeuN – neuronal nuclei
NSCM ‐ neural stem cell media NSCs – neural stem cells NT‐3 – neurotrophin‐3 OCs – organotypic cultures PBS – phosphate buffered saline PCL – Purkinje cell layer
PCs – Purkinje cells PFA – paraformaldehyde PI – propidium iodide PLL – poly‐l‐lysine PN – Purkinje neuron RD – rhodamine dextran RNA – ribonucleic acid RNAi – RNA interference
RT‐PCR – real‐time polymerase chain reaction SC medium – stem cell medium
SCA1 – Spinocerebellar ataxia type 1 SCI – spinal cord injury
SEM – standard error mean SGZ – subgranular zone siRNA – small interfering RNA SVZ ‐ subventricular zone
TPA – tissue plasminogen activator wm – white matter
wt – wild type
1. General Introduction ... 1
1.1. Cerebellum ... 2
1.1.1. Gross anatomical division ... 2
1.1.2. Phylogenetic and functional divisions ... 3
1.1.3. Basic structure of the cerebellar cortex ... 4
1.2. Spinocerebellar ataxia type 1 (SCA1) ... 5
1.3. Stem Cell Biology ... 6
1.3.1. Background ... 7
1.3.2. Adult stem cells ... 9
1.3.3. Neurogenesis in the adult brain ... 9
1.4. Cellular Therapy ... 12
1.5. Interaction of NSCs and brain endothelial cells ... 13
1.6. References ... 15
2. Grafting Neural Precursor Cells promotes functional recovery in an SCA1 mouse model ... 17
Abstract ... 19
Introduction ... 20
Materials and Methods ... 22
Isolation and culture of NPCs ... 22
Immunocytochemistry ... 22
Stereotaxic transplantation ... 23
Motor behavior assessment ... 23
1. Accelerating rotarod test... 23
2. Grip strength test ... 24
Brain processing ... 24
Histology and Immunofluorescence on brain sections ... 24
Confocal imaging and image analysis ... 25
Stereology ... 26
Statistics ... 26
Results ... 27
Isolation and culture of adult murine neural precursor cells ... 27
Transplantation of NPCs improves the motor phenotype of SCA1 mice... 27
Rescue of mutant Purkinje cells and dendritic arbors by grafted NPCs ... 29
Migration and homing of grafted NPCs ... 33
Grafted NPCs restore the basal membrane potential of Purkinje cells ... 34
Discussion ... 39
References ... 42
Supplemental information ... 47
3. Communication via Gap junctions underlies early functional and beneficial interactions between grafted neural stem cells and the host ... 58
Summary ... 60
Introduction ... 61
Results ... 63
Differentiation and integration of grafted NSCs ... 63
Development of membrane properties ... 66
Grafted cells integrate into calcium‐mediated functional network ... 66
Grafted cells establish gap‐junctional couplings with organotypic culture cells ... 72
Grafted human NSCs display similar patterns of gap‐junction mediated interaction ... 76
NSCs improve survival of organotypic striatal cultures and promote a decrease in gliosis ... 76
Blocking gap junction formation and function ... 77
Contact‐dependent rescue of neurons in vivo by NSCs is accompanied by and dependent upon gap junction formation ... 81
Discussion ... 89
NSC integration is beneficial for the host ... 92
Experimental Procedures ... 95
Neural Stem Cells (NSCs) ... 95
Organotypic cultures ... 96
Stem cell grafting into organotypic culture ... 97
Immunohistochemistry ... 97
Electrophysiology ... 97
Calcium imaging ... 98
Dye coupling ... 99
Spinocerebellar ataxia type 1 (SCA1) mouse model ... 100
Nervous (nr) mouse model ... 100
Model of contused cervical spinal cord in adult rats ... 100
RNA interference (RNAi) ... 101
Statistical analysis ... 101
Acknowledgements ... 102
References ... 103
Supplemental material ... 111
4.Blood‐brain barrier promotes differentiation of human fetal neural precursor cells ... 148
Abstract ... 150
Introduction ... 151
Materials and Methods ... 153
Human fetal NPCs isolation and culture ... 153
Multipotentiality and clonal analysis ... 153
Isolation and culture of primary endothelial cells and astrocytes ... 154
Transendothelial migration... 154
Immunocytochemistry and phenotypic analysis ... 155
Results ... 157
Generation of neurospheres from human fetal brain tissues ... 157
Trans‐BBB‐EC migration of hfNPCs and phenotypic profile ... 158
MCP‐1/CCL2 blockade reduces trans‐BBB migration and differentiation of hfNPCs ... 161
hfNPCs did not migrate through but differentiate on human pulmonary artery endothelial cells only after addition of recombinant MCP‐1... 162
Discussion ... 166
References ... 169
Supplemental Data ... 171
5. General Conclusions and Perspectives ... 176
6. Summary ... 180
Acknowledgements ... 183
Curriculum Vitae ... 186
Publications ... 187
General Introduction
1
nlike automobiles, humans get along pretty well for most of their lives with their original parts.
But organs do sometimes fail, and we can’t go to the mechanic for an engine rebuild or a new water pump—at least not yet. Medicine has battled back many of the acute threats, such as infection, that curtailed human life in past centuries. Now, chronic illnesses and deteriorating organs pose the biggest drain on human health and they will only increase in importance as the population ages as tissue regeneration in human is limited. Some other animals possess extraordinary regenerative capabilities.
For e.g. freshwater planarians properly regenerate a head or tail after amputation (Gurley et al., 2008).
Adult vertebrate such as zebrafish regenerate an impressive array of structures, including spinal cord, optic nerve, heart, and fins (Whitehead et al., 2005). Salamander can regenerate its limbs and tail, upper and lower jaws, ocular tissues, and small section of the heart (Brockes and Kumar, 2005). Regenerative capabilities of these animals depend upon stem cells which are key players in regeneration process and are critically involved in the development and maintenance of mammalian tissues.
1.1. Cerebellum
With 100 billion nerve cells and 100 trillion connections, the brain is the most complex biological structure. The cerebellum is a critical component of the brain that plays an important role in the integration of sensory perception, coordination and motor control and contains more neurons and synapses than the rest of the brain. In order to coordinate motor control, there are many neural pathways linking the cerebellum with the cerebral motor cortex (which sends information to the muscles causing them to move) and the spinocerebellar tract (which provides proprioceptive feedback on the position of the body in space). The cerebellum integrates these pathways, using the constant feedback on body position to fine‐tune motor movements.
1.1.1. Gross anatomical divisions
The cerebellum is a large structure at the back of the brain, immediately behind the brainstem and below the occipital cortex and accounts for about 10% of total brain mass. The outer, grey matter or cortex is convoluted into many folia. Three major transverse divisions ‐ lobes ‐ are recognized. The anterior lobe is most rostral, followed by posterior lobe and flocculo‐nodular lobe more caudally. These lobes are divided by the primary fissure and the posterolateral fissure, respectively. Independent of the transverse, lobular arrangement, three major sagittal divisions are recognized. The central longitudinal strip of cortex is the vermis. On each side, the vermis is divided from the lateral cerebellar hemispheres by the narrow pars intermedia. In man, the hemispheres are so highly developed that they obscure pars
U
intermedia and much of the vermis. The cerebellar cortex overlies a core of white matter. At the centre is a collection of nuclei ‐ the deep cerebellar nuclei (DCN). These are the output areas of the cerebellum, receiving inputs from the cortex and projecting out to the thalamus, red nucleus and brainstem. From medial to lateral they are the fastigial, interpositus (in man, this is subdivided into the globose and emboliform nuclei) and dentate nuclei.
1.1.2. Phylogenetic and functional divisions
The cerebellum can also be divided in three parts based on both phylogenetic criteria (the evolutionary age of each part) and on functional criteria (the incoming and outgoing connections each part has and the role played in normal cerebellar function). From the phylogenetically oldest to the newest, the three parts are:
Vestibulocerebellum : The vestibulocerebellum regulates balance and eye movements. It receives vestibular input from both the semicircular canals and from the vestibular nuclei, and sends fibres back to the medial and lateral vestibular nuclei. It also receives visual input from the superior colliculi and from the visual cortex (the latter via the pontine nuclei, forming a cortico‐ponto‐cerebellar pathway).
Lesions of the vestibulocerebellum cause disturbances of balance and gait.
Spinocerebellum : The spinocerebellum regulates body and limb movements. It receives proprioception input from the dorsal columns of the spinal cord (including the spinocerebellar tract) as well as from the trigeminal nerve, as well as from visual and auditory systems. It sends fibres to deep cerebellar nuclei which in turn project to both the cerebral cortex and the brain stem, thus providing modulation of descending motor systems. The spinocerebellum contains sensory maps as it receives data on the position of various body parts in space: in particular, the vermis receives fibres from the trunk and proximal portions of limbs, while the intermediate parts of the hemispheres receive fibres from the distal portions of limbs. The spinocerebellum is able to elaborate proprioceptive input in order to anticipate the future position of a body part during the course of a movement, in a "feed forward"
manner.
Neocerebellum : The neocerebellum is involved in planning movement and evaluating sensory
information for action. It receives input exclusively from the cerebral cortex (especially the parietal lobe) via the pontine nuclei (forming cortico‐ponto‐cerebellar pathways), and sends fibres mainly to the ventrolateral thalamus (in turn connected to motor areas of the premotor cortex and primary motor
area of the cerebral cortex) and to the red nucleus (in turn connected to the inferior olivary nucleus, which links back to the cerebellar hemispheres). The neocerebellum is involved in planning movement that is about to occur and has purely cognitive functions as well.
1.1.3. Basic structure of the cerebellar cortex
Throughout its highly convoluted extent, the cerebellar cortex can be divided into three layers with the same basic neuronal circuitry everywhere, involving five main cell types (Fig. 1). The most conspicuous of these are the Purkinje cells (PCs), which form an orderly monolayer interposed between the granular and molecular layers, extending their planar dendritic trees into the molecular layer above. As these cells are the sole output neurons of the cerebellar cortex, they are central to cerebellar cortical information processing. The granular layer below the PCs derives its name from the small, densely packed granule neurons that send their axons into the molecular layer, where they bifurcate to become parallel fibres (Fig. 1). These run parallel to the long axis of each folium and as a result they intersect the fan‐like dendritic trees of many PCs. Mossy fibre afferents target granule cells and, therefore, excite the PCs indirectly through the granule cell–parallel fibre pathway, which causes the PCs to discharge ‘simple spikes’ (conventionalaction potentials). They also contact various types of interneuron in the cerebellar cortex, both directly and indirectly through the parallel fibres. The other main class of cerebellar afferent is the climbing fibres, which arise exclusively from the inferior olive, a well‐defined complex of sub‐nuclei in the ventral part of the caudal brain stem. In marked contrast to the indirect influence of mossy fibres, the climbing fibres make direct synaptic contact with PCs (Fig. 1). Moreover, each PC receives input from just one climbing fibre but the contact is so extensive that climbing fibres generate the largest depolarizing event seen in any neuron: a highly characteristic burst of impulses known as a climbing fibre response or complex spike (Apps and Garwicz, 2005).
Fig 1. Basic structure of the cerebellar cortex. There are two main afferents to the cerebellar cortex: climbing fibres, which make direct excitatory contact with the Purkinje cells, and mossy fibres, which terminate in the granular layer and make excitatory synaptic contacts mainly with granule cells, but also with Golgi cells. In some cases, the stem axons of climbing and mossy fibres also provide collaterals to the cerebellar nuclei en route to the cerebellar cortex. The ascending axons of the granule cells branch in a T‐shaped manner to form the parallel fibres, which, in turn, make excitatory synaptic contacts with Purkinje cells and molecular layer interneurons — that is, stellate cells and basket cells. Typically, parallel fibres extend for several millimetres along the length of individual cerebellar folia. With the exception of granule cells, all cerebellar cortical neurons, including the Purkinje cells, make inhibitory synaptic connections with their target neurons. Adapted by permission from Macmillan Publishers Ltd: Nat. Rev. Neurosci. Anatomical and physiological foundations of cerebellar information processing, copyright 2005.
Cerebellar degeneration is a disease process in which neurons in the cerebellum deteriorate and die and is most often the result of inherited genetic mutations that alter the normal production of specific proteins that are necessary for the survival of neurons. Dysfunction or loss of neurons within it present as feedback deficits resulting in disorders in fine movement, equilibrium, posture, and motor learning.
1.2. Spinocerebellar ataxia type 1 (SCA1)
The word "ataxia", comes from the Greek word, "ataxis" meaning "loss of order". People with ataxia have problems with coordination because parts of the nervous system that control movement and balance are affected. Ataxia may affect the fingers, hands, arms, legs, body, speech, and eye movements. The cerebellar ataxia is the most common form of ataxia and is caused by dysfunction
either within the cerebellum or in its afferent and efferent pathways. The term Spinocerebellar ataxia (SCA) was originally used to indicate anomalous function of the spinocerebellum, the part of the cerebellar cortex that receives input from the spinal cord. In the current terminology, however, SCA is used to refer to the hereditary forms, and in particular the autosomal dominant forms of degenerative ataxias (Carlson et al., 2009). Although many of these disorders lead to dysfunction of cerebellar PCs, other involves the deep cerebellar nuclei, brainstem nuclei, and spinal sensory as well as spinocerebellar tracts.
Currently, 28 SCAs are recognized. Spinocerebellar ataxia type 1 (SCA1) is an autosomal‐dominant neurodegenerative disorder caused by the expansion of a CAG repeat in the SCA1 gene. The CAG repeat encodes for a polyglutamine tract in the SCA1 gene product, ataxin‐1 (Orr et al., 1993). The number of CAG repeats in ataxin‐1 gene is highly polymorphic in the general population, ranging from 6 to 44, the longer (>21) repeats being interrupted by one to four CAT triplets. SCA1 patients typically have
expansions between 39 and 82 uninterrupted CAG repeats (Chung et al., 1993). The length of the CAG repeat and consequently of the polyglutamine tract is inversely proportional to the age of onset and directly to the severity of the disease (Servadio et al., 1995). SCA1 is also a member of a group of polyglutamine disorders that includes Spinocerebellar ataxia types 2, 3, 6, 7, 17, Huntington’s disease, Spinobulbar muscular atrophy, Dentatorubropallidoluysian atrophy. As described later in chapter 2, SCA1 is usually a late‐onset disorder characterized by progressive cerebellar ataxia, pyramidal, extrapyramidal and oculomotor abnormalities, peripheral neuropathy and cognitive impairment.
Pathologically, loss of cerebellar PCs and of brain stem neurons is hallmark of the disease.
Currently, effective therapeutics is not available to treat patients of SCA1 and other polyglutamine diseases, largely due to the lack of understanding of the functional role of these proteins and pathogenic mechanisms associated with the disease. In mid 1990s, collaborative efforts of Dr Harry Orr and Dr Huda Zoghbi groups generated transgenic mice expressing the human SCA1 gene with an expanded CAG tract (Burright et al., 1995). These mice develop a progressive motor disorder starting at 5 weeks of age, prior to any alteration of cerebellar morphology. By 12 weeks of age, their PCs start to show a shrunken dendritic tree, ectopically located cell bodies in the molecular layer, and an initial decline in number.
Significant PC loss (about a third) occurs by 24 weeks of age. We investigated the potential of our therapeutic strategy for SCA1 by testing it in the B05 transgenic model.
1.3. Stem Cell Biology
Stem cell research has opened one of the most fascinating chapters in the history of biology.
Traditionally belonging to the field of developmental biology, stem cells have become of increasing interest for biomedical research in more recent years. Tissue engineering, therapeutic cloning, transgenic animals and gene therapy are among the most discussed applications.
1.3.1. Background
Stem cells are undifferentiated cells that can divide indefinitely. They can either divide symmetrically, producing two identical daughter cells, or asymmetrically producing one identical and one more differentiated daughter cell (Lin and Schagat, 1997). The least differentiated stem cell type is the omnipotent or totipotent stem cell. It is found in early mammalian embryos (4 – 8 cell stage) and can form any cell type or tissue including the entire fetus and the placenta (Ralston and Rossant, 2005). The inner cell mass of the blastocyst contains pluripotent stem cells (Thomson et al., 1998). These embryonic stem (ES) cells can be maintained in an undifferentiated state in culture, can differentiate in virtually any kind of cell type, but are not capable of forming an entire embryo. ES cells can be differentiated into multipotent stem cells, which are restricted to their specific lineage. These are hematopoietic,
mesenchymal, endodermal or neural stem cells. These lineages follow specific differentiation patterns, with increasingly specialized cells (see Fig. 2). By applying the appropriate clues in a defined order it is possible to direct an ES cell towards a specific cell type (Bibel et al., 2004). However, the controversy over using human embryos as a source of these cells have led to intensified research to find stem cells in adult tissues.
Fig. 2. Characteristics of Embryonic stem cells. Figure courtesy of www.situgen.com
1.3.2. Adult stem cells
For five decades hematopoietic stem cells have been the only adult stem cells known and investigated.
More recently, it was discovered that numerous adult tissues contain stem cells. Normally these cells are involved in the homeostatic self‐renewal and regenerative processes, but are occasionally activated for repair activity (Reya and Clevers, 2005). The lumen of the intestine for example is replaced about once a week. Blood and skin are constantly renewed, hair and nails constantly grow. All these systems depend on small local populations of stem cells, which are highly regulated. If the specific program of proliferation, migration and differentiation fails, the respective tissue will either become dysfunctional or cancerous. The arrangement of these proliferative systems is surprisingly conservative for the different tissues where adult stem cells are found. Generally a population of stem cells is harbored in a defined niche. The stem cells proliferate slowly, maintain the size of population and produce another population of transient amplifying precursor cells. These proliferate at a higher rate, and migrate towards the final destination of the specific mature cell type. This results in a differentiation gradient from the stem cell along to the migratory precursor cell to the fully differentiated cell.
1.3.3. Neurogenesis in the adult brain
It has been a common understanding that in postnatal mammals no new neurons are added to the CNS and that any further changes can only be adopted through rewiring of the synaptic connections. In fact this dogma originates from early works at the end of the nineteenth century describing the
developmental and adult brain of humans and other mammals (Ramon y Cajal, 1999). These
investigators found that the architecture of the brain appears to be fixed soon after birth. At the cellular level, neither mitotic nor developing neurons were observed. Although there were occasional reports on mitotic cells in the brain of adult mammals, the available experimental methods could not convincingly prove that these new cells would differentiate into neurons and be functionally integrated. Using
autoradiography to track 3H Thymidine, incorporated by proliferating cells during mitosis, Joseph Altman published a series of papers in the nineteen sixties showing evidence for adult neurogenesis in the rat and cat (for review see Gross, 2000). The scientific community did not recognize the significance of his results. Although Altman’s experiments were repeated and combined with electron microscopy, and additional evidence for neurogenesis in songbirds and rats was presented (Kaplan and Hinds, 1977;
Nottebohm, 1985), the observation of neurogenesis in the adult brain did not get much attention. In 1992 Reynolds and Weiss (Reynolds and Weiss, 1992) successfully isolated NSCs from adult rodent brain and expanded in vitro. Moreover, new techniques emerged. Instead of tritiated thymidine,
bromodeoxyuridine (BrdU) was used as a proliferation marker. BrdU can easily be labeled with immunohistochemical methods and investigated with brightfield and fluorescence microscopy. In addition specific antibodies against neuronal or glial markers were developed, providing easy methods to distinguish neurons from glia. With the help of these methods adult neurogenesis has been
demonstrated to exist until senescence in numerous mammalian species including humans. In 1998 Evan Snyder and colleagues (Flax et al., 1998) and Ronald Mckay and colleagues (Brustle et al., 1998) independently isolated first human NSCs from aborted fetuses that can differentiate to all cell types found in the brain. Soon in an interesting series of experiments, a team based in California and Sweden (Fred Gage and colleagues) (Eriksson et al., 1998) discovered that hippocampus produces new neurons into old age raising a possibility of inducing patient’s own brain cells to regenerate cells lost to disease.
More recently Steven Goldman and colleagues (Nunes et al., 2003) have isolated multipotent progenitor cells from the adult human brain opening the possibility of implantation strategies of cell‐based
neurological therapy. Since then NSCs garnered the interest of not only developmental community but also neural repair, gene therapy and transplant communities once it was recognized that NSCs can be expanded ex vivo and once reimplanted into the mammalian brain can reintegrate into neural circuitry (Snyder et al., 1992).
Now it is accepted that neurogenesis persists in a mammalian brain throughout adulthood and has been clearly demonstrated at least in two locations under normal conditions: the subventricular zone (SVZ) of lateral ventricles and subgranular zone (SGZ) of the dentate gyrus in the hippocampus (Bonfanti and Peretto, 2007; Ihrie and Alvarez‐Buylla, 2008; Zhao et al., 2008). (see Fig.3)
Fig. 3 Adult Neurogenesis : (A) Depictions of sagittal and coronal views of mouse brain in areas where
neurogenesis occurs. Red areas indicate the germinal zones in the adult mammalian brain: the subgranular zone (SGZ) of the dentate gyrus in the hippocampus and the subventricular zone (SVZ) of the lateral ventricles. Neurons generated in the SVZ migrate through the rostral migratory stream and are incorporated into the olfactory bulb.
(B–E) Neurogenesis revealed by BrdU incorporation in the olfactory bulb (B), rostral migratory stream (C), SVZ (D), and dentate gyrus (E). Inset in (C) is a sagittal view of rostral migratory stream before reaching the olfactory bulb, and inset in (E) is a high‐magnification view of the area indicated by the arrow in (E). Colors indicate the following:
red, BrdU; green, NeuN. (F and G) Newborn neurons in the olfactory bulb and dentate gyrus labeled by retrovirus‐
mediated expression of green fluorescent protein (GFP). Insets are high‐magnification views of the cells indicated by arrows. Colors indicate the following: red, NeuN; green, GFP; blue, DAPI. Reprinted from Cell, 132, 4, Chunmei Zhao, Wei Deng and Fred H. Gage, Mechanisms and Functional Implications of Adult Neurogenesis, 645‐60, Copyright (2008), with permission from Elsevier.
1.4. Cellular Therapy
Cells are the functional unit of the human body. Many of the diseases that afflict humans are caused by cell damage or because cells die and are lost. Many examples include‐‐ Congestive heart failure where heart cells are alive but lost the ability to contract, Type 1 diabetes where insulin producing cells are lost, Alzheimer’s and Parkinson’s disease where particular types of neurons are lost. Muscular
dystrophies are the result of death of muscle cells and tissues. Spinal cord injuries, Retinitis pigmentosa are some other examples. Many diseases which are the product of breakdown in the cell which is not then regenerated, treatment approaches by either stimulating endogenous cell genesis or provide them exogenously are possible to imagine. In SCA1, Purkinje neuron dysfunction is followed by their death.
Cellular therapy using tissue‐specific cells either isolated from patients himself or donors or derived from pluripotent stem cells holds promise for treatment of many devastating diseases and injuries
Adult mammalian brain having limited capacity of regeneration in discrete areas often fail to regenerate the injured or insulted tissue because of an inhospitable environment that contains growth inhibitory proteins and also considerably due to the intrinsic property acquired by precursor cell during
development (Goldberg et al., 2002). Due to lack of inherent ability of the brain to recruit PCs lost during the disease and the poor knowledge of the developmental roadmap for generating PCs from
endogenous stem cells, grafting stem cells in SCA1 mice would be an appropriate therapeutic approach.
Bone marrow contains multipotent hematopoietic stem cells (HSCs) that continuously produce blood cells throughout our lifetime. HSC transplantation (established since 1960s) has proven to be a life‐
saving therapy for diseases refractory to other treatments (Bertz et al., 2002) and have provided “proof of principle” that stem cells can restore loss of function (Parkman, 1986). Patients with extensive burns can be treated with stem cells residing in their own skin. For implantation in B05 mice, it is conceivable to implant NSCs, which can only generate neural and glial cells (Fig. 4) and are safe as compared to pluripotent stem cells which can inflict tumor formation (Roy et al., 2006). Translating this approach is relatively straightforward as it involves autologous transplantation and excludes harmful
immunosuppressive therapy. Moreover, there are limitations and ethical issues involved in isolating NSCs from aborted fetal tissues. In addition, murine adult NSCs transplantation has proven safe and effective in number of animal models of neurological diseases and injury (see chapter 2).
Fig 4. Neural stem cells (NSCs) differentiation pattern. NSCs are undifferentiated cells originating in the central nervous system. They have the potential to give rise to offspring cells that grow and differentiate into neurons and glial cells. Figure courtesy of www.sigma‐aldrich.com
In the first manuscript (chapter 2), we report the data on grafting adult neural precursor cells (NPCs) in a B05 mouse model mimicking human SCA1 and the detailed investigation where we demonstrate that NPCs are neuroprotective if implanted only in animals that had already suffered a significant PC loss at the time of transplantation. Recent published data suggests that neural stem/precursor cells promote recovery in animal models via a number of indirect bystander effects. A comprehensive knowledge of how transplanted NPCs exert their therapeutic effect is still lacking. In the second manuscript (chapter 3), we discuss on the possible mechanism of neuroprotective effect of NPCs in SCA1 mice.
1.5. Interaction of NSCs and brain endothelial cells
Stem cell therapy has been shown to have considerable therapeutic potential for neurodegenerative diseases; however, most experiments in animals have been performed by injecting cells directly into the injured parenchyma with limited migration. Intravascular administration of stem cells would be
attractive to achieve distribution in the whole brain, important especially in diffuse neurodegenerative and neuroinflammatory diseases. In addition, it has several other advantages as it is minimally invasive and avoids post‐surgical trauma and the possibility of repetitive injections. However, it is unknown if systemically injected NSCs can penetrate intact blood‐brain barrier (BBB) endothelium if inflamed parenchyma present an attractive signal. Furthermore, there are several published reports suggesting
endothelium instructing stem cell fate and survival and thus are key players in a stem cell niche (in brain as well as other organs). In this study (chapter 4), we wished to explore whether NPCs can get across an intact BBB endothelium and how their proliferation and differentiation properties are affected by this interaction using an established in vitro model of human BBB (Fig. 4)
Fig. 4. Human in vitro BBB model. Schematic illustration of the preparation of in vitro model of BBB. Temporal lobe specimens from young adults undergoing surgery for the treatment of epilepsy were obtained from the hospital’s operating room. BBB endothelial cells (BBB‐ECs) were isolated and expanded (described in chapter 4) in
endothelial cell medium. For transendothelial migration assay, Boyden chambers were pre‐coated with gelatin and BBB‐ECs were seeded on the membrane in endothelial cell medium and astrocyte conditioned medium and allowed to form a monolayer. In Boyden chamber, upper compartment represent blood and lower compartment represent brain.
Overall, this study focuses on the suitability of adult NSCs for the treatment of cerebellar degenerative diseases, SCA1 in particular and the importance of NSC‐brain endothelium crosstalk for the maintenance of brain stem cell niche.
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Grafting Neural Precursor Cells promotes functional recovery in an SCA1 mouse model
Abbreviated Title: Neural Precursor cells are neuroprotective in an SCA1 mouse model
Satyan Chintawar1, Raphael Hourez2§, Ajay Ravella1§, David Gall2, David Orduz2,
Myriam Rai1, Don Patrick Bishop2, Stefano Geuna 3, Serge N Schiffmann2, Massimo Pandolfo1
1Laboratory of Experimental Neurology, 2Laboratory of Neurophysiology,
Université Libre de Bruxelles, Route de Lennik 808, 1070 Brussels, Belgium.
3Department of Clinical and Biological Sciences and Cavalieri Ottolenghi Scientific Institute,
University of Turin, Orbassano (TO), 10043 Italy
§ contributed equally
J Neurosci. 2009 Oct 21;29(42):13126‐35
2
Correspondence should be addressed to MP.
Prof. Massimo Pandolfo Chef de Service Service de Neurologie Hôpital Erasme ‐ Université Libre de Bruxelles Route de Lennik 808 1070 Brussels Belgium Phone +32‐2‐555‐3429 Fax +32‐2‐555‐3942
Email: massimo.pandolfo@ulb.ac.be
Number of figures : 7 Number of tables : 2
Contents of supplemental material : Methods and figures of qRT‐PCR, western blotting, confocal
profiling image for colocalization, dissector image for stereological PC count, detailed description of experimental procedures and image acquisition tools, softwares used, and 4 videos
Number of pages : 35
Number of words for Abstract : 200
Introduction : 691
Discussion : 1105
Keywords : Neural precursor, Cerebellum, Neuroprotection, Migration, Purkinje cells, Degeneration
Acknowledgements
We thank Dr JM Vanderwinden for expertise and technical assistance to microscopy and we wish to
thank Ana Lopes da Cruz for animal technical assistance. This work was supported by the Belgian
Scientific Research Funds (FNRS).
Abstract
The B05 transgenic SCA1 mice, expressing human ataxin‐1 with an expanded polyglutamine tract in cerebellar Purkinje cells (PCs), recapitulate many pathological and behavioral characteristics of the neurodegenerative disease spinocerebellar ataxia type 1 (SCA1), including progressive ataxia and PC loss. We transplanted neural precursor cells (NPCs) derived from the subventricular zone of GFP‐
expressing adult mice into the cerebellar white matter of SCA1 mice when they showed absent (5 weeks), initial (13 weeks) and significant PC loss (24 weeks). Only in mice with significant cell loss, grafted NPCs migrated into the cerebellar cortex. These animals showed improved motor skills as compared to sham‐treated controls. No grafted cell adopted the morphological and
immunohistochemical characteristics of PCs, but the cerebellar cortex in NPC‐grafted SCA1 mice had a significantly thicker molecular layer and more surviving PCs. Perforated patch clamp recordings revealed a normalization of the PC basal membrane potential, which was abnormally depolarized in sham‐treated animals. No significant increase in levels of several neurotrophic factors was observed, suggesting, along with morphological observation, that the neuroprotective effect of grafted NPCs was mediated by direct contact with the host PCs. We postulate that a similar neuroprotective effect of NPCs may be applicable to other cerebellar degenerative diseases.
Introduction
Spinocerebellar ataxia type 1 (SCA1) is one of the hereditary neurodegenerative disorders caused by the expansion of a CAG trinucleotide repeat, which is translated into a polyglutamine (polyQ) tract in the corresponding protein (Orr and Zoghbi, 2007). It is an autosomal dominant usually late‐onset disorder characterized by progressive cerebellar ataxia associated with variable degrees of pyramidal,
extrapyramidal, and oculomotor abnormalities, peripheral neuropathy and cognitive impairment.
Pathologically, loss of cerebellar Purkinje cells (PCs) and of neurons in the brain stem are typical findings (Watase et al., 2002). The mutated gene in SCA1, AXN1, encodes the ataxin‐1 protein, which is thought to be involved in transcription regulation and RNA processing. Ataxin‐1 is localized in the nucleus of neurons, in the cytoplasm of non‐neuronal cells, and both in the nucleus and cytoplasm of cerebellar PCs. The nuclear localization of ataxin‐1 has been shown to be essential for pathology. The pathogenesis of SCA1 involves a toxic gain‐of‐function effect of the expanded polyQ tract along with dominant‐
negative effects due to altered functionalities of the mutated protein (Bowman et al., 2007).
The first SCA1 mouse model, developed by Burright et. al. in the mid ‘90s (Burright et al., 1995), was based on a multicopy transgene expressing a mutated human ataxin‐1 with an expanded polyQ tract (30 copies of a transgene encoding ataxin‐1 with 82 glutamines in the B05 line, used in the present study) under the control of the PC‐specific Pcp2 promoter. This mouse model develops a progressive motor disorder starting at about 5 weeks of age, before the appearance of any alteration of cerebellar
morphology. Pathological abnormalities of PCs, in the form of a shrunken dendritic tree and migration of the cell body into the molecular layer, become clearly visible by 12 weeks of age, along with an initial decline in PC number. A significant PC loss (about a third) occurs by 24 weeks of age.
No treatment is currently available for SCA1. Patients experience progressive limitations in their
activities, lose the ability to walk, and eventually become bedridden and fully dependent. In the present study, we wished to explore the potential of neural stem cell transplantation as a therapeutic approach for SCA1 by testing it in the B05 transgenic model. We considered that, even if effective treatments will be developed with better understanding of SCA1 pathogenesis, stem cell therapy remains an attractive option, as it may provide additional neuroprotection and possibly promote regeneration. In addition, relatively few studies (5) have addressed the effects of stem cell transplantation in cerebellar disorders as compared to studies in diseases affecting other brain areas, including several neurodegenerative
(Corti et al., 2007 and 2008; McBride et al., 2004; Redmond et al., 2007; Roberts et al., 2006; Yasuhara et al., 2006), neuroinflammatory (Pluchino et al., 2003) and other neurological insults (Jeong et al., 2003;
Cummings et al., 2005; Karimi‐Abdolrezaee et al., 2006; Lee et al., 2007a and 2007b).
For our transplantation experiments, we used neural progenitor cells (NPCs) derived from the subventricular zone (SVZ) of adult mice. NPCs are a mixture of neural stem cells and early progenitor cells that can be isolated from specific regions of the adult mammalian brain (the subventricular zone of the lateral walls of lateral ventricles and the subgranular zone in the dentate gyrus of hippocampus) (Gritti et al., 1996; Taupin and Gage, 2002; Gage 2002; Lie et al., 2004). NPCs possess the potential of generating mature neural and glial progenies (multipotentiality) and their stem cell component has an indefinite self‐renewal property. The donor mice we used have the same strain background as the B05 mice (FBV/N), eliminating any requirement for immunosuppression in grafted animals, and express the green fluorescent protein (GFP) in all their cells, providing a simple marker to trace transplanted cells.
Kidney fibroblasts from the same strain were used in control transplantation experiments to evaluate any general, non NPC‐specific effect of grafted cells.
Our results indicate that transplanted NPCs survived and integrated into the recipient cerebellar cortex only in animals that had already suffered a significant PC loss at the time of transplantation. In these mice, NPCs induced behavioral amelioration, promoted survival of PCs, improved cerebellar
morphology, and restored a normal PC excitability.
Materials and Methods
Isolation and culture of NPCs
NPCs were derived from the SVZ of adult mice as described in Gritti et al. (Gritti et al., 1999) and modified as follows. Four to eight weeks old FVB/N mice expressing GFP under β‐actin promoter (Jackson Lab, USA) were anesthetized by Avertin (i.p.) and sacrificed by cervical dislocation. Brains were removed and the thin layer of tissue surrounding the ventricles was cut, dissected into small pieces and cultured in media containing 20 ng/ml recombinant human EGF and 10 ng/ml recombinant human bFGF (Peprotech, NJ, USA). Big spherical clusters were ready to dissociate 7‐10 days after isolation.
Neurospheres were mechanically dissociated every 4‐5 days and plated in fresh growth medium. They were assessed for self‐renewal and multipotentiality as described earlier (Chintawar et al., 2009) (see supplemental methods). NPCs of passage 4‐8 were used for transplantation. For detailed procedure for isolation and characterization of NPCs, refer to supplementary information.
Immunocytochemistry
Undifferentiated and differentiated NPC cultures seeded on matrigel were fixed with 4%
paraformaldehyde and incubated with 0.1% Triton X 100 for 15 min. Cultures were then incubated overnight at 4°C with primary antibodies: nestin, (Chemicon, USA) as marker for undifferentiated cells, MAP‐2 (Sigma, Belgium) as marker for neurons, GalC (Chemicon, USA) as marker for oligodendrocytes, and GFAP (DakoCytomation, Denmark) as marker for astrocytes. Antibodies were detected using Cy3‐
conjugated IgGs. No antibody was needed for GFP, as the native GFP signal was detectable. In all the cultures, DAPI (4',6‐diamidino‐2‐phenylindole) was used to counterstain cell nuclei. Slides were mounted using Fluorsave (Calbiochem, Germany).
