The Pennsylvania State University
The Graduate School
Department of Neural and Behavioral Sciences
A REGENERATIVE RESPONSE OF ENDOGENOUS NEURAL STEM CELLS
TO PERINATAL HYPOXIC/ISCHEMIC BRAIN DAMAGE
A Thesis in
Neuroscience
by
Ryan J. Felling
© 2006 Ryan J. Felling
Submitted in Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy
May 2006 ii The thesis of Ryan J. Felling was reviewed and approved* by the following:
Steven W. Levison Professor of Neurology and Neurosciences Thesis Advisor Co-Chair of Committee
Teresa L. Wood Associate Professor of Neural and Behavioral Sciences Co-Chair of Committee
Sarah K. Bronson Assistant Professor of Cell and Molecular Biology
Charles Palmer Professor of Pediatrics
James R. Connor Professor and Vice-Chair Department of Neurosurgery; Director, G.M. Leader Family Laboratory for Alzheimer's Disease Research
Robert J. Milner Professor of Neural and Behavioral Sciences Head of Neuroscience Graduate Program
*Signatures are on file in the Graduate School
iii ABSTRACT
Hypoxic/ischemic (H/I) insults are the leading cause of neurologic injury during the perinatal period, affecting 2-4 per 1000 term births as well as a high percentage of premature infants. The ensuing sequelae are devastating and include cerebral palsy, epilepsy and cognitive deficits. Despite astounding advances in perinatal care, the incidence of cerebral palsy has changed little over the last 50 years. This demands that we pursue alternative therapeutic strategies that will reduce the significant morbidity associated with perinatal H/I encephalopathy.
The revelation that the brain retains populations of neural stem cells throughout life offers the promise of endogenous regeneration following brain injury. Already extensive data have demonstrated that new neurons are born after cerebral ischemia and migrate to sites of injury. The existence of multipotential, self-renewing neural stem and progenitors, however, suggests that this response could be much more significant. This thesis offers data indicative of a mobilization of this population following perinatal H/I.
Using a clonal assay to quantify neural stem and progenitors (NSPs), we
demonstrated almost a doubling in the numbers of NSPs following perinatal H/I. This
was accompanied by the generation of larger neurospheres and increased symmetric
proliferative divisions in vitro upon EGF and FGF-2 stimulation. Furthermore, a higher
proportion of the clonal neurospheres retained the capacity to generate all 3 neural cell
types upon differentiation. These effects were preceded by an upregulation of genes
important in the regulation of NSP maintenance and proliferation, including notably the
EGF receptor and Notch1. iv Further investigation demonstrated that the larger size of neurospheres following perinatal H/I was due to increased proliferation, specifically in response to EGF stimulation. These effects as well as the induction of Notch1, were dependent upon signals from mature cells as demonstrated by an in vitro paradigm of H/I injury. Other components of the Notch signaling cascade were also induced specifically within the
NSP niche of the subventricular zone (SVZ). Pharmacological inhibition of this pathway using a well-characterized inhibitor of γ-secretase that prevents release of the intracellular mediator, the Notch intracellular domain (NICD) effectively reduced both the increase in numbers of NSPs as well as the increase in tripotency of the neurospheres.
These data provide evidence of a mobilization of the NSP population in response to perinatal H/I. A better understanding of the molecular mechanisms underlying these results will identify therapeutic targets for enhancing this response. It is no longer acceptable to settle for just managing neurologic injuries such as perinatal H/I encephalopathy; we must pursue strategies to repair the brain after such damage. This thesis demonstrates the basis for endogenous regeneration of the CNS following brain injury and describes several potential molecules that may be used to manipulate this response.
v TABLE OF CONTENTS
LIST OF FIGURES ...... ix
LIST OF TABLES...... xi
ACKNOWLEDGEMENTS...... xii
Chapter 1 Literature Review...... 1
Stem Cells...... 1 General considerations ...... 1 Neural stem cells ...... 4 Maintenance of NSCs...... 8 Mitotic spindle orientation ...... 9 Asymmetric inheritance of cell fate determinants...... 11 Notch Signaling...... 12 EGF Receptor...... 13 Emx2 ...... 14 Pax6...... 15 Bmi-1...... 16 Enhanced neurogenesis following ischemic injury ...... 17 Proliferation of SVZ cells following ischemic injury ...... 18 Migration of new cells following ischemic injury ...... 20 Differentiation of new cells after ischemic injury...... 21 Perinatal hypoxic/ischemic encephalopathy...... 23 Clinical Background...... 23 Effects of perinatal H/I on the SVZ ...... 24 Specific Aims of the Thesis...... 25
Chapter 2 Summary of Results ...... 26
NSPs are more abundant in the rodent SVZ following perinatal H/I...... 26 Perinatal H/I promotes increased Notch signaling in the SVZ...... 30 Changes observed in NSPs following perinatal H/I depend on Notch signaling ...... 33 Changes in the NSP niche underlie the effects of perinatal H/I...... 34
Chapter 3 Discussion of Results ...... 36
Neural stem cells are more abundant following perinatal H/I...... 36 NSPs respond to the injured environment ...... 37 The NSP population actively expands following perinatal H/I...... 38 Increased EGF signaling is an important contributor to NSP expansion following perinatal H/I ...... 39 vi Notch signaling is an important contributor to NSP expansion following perinatal H/I...... 40 Realizing Cajal’s vision – reflections on the future of regenerative medicine ...... 42
Chapter 4 Conclusions and Future Directions ...... 44
Chapter 5 Neural stem/progenitor cells initiate a regenerative response to perinatal hypoxia/ischemia ...... 47
Introduction ...... 47 Methods ...... 49 Perinatal hypoxia/ischemia model ...... 49 Tissue fixation and BrdU labeling ...... 50 Antibodies and Immunohistochemistry ...... 51 In situ hybridization ...... 53 Primary neurosphere propagation ...... 54 Secondary neurosphere propagation ...... 55 Neurosphere quantitation ...... 56 Neurosphere immunohistochemistry...... 57 RNA isolation...... 58 Real-time PCR...... 59 Statistics ...... 60 Results ...... 60 Markers of cell proliferation increase in the medial SVZ following perinatal H/I ...... 60 More neurospheres can be isolated from the SVZ following perinatal H/I ...... 65 NSPs divide symmetrically more often following perinatal H/I...... 68 A greater proportion of neurospheres are multipotent following perinatal H/I ...... 69 Perinatal H/I induces the expression of Notch1, gp130, and EGFR mRNA ...... 70 Discussion...... 73 Acknowledgements and support...... 79
Chapter 6 Increased EGF responsiveness following perinatal H/I alters proliferation of neural stem and progenitor cells...... 80
Introduction ...... 80 Methods ...... 81 Perinatal hypoxia/ischemia model ...... 81 Primary neurosphere propagation ...... 83 3H-thymidine Assay ...... 84 In vitro H/I insult...... 84 Co-culture experiments ...... 85 vii Results ...... 86 Larger neurospheres are observed following perinatal H/I in the presence of EGF, but not in the presence of FGF-2 alone...... 86 Proliferation is increased in ipsilateral neurosphere cultures...... 87 Neurospheres require external signals to exhibit increased proliferation following H/I...... 89 Discussion...... 91
Chapter 7 Induction of Notch1 promotes increased neural stem/progenitors following perintatal H/I ...... 94
Introduction ...... 94 Methods ...... 96 Perinatal hypoxia/ischemia model ...... 96 DAPT injections...... 97 In situ hybridization ...... 98 Primary neurosphere propagation ...... 99 Neurosphere quantitation ...... 100 Neurosphere immunohistochemistry...... 100 In vitro H/I...... 102 Western Blot...... 103 RNA isolation...... 104 Real-time PCR...... 104 Results ...... 106 Notch1 and Delta expression increase within the first 2 days of recovery from perinatal H/I...... 106 Pharmacologically inhibiting Notch1 in vivo reduces the increase in NSPs following perinatal H/I...... 111 Mixed brain cells subjected to H/I in vitro stimulate Notch1 expression in neurospheres ...... 113 Discussion...... 115
Bibliography ...... 121
Appendix A Altered Expression of NSC-Related Genes After Perinatal H/I ...... 146
viii LIST OF FIGURES
Figure 1.1: Two models of a stem cell lineage...... 3
Figure 1.2: Diagrammatic representation of the development of the adult SVZ...... 6
Figure 1.3: Modes of stem cell division...... 9
Figure 2.1: More neurospheres can be cultured after perinatal H/I...... 27
Figure 2.2: Neurospheres grow larger following perinatal H/I...... 28
Figure 2.3: NSPs proliferate more rapidly in the presence of EGF following perinatal H/I...... 29
Figure 2.4: EGFR is induced following perinatal H/I...... 30
Figure 2.5: Notch1 receptor and the Notch ligand, Delta-like 1, are induced following perinatal H/I ...... 31
Figure 2.6: Hes5 expression is induced within the NSP niche following perinatal H/I...... 32
Figure 2.7: Inhibition of Notch activity reduces the effects of perinatal H/I on NSPs...... 34
Figure 2.8: NSPs respond to signals from injured tissue following perinatal H/I...... 35
Figure 5.1: Diagram of the brain regions analyzed...... 53
Figure 5.2: Hypoxia-ischemia increases the number of BrdU+ cells adjacent to or among the ependymal lining in the ipsilateral SVZ...... 62
Figure 5.3: Hypoxia-ischemia increases the number of PCNA+ and PCNA+/Nestin+ cells adjacent to or among the ependymal lining in the ipsilateral SVZ...... 64
Figure 5.4: Hypoxia-ischemia increases the number of colony-forming NSPs and the self-renewal of NSPs in the ipsilateral SVZ...... 67
Figure 5.5: Hypoxia-ischemia increases the proportion of multi-potential SVZ- derived neurospheres...... 70
Figure 5.6: Receptors involved in control of NSP fate are induced by perinatal H/I...... 72 ix Figure 6.1: Neurospheres grow larger in the presence of EGF, but not FGF-2...... 87
Figure 6.2: NSPs proliferate more rapidly in the presence of EGF following perinatal H/I...... 88
Figure 6.3: NSPs respond to signals from injured tissue following perinatal H/I. ...90
Figure 7.1: Components of the Notch signaling pathway are induced following perinatal H/I...... 108
Figure 7.2: Notch1 receptor and its ligands are induced within the NSP niche following perinatal H/I...... 110
Figure 7.3: Inhibition of Notch activity reduces the expansion of NSPs following perinatal H/I...... 112
Figure 7.4: Inhibition of Notch activity after perinatal H/I reduces the frequency of tripotent neurospheres...... 113
Figure 7.5: Signals from NSP niche triggers Notch1 induction following exposure to H/I...... 115
x LIST OF TABLES
Table 5.1: Primers used in qRT-PCR ...... 59
Table 7.1: Primers used in qRT-PCR ...... 106
xi ACKNOWLEDGEMENTS
This thesis represents the culmination of my efforts and devotion toward this
research project. Despite the hard work that I put forth over the last several years, the
fact remains that this would never have been possible without the support of many
important people. The simple words dedicated to them in these acknowledgements are
not sufficient to reflect how important their contributions have been, but I feel it is
essential to recognize the roles they have played in this achievement.
Practicalities of life dictate that nothing is free, and a project of this magnitude
requires significant investment of resources. For this reason I am compelled to thank the
MD/PhD program at the Penn State College of Medicine and the National Institutes of
Health. These organizations supported me financially throughout this project via a
Medical Scientist Training Program fellowship and a Ruth-Kirchstein Predoctoral F31 fellowship, respectively.
Throughout the course of this project, the bureaucratic matters of academia
frequently pushed me to the edge of sanity. Fortunately I had a wonderful staff to lead
me, and sometimes drag me, through these issues. Dee, Karen, and Rachel in the Neural
and Behavioral Sciences Office and Barb in the MD/PhD program were incredible in
both their ability and patience in dealing with the day to day management of my
education. Without them I would likely have tripped over quite a few speed bumps along
the way.
I was also fortunate to find a truly wonderful laboratory to work in. The members
of the Levison lab were terrific in helping me along throughout my time there. There was xii always an open relationship with everyone in the lab so that it was never uncomfortable to seek help. Mike and Christine, two other graduate students were very helpful in orienting me and giving me a jumpstart in my research. Colleen and Ray were always willing to help, and I appreciate their assistance throughout the project. I also need to extend a particular thanks to Kyle, whose diverse expertise educated me both in the field of molecular biology and in my short game on the golf course (outside of work hours, of course).
My thesis committee was an important source of ideas during the evolution of this project. Dr. Connor, Dr. Bronson, Dr. Wood, and Dr. Palmer were always available for discussion of my work and provided valuable feedback at all stages of the project. I truly appreciate their input, as it helped strengthen every part of my work and my education.
My thesis advisor, Dr. Steve Levison, is clearly the one person most directly responsible for the success of this project. He deserves my thanks for not only bringing me into his laboratory, but for giving me the latitude to develop this project according to my own interests and goals. I came into graduate school with good experience conducting research, but Dr. Levison must be credited with teaching me to be a scientist.
His guidance both in an out of the lab has given me the foundation and confidence necessary to take the next step toward becoming an independent investigator. I can only hope that my path will allow me to collaborate with him in the future.
While many of the people recognized above had clear contributions to this project, I must also recognize two people in particular who influenced this project long before it began. Dr. Ronald Weiss was my first research mentor in high school at the
Army Research Laboratory, and he opened the doors to the realm of science for me. He xiii taught me how important asking questions was and provided the fundamentals necessary to seen the answers to those questions. Secondly, Dr. Richard Ordway at Penn State was my undergraduate thesis advisor. It was in his lab that I first gained a love for neuroscience and without his guidance I would likely have never made it to the next level.
Family has always been a source of both support and perspective for me. My parents, while often wondering if I will ever get a “real job,” have always been behind me, ready to help in whatever way they could. I cannot adequately express my appreciation and love for them. I also want to thank my wife, Erin, for making these years truly joyful. She endured all the difficult schedules and hardships of life that go with being a struggling graduate, fully supporting me the entire time. She is the reason for everything I do, and each time I see her reminds me of the truly important parts of life.
Of course this list does not come close to naming all the people who have helped me along the way. Many other friends and family were there both in and out of school. I apologize for not being able to name everyone here, but you all know how important you are to me. Without you all, my achievements would still be dreams. Thank you, and I hope that someday I will have repayed you for all that I owe.
xiv
“In adult centres the nerve paths are something fixed, ended, immutable. Everything
may die, nothing may be regenerated. It is for the science of the future to change, if
possible, this harsh decree”
Santiago Ramon y Cajal (1913)
Estudios sobre la degeneracion y regeneracion del sistema nervioso
Chapter 1
Literature Review
Stem Cells
General considerations
“Omnis cellula e cellula” (Latin - “Cells stem from existing cells”). This observation by the early cell theory proponent Rudolf Virchow could be considered the first step in the recognition of a stem cell. Cells are born through the process of cell division and mature through a series of events collectively termed differentiation. Stem cells reside at the origin of this differentiation process representing the founders of cell lineages. Their potential to give rise to multiple cell types is the primary reason stem cells have garnered intense interest in the scientific community as a therapeutic strategy and, unfortunately, the reason they have ignited political and ethical debate in many arenas.
The definition of a stem cell is one of function rather than identity. The prototypical stem cell is the early embryonic stem cell which gives rise to all the different cells and tissues of the body; however, we also identify multiple classes of tissue-specific stem cells that persist throughout life. There are specific criteria which a cell must fulfill 2 in order to be labeled a stem cell: 1.) Multipotency, defined as the ability of a cell to generate all the basic cell types of the tissue in which the stem cell resides; 2.) Self- renewal, defined as the ability of a cell to maintain itself through indefinite rounds of cell division; 3.) Production of large numbers of differentiated progeny. The nature of this definition presents a difficulty in identifying stem cells in vivo – their identity must be verified through confirmation of their functional capabilities.
Adult stem cells exist as discreet populations of cells in various tissues. Their physiological purpose is to provide a continuous supply of new cells in tissues that exhibit turnover such as the skin, gut, and hematopoietic system. They also provide a reserve for the replacement of cells in the event of injury. A stem cell is an uncommitted, slowly cycling precursor that represents the earliest stage in a cell lineage (Figure 1.1).
Through unique patterns of cell divisions, stem cells generate pools of rapidly dividing cells with finite lifespans called transit-amplifying precursors. As they continue to divide and mature, transit-amplifying cells gradually give rise to committed progenitors which mature into functional non-cycling cells. 3
A
Stem cell Transit-amplifying Lineage-restricted Mature cell cell progenitor
B Differentiation Proliferative potential
Figure 1.1: Two models of a stem cell lineage. (A) Simplified depiction of discreet phase model of cell development. (B) Continuum model of cell development depicting gradual loss of proliferation potential in favor of differentiated functionality.
Although the traditional dogma was that adult stem cells were restricted to producing cells of the tissue in which they reside, recent evidence indicates a much higher degree of plasticity (Verfaillie et al., 2003; Lakshmipathy and Verfaillie, 2005).
The antigenic heterogeneity of stem cells, even within a single tissue, lend credence to the view of a stem cell as a functional characteristic rather than a specific cellular entity.
This presence of “stem cellness” is determined by many factors including both cell- autonomous features and the influence of the cell’s niche (Blau et al., 2001). It has even been proposed that all the various characteristics described by investigators for a given stem cell may be correct and simply reflect different functional states as that cell fluctuates along a continuum (Quesenberry et al., 2002; Quesenberry et al., 2005).
Clearly it is essential to remain open-minded and recognize the diversity of stem cell populations.
4 Neural stem cells
The central nervous system was long thought to be a static entity following
development despite astounding evidence to the contrary. Mitotic activity was repeatedly reported in studies of the postnatal and even adult brain in the early part of the 20th
century (Allen, 1912; Messier et al., 1958; Smart, 1961). These cell divisions were generally considered abortive because techniques available at the time were insufficient for tracking the fates of these cells; however, a series of subsequent experiments definitively demonstrated persistent postnatal neurogenesis in various brain regions
(Altman and Das, 1965, , 1966; Altman, 1969a, 1969b). The basis of this inherent plasticity was finally revealed in the early 1990’s when individual cells cultured in vitro were shown to self-renew and produce progeny capable of generating the 3 basic cell types of the nervous system: neurons, astrocytes, and oligodendrocytes (Reynolds et al.,
1992; Alvarez-Buylla and Lois, 1995; Gritti et al., 1995). The characteristic colonies generated by these cells inspired the term “neurosphere assay (NSA),” for this process which remains the gold standard for identifying these bona fide NSCs.
The NSA relies on the fact that NSCs proliferate in the presence of epidermal
growth factor (EGF) and/or fibroblast growth factor (FGF)-2. During brain development,
there is a sequential appearance of NSCs responsive to FGF alone followed by the
emergence of EGF-responsive NSCs (Tropepe et al., 1999; Martens et al., 2000;
Ciccolini, 2001). The primary NSC in the adult brain is responsive to both EGF and
FGF-2, and it has been shown that exposure of early development NSCs to FGF-2 can 5 promote acquisition EGF-responsiveness, suggesting that these may represent 2 phases of a continuum of NSC development (Ciccolini and Svendsen, 1998; Gritti et al., 1999).
Since their initial discovery, NSCs have been isolated from multiple brain regions
(Picard-Riera et al., 2004). One important region, the subventricular zone (SVZ), represents the remnant of the secondary embryonic germinal matrix, (Figure 1.2). After development, the primary germinal matrix or ventricular zone (VZ) regresses, but the
SVZ persists as a thin region along the walls of the lateral ventricles subjacent to the ependyma. Although SVZ is the more developmentally correct name, the term
“subependymal zone” is also seen in the literature, persisting from early studies before there was a distinction between the SVZ and VZ (Kreshman, 1938; Boulder Committee,
1970). The SVZ remains active in the postnatal brain, producing both neuroblasts that migrate along the rostral migratory stream, contributing to neurogenesis in the olfactory bulb, as well as glial cells (Levison and Goldman, 1993; Lois and Alvarez-Buylla, 1993;
Luskin, 1993). 6
A B LV
SVZ CTX LGE STR MGE VZ
C
Figure 1.2: Diagrammatic representation of the development of the adult SVZ. (A) Depiction of a E15 rodent brain showing the primary germinal matrix (VZ, dark gray) and the secondary germinal matrix (SVZ, black). MGE-medial ganglionic eminence, LGE-lateral ganglionic eminence. (B) Depiction of the adult rodent brain showing the persistent SVZ (black) around the lateral ventricles. (C) Depiction of the 3 compartements of the adult dorsolateral SVZ.
The SVZ is a heterogeneous mosaic of cells, and detailed analyses of the region in the adult rodent brain have established a cytoarchitecture consisting of Type A, B, and C cells (Doetsch et al., 1997). Ultrastructural characteristics and cell cycle kinetics established a hierarchy in which Type A cells represent migrating neuroblasts; Type C cells represent their immediate precursor, likely a transit-amplifying cell; and Type B 7 cells represent glial fibrillary acidic protein (GFAP)-expressing, comprising both immature SVZ astrocytes and putative NSCs. The theory that the Type B cells contained
NSCs arose when, after ablation of highly mitotic precursors, Type B cells remained in the SVZ and were able to reconstitute the structure described above (Garcia-Verdugo et al., 1998; Doetsch et al., 1999). The idea that NSCs express GFAP is supported by an elegant study showing that selective ablation of GFAP-expressing cells effectively eliminates neurosphere-forming cells from the rodent forebrain (Morshead et al., 2003).
Despite intense efforts at finding markers specific for NSCs, the in vivo identity of NSCs has remained elusive. There are compelling reasons to suspect that they represent a subset of glia (Doetsch, 2003; Goldman, 2003). Abundant evidence indicates that radial glia, cells whose soma resides in the VZ with processes maintaining contact with both the ventricular and pial surfaces, serve as NSCs during embryonic neurogenesis
(Malatesta et al., 2000; Tamamaki et al., 2001; Anthony et al., 2004). Evidence now shows that progeny of radial glia continue to generate neurons, astrocytes, and oligodendrocytes, and radial glia-derived cells at all ages are able to generate self- renewing, multipotent neurospheres in vivo (Merkle et al., 2004).
Despite tremendous successes in identifying which cell populations represent true
NSCs, there is still no all-encompassing marker or set of markers that selectively labels all NSCs. In fact, despite all the evidence described above indicating that NSCs express
GFAP, studies have shown that non-GFAP expressing cells are also capable of generating self-renewing, multipotent neurospheres. These include Lex+ and NG2+ cells
(Capela and Temple, 2002; Aguirre et al., 2004). The intermediate neurofilament protein
Nestin has traditionally been used to label NSCs, but this protein is also present in non- 8 stem cell precursors (Dahlstrand et al., 1995; Kawaguchi et al., 2001). Furthermore it is difficult to differentiate between true NSCs and multipotent transit-amplifying cells as they are both capable of forming neurospheres in vitro (Doetsch et al., 2002; Reynolds and Rietze, 2005). The term neural stem and progenitors (NSPs) has been adopted in this thesis to encompass both true NSCs and other undifferentiated, neurosphere-forming precursors. Despite these obstacles to definitively identify NSCs, it is clear that the brain retains tremendous potential for cell production long after development is complete.
Maintenance of NSCs
Understanding cell cycle regulation in NSCs is critical to the development of stem cell-based therapies for neurological disease. Self-renewal, which is essential for the maintenance of a functional stem cell pool throughout life, relies on a balance between asymmetric and symmetric cell divisions (Figure 1.3). Symmetric divisions can be either
proliferative (resulting in 2 new stem cells) or terminal (resulting in 2 downstream progenitors). During CNS development, proliferative symmetric divisions initially expand the NSC pool. Following this expansion, asymmetric divisions sustain the NSC pool while at the same time providing progenitors to populate the developing brain.
Asymmetric cell divisions produce 2 daughter cells that differ morphologically or functionally and also fall into 2 classes: self-renewing (resulting in 1 new stem cell and 1 downstream progenitor) or differentiating (resulting in 2 distinct downstream progenitors). The concept of self-renewal is not necessarily tied to asymmetric cell 9 division, however, because a population undergoing all symmetric divisions can be self- renewing (i.e. maintain a constant size) if half of those symmetric are proliferative
(Potten and Loeffler, 1990). The pathways regulating these different types of division are central to understanding how stem cells function normally and how they adapt to changes in their environment.
A B
Figure 1.3: Modes of stem cell division. (A) Asymmetric division can result in renewal of the stem cell and generation of a new downstream precursor or generation of 2 distinct daughters. (B) Symmetric division produces either 2 new stem cells (top) or 2 new downstream precursors (bottom), thereby increasing or decreasing the size of the stem cell pool, respectively.
Mitotic spindle orientation
The developing Drosophila CNS provides an excellent model for studying the
fundamental mechanisms coordinating asymmetric division within a cell. Following
delamination from the neuroepithelium precursors called neuroblasts undergo asymmetric
divisions to self-renew and produce smaller progenitors that gives rise to neurons and glia. Underlying these divisions is cellular polarity created by the localization of a 10 protein complex involving Inscuteable, Pins, Par3/Bazooka, Par6, and atypical protein kinase C (aPKC) to a crescent at apical pole of the cell (Wang and Chia, 2005). This apical complex orients the metaphase plate in the apical-basal direction and generates asymmetry in the geometry of the spindle, resulting in different daughter cell sizes (Cai et al., 2003). This apical complex forms during interphase, but it is dependent on the cell cycle regulatory protein cdc2 for maintenance through the cell cycle (Tio et al., 2001).
Thus all asymmetric divisions of Drosophila neuroblasts occur with a cleavage plane parallel to the neuroepithelium and the apical daughter cell remaining a neuroblast while the basal daughter progresses down the lineage.
Homologues of the proteins involved in asymmetric division of Drosophila neuroblasts have been identified in mammals. In accordance with the Drosophila model of asymmetric cell division, there is evidence suggesting that the orientation of cell cleavage relative to the ventricular surface is important in mammalian neurogenesis, with horizontal cleavages (parallel to the ventricular surface) being asymmetric and vertical cleavages (perpendicular to the ventricular surface) being symmetric (Chenn and
McConnell, 1995). Studies of movement of the mitotic spindle during precursor division also show similarities to Drosophila, substantiating this scheme and further suggesting that active rotation of the mitotic spindle is necessary to establish a horizontal cleavage; whereas, the vertical cleavage plane may represent a default state (Haydar et al., 2003).
The importance of this scheme is not firm, however, as other studies have shown that vertical cleavages often result in asymmetric daughter cell fates due to the selective inheritance of a basal fiber with which the precursor maintains contact with the pial surface (Weissman et al., 2003). Probably more relevant than its relationship to the 11 ventricular surface is the role of mitotic spindle orientation in the asymmetric distribution of cell fate determinants to daughter cells.
Asymmetric inheritance of cell fate determinants
The same protein complex that coordinates the orientation of the mitotic spindle
performs another crucial step in the process of asymmetric cell division,
compartmentalization of cell fate determinants. It is joined in this task by a basal protein
complex that includes the tumor-suppressor proteins Discs-large (Dlg) and Lethal giant
larvae (Lgl). During mitosis these complexes direct specific proteins to a crescent at the basal pole of the cell including the cell fate determinants Prospero and Numb (Rhyu et al., 1994; Knoblich et al., 1995). The interaction between Numb and the cell fate regulatory receptor Notch, described further in the next section, is the prototypical model for how this compartmentalization of cell fate determinants can contribute to asymmetric cell division. Numb is a known inhibitor of Notch signaling; therefore, when a large
amount of Numb protein is inherited by a single daughter, Notch is unable to exert its control over cell fate within that daughter cell while the other daughter will remain responsive to Notch signals. This mechanism for influencing cell fate decisions is highly conserved in all species including humans.
12 Notch Signaling
Importantly, this mechanism is shared by the mammalian brain, although it
appears more complex than in Drosophila (Zhong et al., 1996; Shen et al., 2002).
Originally identified in Drosophila, Notch signaling is an evolutionarily conserved
mechanism for regulating patterning and cell fate decisions (Artavanis-Tsakonas et al.,
1999). Notch signals are mediated through cell-cell contact via the Delta, Serrate, and
Lag-2 (DSL) family of transmembrane ligands. Once bound by its ligand, the Notch receptor undergoes a series of cleavage events to release the intracellular mediator, the
Notch intracellular domain (NICD). NICD then translocates to the nucleus where it interacts with CSL proteins to trigger upregulation of downstream targets including the
Hairy/Enhancer of Split (Hes) family of transcription repressors, particularly Hes1 and
Hes5 (Mumm and Kopan, 2000).
There are 4 mammalian homologs of the Notch receptor (Notch1-4). In the brain,
Notch1 is essential in maintaining the NSC population by promoting self-renewal. Mice
genetically deficient in Notch1 or other pathway components, including presenilin (γ-
secretase) or RBP-Jκ, exhibit a progressive loss of NSPs as reflected by declining ability
to generate neurospheres, a defect that can be rescued with the introduction of activated
Notch1 (Hitoshi et al., 2002). Inhibition of Notch activity in neurospheres in vitro, either
pharmacologically or genetically, also reduces the self-renewal capacity of NSPs to
generate secondary neurospheres (Chojnacki et al., 2003). The in vivo role of Notch,
however, is strongly context-dependent. Overexpression of an active form of Notch1 in
cortical precursors just prior to embryonic neurogenesis promotes a radial glial 13 phenotype, while overexpression in early postnatal SVZ cells inhibits differentiation and delays emergence from the SVZ (Gaiano et al., 2000; Chambers et al., 2001). As radial glia function as embryonic NSCs, these 2 functions may represent stage-dependent variations of a common theme: Notch signals promoting a stem cell state.
EGF Receptor
Signaling through the EGFR is critical to NSC function. Mice lacking TGFα, an
important in vivo EGFR ligand, have reduced numbers of NSCs (Tropepe et al., 1997).
New evidence suggests that asymmetric inheritance of this receptor may play a role in specifiying cell fate in the developing mammalian brain. A subset of cells from the embryonic VZ, and to a lesser extent from the SVZ, exhibit asymmetric localization of
EGFR during mitosis both in vivo and in vitro (Sun et al., 2005). This phenomenon is
highly correlated to numb asymmetry in earlier phases of development (E13) but not at
later stages (E16-E17); however, EGFR asymmetry is independent of Numb as there
were no differences observed in Numb and Numblike double mutants. Daughters
inheriting high levels of EGFR consistently migrate further and express markers typical
of NSCs or radial glia (LeX, GLAST, RC2) (Sun et al., 2005). These results suggest a
possible role for localization of EGFR in asymmetric cell division in the developing
brain, but it remains unknown whether this mechanism persists into the adult. 14 Emx2
Emx2 is a vertebrate homologue of the Drosophila homeobox gene empty spiracles (Simeone et al., 1992). Anomalies in Emx2-/- mice and schizencephalic humans
with Emx2 mutations demonstrate the importance of this gene in patterning and
morphogenesis of the CNS as many brain structures including the hippocampus, olfactory
bulbs, and cortex fail to form properly (Pellegrini et al., 1996; Yoshida et al., 1997; Tole
et al., 2000). Interestingly Emx2 expression is restricted to the VZ of the developing
brain, and the size of this proliferative region is actually increased in Emx2-/- mutants,
suggesting deficits in cell differentiation and migration (Mallamaci et al., 1998;
Mallamaci et al., 2000). Emx2 expression persists in proliferative regions of the adult
brain including the dentate gyrus and SVZ, and it is highly expressed in adult NSCs
cultured as neurospheres but it is downregulated as the cells differentiate (Gangemi et al.,
2001; Galli et al., 2003). These observations have driven the proposal that Emx2
regulates the proliferation of neural precursors.
Gain and loss of function analyses in adult NSCs suggest that Emx2 inhibits
proliferation in this population. Over-expressing cells incorporate less 3H-thymidine and generate fewer cells while ANSCs from Emx2-/- mice incorporate twice as much 3H- thymidine as control cells and display a steeper growth curve (Gangemi et al., 2001; Galli et al., 2003). Furthermore FACS analysis 1 week following BrdU incorporation by either control or Emx2 overexpressing cells demonstrated that a higher percentage of the overexpressing cells retained BrdU. This result suggests that control cells continue to divide over the observed time period and therefore dilute the BrdU label. By contrast, in 15 cultures that overexpress Emx2, more cells cease dividing and therefore retain BrdU.
Secondary neurosphere cultures indicate that the absence of Emx2 promotes proliferative symmetric divisions (higher rate of secondary neurosphere formation) while enhanced expression increases the rate of terminal symmetric divisions (lower rate of secondary neurosphere formation). In both cases there was no difference in the survival or multipotentiality of the neurospheres (Galli et al., 2003). The interpretation of these data is that Emx2 shifts the balance of cell division mode toward terminal symmetric divisions.
Pax6
The paired box (Pax) family of genes are related to the homeobox genes and highly involved in regulating organogenesis (Tremblay and Gruss, 1994). Of these genes
Pax6 is one of the earliest to appear in the developing brain, and unlike others, its
expression persists throughout much of development in mitotically active cells of the VZ
as well as in some other brain regions (Walther and Gruss, 1991). Mutations in this gene
cause a variety of developmental brain abnormalities in vertebrates including humans,
presumably due to alterations in cellular adhesion and migration (Stoykova et al., 1997).
During embryonic neurogenesis Pax6 expression is highly localized to radial glia
identified by the coexpression of RC2 and Nestin. In Pax6-deficient mice there is an
increase in the number of RC2+ cells, but they exhibit marked morphological
abnormalities both in vivo and in dissociated cell cultures (Gotz et al., 1998). 16 Analysis of radial glia in Pax6-deficient mice provide direct evidence of a role for
Pax6 in NSC cell cycle regulation. Pax6-deficient radial glia show abnormal interkinetic migration (a feature typical of wild-type VZ precursors) and a higher labeling index in
BrdU pulse labeling experiments, suggesting an alteration of cell cycle characteristics
(Gotz et al., 1998). Pax6-deficient mice show an accelerated transition from symmetric to asymmetric divisions, as defined by cleavage orientation, with concurrent increases in the expression of differentiating markers by dividing cells (Estivill-Torrus et al., 2002).
Cell cycle analyses in these mutants demonstrate that early in neurogenesis Pax6- deficient mice exhibit shorter cell cycles, but by mid-neurogenesis the cell cycle lengthens beyond that of wild-type cells with S-phase occupying a larger proportion of the entire cycle (Estivill-Torrus et al., 2002). These results suggest that Pax6 may be a component of an intrinsic timing mechanism that regulates a NSC’s transistion from symmetric to asymmetric divisions during development.
Bmi-1
Bmi-1 is a polycomb transcriptional repressor that promotes entry into S phase via
p16ink4a inhibition and decreases apoptosis by depletion of p53 through repression of p19arf (Jacobs et al., 1999a; Jacobs et al., 1999b). Loss of Bmi-1 in vivo causes
progressive growth retardation as well as neurologic abnormalities including ataxia and
seizures. Both embryonic and adult NSCs are dependent upon Bmi-1 for self-renewal as
reflected by the observation that fewer neurospheres form from the SVZs of Bmi-1-/- 17 mice. Self-renewal of Bmi-1-/- neurospheres is also severely reduced, a phenomenon that
is mimicked by depleting Bmi-1 in vitro with siRNA (Molofsky et al., 2003; Zencak et
al., 2005). These changes are also accompanied by smaller neurospheres and fewer
cycling cells both in vitro and in vivo as indicated by BrdU incorporation (Molofsky et
al., 2003; Zencak et al., 2005). Importantly, these differences are restricted to
multipotential colonies. There were no changes in the characteristics of adherent neuron-
only or glia-only colonies, indicating that Bmi-1 selectively plays a role in NSCs and not
in lineage-restricted progenitors (Molofsky et al., 2003). In addition to the described
effects on NSCs, loss of Bmi-1 causes an increase in GFAP+ cells at birth and
generalized gliosis at older ages, suggesting that Bmi-1 may restrict cells from an
astroglial phenotype (Zencak et al., 2005).
Enhanced neurogenesis following ischemic injury
The revelation that the adult brain contained NSCs soon allowed the discovery of
a previously unrecognized capacity of the CNS for adaptation to injury. Recent evidence
has demonstrated that new cells are generated in both the adult and perinatal SVZ following stroke (Felling and Levison, 2003). Much of the research in this area has focused on neurogenesis, but the presence of putative NSCs in the SVZ suggests that other cell types may also contribute to the newborn population.
18 Proliferation of SVZ cells following ischemic injury
While the data regarding cell proliferation and neurogenesis in the SVZ following
perinatal H/I is sparse, many investigators have demonstrated such a phenomenon in
adults. Most of these data indicate that proliferation in the ipsilateral SVZ following
MCAo peaks at seven days and persists to at least 14 days of recovery (Zhang et al.,
2001; Li et al., 2002). Another report places the peak at 14 days after a similar insult (Jin
et al., 2001). This same experiment demonstrated a comparable increase in proliferation
in the contralateral SVZ despite an absence of damage in that hemisphere. The bulk of
these studies used pulse labeling paradigms to examine specific timepoints; therefore,
they do not adequately describe the total amount of proliferation that occurs following
stroke.
Cumulative BrdU labeling has define the total number of proliferating cells
during the first 2 weeks after MCAo. Their results demonstrate approximately a 37%
increase in the number of proliferating cells in the ipsilateral SVZ compared to both the
contralateral SVZ and nonischemic controls. No difference was observed at four weeks
of recovery (Zhang et al., 2001). As the investigators only injected BrdU once daily
during the recovery interval, the results of this analysis may underestimate the absolute level of proliferation due to a lack of constantly available BrdU and rapidly cycling mitosis.
One concern with using BrdU to examine cell proliferation is that DNA repair can
lead to BrdU incorporation. One study failed to show overlap of BrdU incorporation
with labeling for PCNA, a cell-cycle dependent protein marker of cell proliferation, in the 19 SVZ (Jin et al., 2001). The failure to confirm the BrdU results with PCNA may be a technical artifact, as two studies have demonstrated that administration of Ara-C, a toxin for dividing cells, combined with BrdU administration eliminates the observed increases in the number of BrdU+ cells (Arvidsson et al., 2002; Nakatomi et al., 2002), supporting
the claim that the increases in BrdU labeling are primarily due to cell division following
H/I.
Direct evidence indicates that progenitor cells comprise the majority of the
proliferating population. In one study BrdU+ cells in the rat SVZ did not label with
NeuN at one week of recovery from MCAo. Rather, the BrdU+ cells reside subjacent to
regions of high NeuN positivity. Furthermore, these BrdU+ cells do not express another
neuronal marker, Hu, providing additional support for the hypothesis that progenitor cells
rather than mature cells proliferate in response to ischemic injury and that BrdU is not
being incorporated into dying cells (Jin et al., 2001). While these data demonstrate a lack
of mature cell markers among the proliferating population of cells, other experiments
have shown that these cells express markers specific for immature cells. Doublecortin
(DCX) is a marker of early neuronal progenitors, and many BrdU+ cells in the SVZ and
striatum also label for DCX two weeks after MCAo (Arvidsson et al., 2002). Other
markers of immaturity including Pax6, Emx2, and Mash1 are also elevated in the
posterior SVZ following transient global ischemia in rats (Nakatomi et al., 2002).
Little work has addressed the effects of ischemia on cell proliferation in the
perinatal SVZ, a primary subject of this thesis. Data available using doublecortin (DCX)
to identify migrating neuronal precursors have demonstrated significantly more immature
neurons 1 week following a perinatal H/I insult (Plane et al., 2004). This appears to be 20 consistent with results obtained in the adult brain reviewed above. While it appears clear that there is cell proliferation in the SVZ following ischemic injury to the perinatal brain, the significance and long term fate of the newborn cells is still unclear.
Migration of new cells following ischemic injury
The data reviewed above provide evidence of a significant proliferative response
of SVZ NSPs to ischemic injury. This response, however, is only advantageous if the
new cells ultimately repopulate brain regions depleted of cells as a consequence of stroke.
Indirect evidence suggests that newly generated cells migrate away from the SVZ.
Examination of the ipsilateral hemisphere after MCAo in adult rats reveals BrdU+/DCX+ progenitors with the morphology of migrating neurons streaming from the SVZ to the striatum. A gradient of migrating profiles is seen, with the greatest number of spindle- shaped DCX+ cells located up to 0.5 mm lateral to the SVZ, but with scattered cells
located as far as 2 mm from the SVZ. In the contralateral hemisphere, DCX
immunoreactivity is restricted to the SVZ and to rare single cells of the striatum
(Arvidsson et al., 2002). Although indirect, these observations suggest that newly
generated neuroblasts are migrating into damaged areas of the brain.
Another study looked at the expression of PSA-NCAM after MCAo in adult rats.
They found increased expression of PSA-NCAM in the ventral region of the SVZ 3 days after the injury, and immunostaining revealed an absence of GFAP in these cells (Sato et al., 2001). While this result is consistent with the conclusion that newly formed cells are 21 migrating out of the germinal zones, PSA-NCAM is also highly expressed by reactive astrocytes that are certainly present in the ischemic brain (Stoll et al., 1998). Another study reported an elevated number of PSA-NCAM+ cells in the dorsolateral SVZ as well,
but no effort was made to determine whether cells expressing PSA-NCAM were reactive
astrocytes (Zhang et al., 2001).
It is currently unknown whether cells detected in damaged regions following H/I
insult represent proliferating cells resident in the parenchyma or cells that have migrated
from proliferative regions. While the observation of DCX+ cells with spindle-shaped profiles hundreds of microns from the SVZ is consistent with migration, fate-mapping studies using stereotactically-injected retroviruses or time-lapse video microscopy studies are needed to define where the cells that proliferate and migrate from the SVZ early after an insult ultimately reside. These types of data would help to define the migratory
patterns of progenitors after ischemic injury to verify that progenitor cells repopulate
areas where they are needed.
Differentiation of new cells after ischemic injury
Several studies have addressed the long-term fate of cells that proliferate early
after an ischemic insult in an adult stroke model. Four weeks after ischemia with BrdU
administered during the first week of recovery, investigators reported a 31-fold increase 22 in the absolute number of BrdU+/NeuN+ cells in the ipsilateral striatum compared to the
contralateral striatum and sham-operated controls. The density of BrdU+/NeuN+ cells is nearly 10 times greater at 4 weeks of recovery than at 2 weeks, indicating that progenitor cells continue to mature over the 4 week recovery period (Arvidsson et al., 2002).
Consistent with the interpretation that newly generated neurons were maturing within the damaged striatum, BrdU+/DCX+ cells stained for Meis2 and Pbx, two markers that are expressed by developing striatal cells. Furthermore, at 5 weeks of recovery, 42% of the
BrdU+/NeuN+ cells were also positive for DARPP-32, a marker of mature medium-spiny
neurons characteristic of the striatum (Arvidsson et al., 2002). Importantly, growth
factor-treatment significantly enhances both dendritic branching and synapse formation
after ischemia. These data suggest that a significant proportion of the newly generated
cells in the striatum develop in a regionally appropriate manner.
After an ischemic insult, some newly generated cells differentiate into a mature
neuronal phenotype, but survival must be investigated at longer recovery intervals.
Although four weeks may be enough recovery time for cells to mature, any therapeutic
role for these cells requires that they maintain a functional presence in the local networks
for extended periods of time if not permanently. While current literature focuses on the
generation of neurons, an involvement of putative NSCs in the neurophysiological
response to ischemia suggests that other cell types may be affected during recovery. The
astrocytic response has been thoroughly investigated (Stoll et al., 1998), but little is
known about oligodendrocyte production following stroke. One group demonstrated an
accumulation of oligodendrocytes in both the border and core of the infarct after an initial
loss of proteolipid protein (a component of myelin) mRNA expression (Mandai et al., 23 1997). Another group has shown a significant increase in the number of NG2+
oligodendrocyte progenitors surrounding the infarct within 2 weeks after MCAo, but the
fate of these cells has not been determined (Tanaka et al., 2003).
Perinatal hypoxic/ischemic encephalopathy
Clinical Background
Perinatal hypoxic/ischemic (H/I) insults are the leading cause of neonatal encephalopathy with a incidence between 1-4/1000 live term births (Thornberg et al.,
1995; Ekert et al., 1997; Smith et al., 2000; Carli et al., 2004). They are also the primary cause of morbidity and mortality in preterm infants with incidence correlating to the degree of prematurity and approximately 50% of survivors sustaining permanent brain damage (Volpe, 2001; Himmelmann et al., 2005). The major neurological sequela of this class of injuries is the group of motor deficits collectively termed cerebral palsy, but cognitive deficits and seizures are also common (Volpe, 2000). Among the causes of perinatal H/I are birth asphyxia, postnatal cardiac insufficiency, intrauterine exposure to infection (chorioamnionitis), and prematurity (Hagberg and Mallard, 2000; Garnier et al.,
2003). As the occurrence of these causes is often impossible to predict and treat acutely, strategies must be pursued to address repair of the brain after an insult has occurred.
24 Effects of perinatal H/I on the SVZ
The course of injury following perinatal H/I has been established using a model of
unilateral carotid artery ligation followed by systemic hypoxia in the 7-day-old rat (Rice
et al., 1981; Vannucci and Vannucci, 1997). This model produces a unilateral infarct
primarily in the territory of the middle cerebral artery. During the acute recovery period
following this insult, a transition occurs in the type of cell death within the SVZ. Initially
necrotic cell death predominates, but by 12 hours of recovery apoptotic and hybrid deaths are more common (Romanko et al., 2004). Importantly, although some restricted progenitors (PSA-NCAM+ cells) within the SVZ underwent apoptosis following
perinatal H/I, the medial SVZ where putative NSPs reside exhibits very little cell death
(Romanko et al., 2004).
As described previously, much effort has been exerted in characterizing the
generation of new neurons following adult models of stroke. The perinatal brain is still a
developing organ and as such represents an entirely different environment than the adult
brain. In fact the cells most susceptible to damage during H/I in the perinatal rodent
brain appear to be late oligodendrocyte progenitors in the white matter (Ness et al., 2001;
Back et al., 2002). In humans, the most prevalent pathological form of perinatal H/I is
periventricular leukomalacia which consists of a combination of focal cystic and diffuse
white matter damage, and the window of susceptibility to this injury coincides with the
appearance of these oligodendrocyte progenitors (Back et al., 2001). Thus it is clear that
for effective repair following perinatal H/I, any therapeutic strategy will have to address
all the cell types that comprise the nervous system, and not simply neurons. 25
Specific Aims of the Thesis
Our laboratory has been interested in the effects of perinatal H/I on the SVZ, a region known to harbor NSCs throughout life. Based upon previous descriptions of neurogenesis following stroke, we hypothesized that the NSP population of the SVZ would be mobilized following perinatal H/I to provide an increased pool of precursors.
Three specific aims were formulated to address this hypothesis:
Specific Aim 1: Define changes in NSP population following perinatal H/I. We
hypothesized that the NSP population would expand to support increased neurogenesis
following the injury.
Specific Aim 2: Determine the role of the EGF receptor in regulating the NSP response
to perinatal H/I. We hypothesized that increased expression of the EGF receptor would promote sensitivity to EGF and increased proliferation of NSPs.
Specific Aim 3: Determine the role of Notch1 in regulating the NSP response to
perinatal H/I. We hypothesized that Notch1 induction following perinatal H/I would
function to maintain the NSPs and aid in the expansion of the population.
Chapter 2
Summary of Results
NSPs are more abundant in the rodent SVZ following perinatal H/I
A specific marker that labels all NSCs remains elusive, and the gold standard for quantifying NSCs is the neurosphere assay. In this assay SVZ cells are cultured in vitro at low cell density in the presence of epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) where they form free-floating clonal clusters (spheres). These neurospheres are derived from a single NSP and, therefore, reflect the number of NSPs originally plated. We employed this method to assess the number of NSPs between 1 and
3 days of recovery following perinatal H/I and revealed a doubling in the population of
SVZ NSPs between 2 and 3 days of recovery (Figure 2.1). This degree of expansion was not observed in the contralateral hemisphere relative to a normal control animal. 27
Figure 2.1: More neurospheres can be cultured after perinatal H/I. Control and experimental animals were sterilely decapitated at the designated timepoints and the SVZs were dissociated into single cell suspensions. These suspensions were cultured in the presence of FGF-2 (10 ng/mL) and EGF (20 ng/mL). Each replication consisted of pools of 2-3 animals per group. The number of NSPs per hemisphere for controls was compared to H/I ipsilateral (A) and H/I contralateral (B) hemispheres.
Neurospheres were grown in vitro for 6 days in the presence of EGF and FGF-2.
After 6 days in vitro, neurospheres from the injured hemisphere after 3 days of recovery were noticeably larger in diameter than those from contralateral or control hemispheres
(Figure 2.2). This increase in diameter translated to more than a 3-fold increase in
volume compared to sham neurospheres and more than a 2.3-fold increase compared to
contralateral neurospheres. Bulk dissociation of these spheres revealed that the increase
in volume was due to increased numbers of cells and not just increased size of cells. 28
Figure 2.2: Neurospheres grow larger following perinatal H/I. Representative neurospheres from ipsilateral H/I (C), contralateral H/I (D), and control (E) SVZs demonstrate the larger size of spheres from the ipsilateral SVZ. Scale bar in C represents 20 μm. The diameter of 10 neurospheres from each of 4 animals per condition was measured (F, error bars represent a 95% confidence interval).
As the NSPs had been cultured in the presence of both FGF-2 and EGF we evaluated whether these cells were differentially responsive to either FGF-2 or EGF .
These experiments revealed that the NSPs produced larger spheres only when stimulated with EGF; stimulation with FGF-2 alone did not produce this effect. As they were all cultured in vitro for identical periods of time and under identical culture conditions, this result implied that the cells possessed greater sensitivity to growth factors and a shorter cell cycle time. We used a cummulative 3H-thymidine incorporation assay to quantify proliferation in these cultures. As cells synthesize DNA as they proliferate, they 29 incorporate the 3H-thymidine from the culture media. The amount of 3H-thymidine incorporated is directly related to the amount of proliferation. Cells from the ipsilateral hemisphere incorporated 3H-thymidine at a higher rate than either contralateral or control cells, suggesting increased proliferation within the ipsilateral cultures. Moreover, a greater amount of thymidine was incorporated by NSPs from the damaged hemisphere suggesting that a greater proportion of SVZ cells are proliferating ( Figure 2.3 ).
4500 4000 3500 3000 2500 2000 1500 1000 DPM (% of 4h Control) 4h of (% DPM 500 0 4 8 12 16 20 Hours
Figure 2.3: NSPs proliferate more rapidly in the presence of EGF following perinatal H/I. Animals were sacrificed 3 days after H/I and cultured in 20 ng/mL EGF overnight. The following morning 3H-thymidine was added to the culture medium (8 µCi/mL). At 4 h intervals, cells were collected on Whatman filter discs by vacuum, the DNA was precipitated with TCA and 3H-thymidine incorporation was quantified. This figure depicts one representative experiment from 3 repetitions, normalized to the 4 h control. Diamonds represent ipsilateral, squares represent contralateral hemisphere and triangles represent controls. Values are averages ± SEM
Molecular changes within the SVZ could support the increase in proliferation that
we observed in vitro. Analyses of gene expression including microarray analysis and
confirmation by quantitative real-time PCR (qRT-PCR) demonstrated increased 30 expression of the EGF receptor (EGFR) at 48 hours after the injury ( Figure 2.4 ). These
data suggest that perinatal H/I causes an increased sensitivity to one of the critical
mitogens for NSPs that increases their rate of proliferation and recruits quiescent cells into the cell cycle.
Figure 2.4: EGFR is induced following perinatal H/I. RNA was isolated from ipsilateral, contralateral, and sham SVZs 48 h following perinatal H/I and amplified by qRT-PCR. *=p<0.05 vs sham by REST
Perinatal H/I promotes increased Notch signaling in the SVZ
Using qRT-PCR, we determined that Notch1 expression in the ipsilateral hemisphere increased to approximately 2-fold relative to the contralateral hemisphere at
48h ( Figure 2.5 ). Additionally, immunostaining for Notch1 revealed pockets of cells
within the NSP niche of the ipsilateral hemisphere that labeled more intensely than cells
in the contralateral or control SVZs. Further investigation of Notch ligands revealed 31 induction of Delta-like 1 (DLL1) in both hemispheres at 48h of recovery. This suggests that DLL1 may be influenced by hypoxia alone as it was elevated in the contralateral hemisphere; whereas, Notch expression may require signals from the surrounding injured tissue.
Figure 2.5: Notch1 receptor and the Notch ligand, Delta-like 1, are induced following perinatal H/I. RNA was isolated from ipsilateral (solid bars), contralateral (white bars), and sham (line) SVZs 48 h following perinatal H/I and amplified by qRT-PCR. *=p<0.05 vs sham by REST; †=p<0.05 vs. contralateral by REST.
We also examined downstream effectors of Notch signaling. By real-time PCR,
Hes1 expression remained unchanged 48h following perinatal H/I. Hes5 expression was reduced 2-fold in the ipsilateral hemisphere and unchanged in the contralateral hemisphere. However, localization of gene expression using in situ hybridization revealed an intense region of Hes5 expression within the NSP niche (very close to the lateral ventricles) of the ipsilateral hemisphere ( Figure 2.6 ). The reduced expression 32 detected using PCR corresponded to a more generalized decrease in Hes5 expression in the more lateral regions of the SVZ.
Figure 2.6: Hes5 expression is induced within the NSP niche following perinatal H/I. In situ hybridization was performed on cryostat sections of contralateral (A), ipsilateral (B), and control (C) hemispheres using a digoxygenin-labeled RNA probe for Hes5. Arrows in E delineate region of increased Hes5 expression. V = ventricle, cp = choroid plexus. Scale bar in F represents 10 μm.
33
Changes observed in NSPs following perinatal H/I depend on Notch signaling
To determine whether Notch signaling is essential for the expansion of the NSP population, we pharmacologically inhibited the activation of the Notch receptor in vivo during recovery from perinatal H/I by administering the γ-secretase inhibitor DAPT subcutaneously. This drug inhibits the enzyme responsible for processing the full length
Notch receptor into an intracellular fragment (NICD) to convey a signal into the nucleus.
DAPT administration decreased the ratio of NICD to its immediate precursor by ~ 50% compared to vehicle injected animals ( Figure 2.7A ). The decreased production of NICD
correlated with a significantly reduced expansion of the NSPs from ~ 60% increase in the
ipsilateral hemisphere compared to the contralateral hemisphere to ~ 30% increase
(Figure 2.7B). Neurospheres were also differentiated in the presence of serum and stained neural cell markers. The inhibition of Notch activity also correlated with a reduced proportion of tripotential neurospheres (Figure 2.7C). 34
Figure 2.7: Inhibition of Notch activity reduces the effects of perinatal H/I on NSPs. A gamma secretase inhibitor was administered subcutaneously during the first 3 days of recovery from perinatal H/I. (A) The active NICD is reduced in animals injected with inhibitor compared to animals injected with vehicle. (B) There was less than a 30% increase in NSPs following injection of DAPT compared to a 60% increase following injection of vehicle, n=10 DAPT, 9 Vehicle, *=p<0.05 by Student’s t-test, DAPT vs. Vehicle. (C) Fewer neurospheres contained all 3 cell types after injection of DAPT vs. vehicle, n=3 animals per group, p<0.03 by Student’s t-test for tripotential groups.
Changes in the NSP niche underlie the effects of perinatal H/I
In the context of injury, 2 possibilities exist for changes in cell behavior. First, the injury itself could directly induce changes within the cells. Alternatively, the injury could primarily affect the environment surrounding the cells of interest, and changes in this niche could trigger secondary changes in a select population of cells. We 35 hypothesized that the changes we observed in NSPs following perinatal H/I were a secondary effect resulting from the influence of the NSP niche. To test this hypothesis we devised a scheme for inducing an H/I insult in vitro.
When NSPs (in the form of neurospheres) were subjected to in vitro H/I, proliferation was actually reduced compared to control cells as indicated by 3H- thymidine incorporation assays. By contrast, if we first subject a culture of mixed brain cells to in vitro H/I, and subsequently culture normal neurospheres in the presence of these “injured” cultures, we replicate the effect of in vivo H/I on the subsequent in vitro proliferation of NSPs. NSPs cultured with injured brain cells proliferated more than
NSPs grown with undamaged brain cells, and there was an induction of Notch1 expression within these neurospheres ( Figure 2.8 ).
Figure 2.8: NSPs respond to signals from injured tissue following perinatal H/I. Normal neurospheres were subjected to in vitro H/I alone, or neurospheres were cultured in transwells exposed to media from mixed brain cells that had been subjected to in vitro H/I. (A) Neurospheres proliferate more when exposed to mixed brain cells that had been subjected to in vitro H/I, but not when neurospheres are subjected to H/I themselves. White bars=control, black bars=in vitro H/I; *=p<0.05 vs. control by Student’s t-test. (B) Notch is induced in neurospheres following co-culture with mixed brain cells subjected to H/I, but not when neurospheres were subjected to H/I. Values are normalized to controls. 36
Chapter 3
Discussion of Results
Neural stem cells are more abundant following perinatal H/I
Using this neurosphere assay, we have demonstrated that there are nearly twice as many NSPs present in the brain by 3 days after perinatal H/I. Controversy has arisen regarding which cells in vivo are actually capable of generating neurospheres in vitro, specifically whether they are stem cells or transit-amplifying cells (Doetsch et al., 2002;
Reynolds and Rietze, 2005). Additional data from our studies can address this concern
(see Chapter 7). Following bulk passaging of primary neurospheres, there is a higher rate of secondary sphere formation. This demonstrates not only self-renewal, but also suggests an increase in the occurrence of symmetric, expansive divisions. Furthermore, neurospheres isolated after perinatal H/I more frequently give rise to all 3 neural cell types following differentiation. Finally, the 3H-cumulative thymidine incorporation experiment showed that perinatal H/I increased the proportion of proliferating cells, suggesting that this insult recruits quiescent NSCs to cycle more rapidly. Together, these data suggest that the increase in neurospheres following perinatal H/I is highly specific to multipotential, self-renewing neural precursors.
An uncertainty that still remains is the extent to which “restricted” cells can revert to function as stem cells. Recent discoveries have shown unprecedented plasticity within 37 adult stem cell populations, leading to the suggestion of a “stem cell continuum”
(Quesenberry et al., 2005). With specific regard to NSCs, evidence shows that EGF stimulation can promote stem cell-like behavior in transit-amplifying type C cells
(Doetsch et al., 2002). Our studies have shown that cells within the SVZ exhibit increased sensitivity to EGF signaling due to induced expression of the EGFR after perinatal H/I, suggesting that this may be an example of a condition promoting acquisition of a stem cell phenotype by these transit-amplifying cells.
NSPs respond to the injured environment
An important consideration in these studies is that the observed effects are specific to the ipsilateral hemisphere, suggesting a requirement for injury. In this model, the contralateral hemisphere is not rendered ischemic and remains undamaged despite transient exposure to systemic hypoxia. Although increased proliferative markers were seen within the contralateral medial SVZ, there was no significant increase in neurosphere formation, nor in Notch1 and EGFR expression (see Chapter 7).
Furthermore, the neurosphere preparations from the contralateral hemisphere did not exhibit the same increase in proliferation as neurospheres from the ipsilateral hemisphere
(see Chapter 8).
It is likely that NSPs respond to diffusible signals produced during recovery from injury because the NSP compartment itself is relatively resilient in this injury model compared to the rest of the SVZ (Romanko et al., 2004). Previous work in adult models 38 of stroke has shown that newly generated cells can migrate to areas of injury, suggesting the capacity to respond to chemoattractants (Arvidsson et al., 2002; Parent et al., 2002).
Support for this theory is also garnered by the fact that exposure to H/I conditions in vitro will not directly induce these changes in neurospheres, but exposure of normal neurospheres to mixed brain cells that had previously been exposed to H/I can alter the neurospheres’ function. These data suggest that signals from the NSP niche trigger changes in these precursors in response to injury, a promising consideration for future therapeutic developments.
The NSP population actively expands following perinatal H/I
The increase in NSPs following perinatal H/I could represent recruitment of quiescent NSPs without significantly altering the intrinsic properties of these cells; however, our data suggest that the explanation is not that simple. First, when grown as neurospheres and passaged, NSPs exhibit a higher rate of secondary sphere formation after perinatal H/I (see Chapter 7). This assay is indirectly reflective of the rate of symmetric versus asymmetric cell division occurring within the population (Reynolds and Weiss, 1996). The higher rate of secondary neurosphere formations indicates more frequent proliferative symmetric divisions. This is the same mechanism by which the neuroepithelial stem cell population initially expands during development before switching to predominantly asymmetric cell divisions (Caviness et al., 1995). Secondly, the proliferative activity of NSPs following perinatal H/I is much higher as reflected by 39 the growth of larger neurospheres and the increased rate of 3H-thymidine incorporation.
Together these data support an active model of population expansion following perinatal
H/I rather than only recruitment of quiescent NSPs.
Increased EGF signaling is an important contributor to NSP expansion following perinatal H/I
The EGFR signaling pathway is an essential mitogenic signal for NSPs in vivo.
Infusion of exogenous EGFR ligands, EGF or TGFa, into the brain significantly increases proliferation within the SVZ (Craig et al., 1996; Kuhn et al., 1997). Furthermore, mice deficient in either EGF or transforming growth factor alpha (TGFα) exhibit substantially compromised proliferation within the SVZ and fewer neurosphere forming cells (Tropepe et al., 1997). More recent evidence actually suggests that transit-amplifying cells can possess the capacity to behave as stem cells under EGF stimulation (Doetsch et al.,
2002). Our data demonstrated increased EGFR expression in the SVZ 48 hours after perinatal H/I. We furthermore showed functional consequences of this increased expression, including growth of larger neurospheres in vitro and increased prolilferation.
These data suggest that changes in the sensitivity to EGF signaling play an important role in stimulating the expansion of the NSP population following perinatal H/I.
Interestingly, the increase in EGFR expression appears to be dependent upon signals from differentiated cells during recovery from injury. Based on our analysis of
3H-thymidine incorporation following in vitro H/I neurosphere proliferation cannot be
directly increased by H/I injury, but it can be stimulated by exposure to injured mixed 40 brain cells. Studies have shown that FGF-2 stimulation can induce EGFR expression within NSPs (Ciccolini and Svendsen, 1998). Many studies have shown that FGF-2 is strongly induced following brain injury, primarily within glial cells (Alzheimer and
Werner, 2002). This suggests a link between increased levels of FGF-2 and the induction of EGFR expression in this model, and this is supported in part by our demonstration that the change in proliferative behavior of NSPs following H/I requires some stimulation from other injured brain cells.
Notch signaling is an important contributor to NSP expansion following perinatal H/I
Previous studies have firmly established the importance of Notch signaling in the maintenance of NSPs. Mice genetically deficient in Notch1 or other pathway components, including presenilin-1 (γ-secretase) or RBP-Jκ, exhibit a progressive loss of
NSPs as reflected by declining ability to generate neurospheres, an defect that can be rescued with the introduction of activated Notch1 (Hitoshi et al., 2002). Inhibition of
Notch activity in neurospheres in vitro, either pharmacologically or genetically, also reduces the self-renewal capacity of NSPs to generate secondary neurospheres
(Chojnacki et al., 2003). These effects are likely dependent upon Hes1 and Hes5 activity because the lack of both these genes causes a similar deficiency, although loss of only one can be compensated for by the other (Ohtsuka et al., 2001; Kageyama et al., 2005). 41 We have shown increased Notch1 expression within the SVZ 48 hours following perinatal H/I, just prior to the expansion of NSPs. This is accompanied by concomitant increased expression of the Notch ligands (Dll1 and Jgd1) and targets of Notch signaling
(Hes1 and Hes5). We effectively reduced the expansion of the NSP population following perinatal H/I to numbers equivalent to the contralateral hemisphere by transiently reducing Notch signaling with a γ-secretase inhibitor. These data indicate that significant changes in Notch signaling within the SVZ following perinatal H/I are at least partly responsible for the increased numbers of NSPs that we have observed. If this is true, the
Notch pathway would be a promising target for therapeutic manipulation to restore development after injury.
Interestingly, the level of Notch inhibition achieved in these studies is insufficient to appreciably reduce the number of NSPs in the normal brain, but it produces a significant reduction in the number of new NSPs generated in response to the injury. One reason for this could be the short length of treatment. NSCs are generally considered to be slowly cycling populations, and therefore unlikely to be dramatically affected by a brief treatment (Morshead et al., 1994). Upon stimulated proliferation, as demonstrated by our data in the post-perinatal H/I SVZ, they are more likely to encounter cell fate decisions during the treatment course. In this case, with reduced Notch activity, the NSP population would differentiate rather than self-renew or expand. This interpretation is further supported by the demonstration that fewer neurospheres are tripotential following administration of DAPT.
A concern with the use of DAPT is that γ-secretase is involved in other functions in the brain besides Notch cleavage, most notably the processing of amyloid precursor 42 protein (APP). This leaves the possibility that the effect of the inhibitor that we have seen is not due to decreased Notch activity, but rather to altered APP processing. In fact, a recent study has shown that the soluble form of APP (sAPP), generated mostly through cleavage by an α-secretase, actually stimulates proliferation of cells within the SVZ
(Caille et al., 2004). By inhibiting γ-secretase, we would expect a larger amount of APP to be processed via the α-cleavage, thus generating more sAPP and stimulating proliferation within the SVZ. Instead, we see a decrease in these precursors with the inhibition of γ-secretase, suggesting that our results are not due to altered processing of
APP.
Realizing Cajal’s vision – reflections on the future of regenerative medicine
Nearly a century ago, Santiago Ramón y Cajal declared that the brain was a fixed, unchangeable organ without the capacity for regeneration, but knowing the limits of his own capabilities he also threw down a gauntlet calling for future scientists to prove him wrong (Ramón y Cajal, 1928). The time has now come for neuroscience to accept that challenge. While the severity and relative permanence of neurologic injuries still provides fodder for skeptics of brain repair, we now know that stem cells, a necessity for a regenerating organ, exist in the brain and provide the foundation for regeneration to be a reality. Many studies have shown the production of all neural cell types throughout life and enhanced neurogenesis following injuries. Questions still persist as to why these impressive phenomena lead to so little functional recovery after brain damage. As we 43 continue to understand the biology behind NSCs, the answers to these questions will become apparent. More importantly, the means by which we can improve these effects therapeutically will be revealed. Fortunately we can build upon a wealth of knowledge from studies of stem cells in other organ systems, determining both the commonalities and differences between these cells and NSCs. Already, attempts at manipulating the responses of NSCs to various brain insults are yielding promising results. With continued diligence, we will be able to show that a vision of brain regeneration offered by a pioneering neuroscientist long ago is not a dream, but a reality.
44
Chapter 4
Conclusions and Future Directions
Perinatal H/I is a devastating injury resulting in severe neurological compromises in children. It is estimated that up to 90% of these insults are ante- or intra-partum.
Given the difficulty of intrauterine diagnosis and treatment, prevention of these injuries may not always be possible. This necessitates alternative options to pursue after an insult has occurred to address the long-term consequences. This thesis demonstrates a basis for regenerative therapy using endogenous NSPs. Perinatal H/I increases the size of this invaluable cell population during the acute recovery period, and this work has identified strong candidates for molecular mechanisms regulating this response.
The promise of these results is tempered by the extent of what we still do not know regarding the response of NSPs to brain injury. First and foremost, it is necessary to define specifically which cells provide the basis of this expansion. Do putative NSCs initiate the response, or is this predominantly an effect on the transit amplifying population. This may have implications on the duration this response can last because transit amplifying cells typically have a finite limit of self-renewal; whereas, true NSCs are indefinitely self-renewing. One obstacle to this study is a general lack of consensus within the field as to which cells constitute which population. This problem will be resolved as better and more specific, markers are developed.
This thesis presents a glimpse of an expansion of NSPs during the first 3 days after perinatal H/I. This is a very short period, and it will be useful to determine what 45 happens to this response over a longer time span. Our laboratory is in the process of investigating this aspect, and we have data that this response is sustained for at least 1 week after the insult. Furthermore, it is essential to determine what happens to the additional cells that are generated. As described throughout this thesis, the birth of new neurons is a common event following brain injury. This is likely one fate of these new
NSPs, but they may also contribute to massive gliosis and the formation of an astrocytic scar. Furthermore in light of the prominence of white matter injury in perinatal H/I, it is important to know the extent to which the expanded population of NSPs is capable of producing new oligodendrocytes. This knowledge will help to determine whether this response is truly a beneficial attempt at regeneration or merely a prelude to scarring that will ultimately hinder repair.
We have reduced the expansion of NSPs by pharmacologically inhibiting the activation of Notch1; however, the compound used was an inhibitor of gamma secretase, an enzyme that has functions independent of its action on Notch signaling. Analysis of neurosphere numbers after perinatal H/I in mice genetically deficient in Notch1 or other downstream components of the pathway would selectively target this pathway and provide better support for a role for Notch signaling in the expansion of NSPs. One problem with this strategy is that Notch1-/- mutants do not survive and exhibit severely abnormal brain development; therefore, these studies would require the use of heterozygotes or inducible knockouts of Notch. It also may be possible to inhibit specific aspects of Notch signaling via RNAi approaches or antibody blockade of individual ligands. These studies would further define how Notch signals promote expansion of the
NSP population. 46 The ultimate hope is that this research can be translated into a therapeutic strategy to promote regeneration. One means by which this may be accomplished is to enhance the expansion of NSPs following injury. Our evidence that Notch is responsible for this expansion provides a promising target to achieve this goal. Data have shown that gp130 signaling can induce Notch signaling, thereby promoting self-renewal of NSCs. LIF is a cytokine that signals through a complex involving gp130; therefore, administration of
LIF could further increase the expression of Notch receptors. Alternatively, soluble DSL ligands conjugated to antibodies are capable of activating Notch receptors and could be used to increase Notch activity. 47
Chapter 5
Neural stem/progenitor cells initiate a regenerative response to perinatal hypoxia/ischemia
Introduction
Perinatal hypoxia/ischemia (H/I) is a disruption of the delivery of blood and
oxygen to the brain and represents a primary cause of neurologic injury during the
perinatal period. Perinatal H/I is estimated to occur in 1-2/1000 live term births, and
approximately 50% of surviving preterm infants sustain permanent brain damage from
this type of injury (Volpe, 2000). The neurologic sequelae of the injury include cerebral
palsy, epilepsy, and cognitive deficits. Current therapeutic strategies are aimed at
preventing brain damage, but at present, there are no effective means to repair the brain
once damage has occurred. Moreover, as there is accumulating evidence that most of
these insults occur in utero, prevention may prove difficult, demanding that regenerative
strategies be pursued to reduce the morbidity associated with these events.
Many studies have shown that the brain possesses a limited capacity for
endogenous regeneration following various brain insults including ischemia, traumatic
brain injury and focal apoptosis (Magavi et al., 2000; Arvidsson et al., 2002; Parent et al.,
2002; Goings et al., 2004). Most of these investigations focused on neuronal production following injury to the adult brain, but perinatal H/I occurs during a critical period of brain growth and development. Neurogenesis alone would, therefore, be insufficient to properly restore development. In particular, the white matter and its resident 48 oligodendrocyte progenitors are especially vulnerable to perinatal brain injuries (Ness et al., 2001; Back et al., 2002). It is now well established that neural stem cells (NSCs) as well as multiple classes of transit-amplifying progenitors, some of which express qualities of a true stem cell under certain conditions, reside in the subventricular zone
(SVZ) of the mammalian brain throughout life (Reynolds et al., 1992; Palmer et al., 1995;
Sanai et al., 2004). As it is still not possible to discern NSCs from multipotential progenitors that possess limited self-renewal, we have adopted the term neural stem/progenitor cells (NSPs) to encompass both populations. These NSPs provide the greatest potential to regenerate not only neurons, but also the various macroglia required to reconstitute a fully functional brain.
The demonstration that neurogenesis occurs following ischemic injury presents 2 possible alternatives: 1) Ischemic injury directly affects committed neuroblasts which then expand and mature into neurons; or 2) Ischemic injury affects unrestricted precursors that subsequently produce new neuroblasts. We hypothesized that ischemic injury first stimulates tripotential precursors. Should this prove true, it would provide additional hope that a broader range of cells could be replaced, a necessity for achieving functional recovery after an ischemic insult.
To test our hypothesis we analyzed the precursor population at early recovery time-points using a rat model of perinatal H/I. Here we demonstrate that perinatal H/I increases the proliferation of cells that are positionally and phenotypically NSPs. We show that twice as many NSP-derived neurospheres can be generated from the affected
SVZ, that precursors within these neurospheres exhibit more frequent symmetric divisions, and that a greater proportion of the spheres generated from the damaged 49 hemisphere are multipotential. Finally, we find that perinatal H/I induces the expression of multiple genes within SVZ known to influence stem-cellness. These results encourage the pursuit of therapies that may amplify the compensatory response initiated by the endogenous tripotential, self-renewing precursors of the SVZ.
Methods
Perinatal hypoxia/ischemia model
Timed pregnant Wistar rats (Charles River, Wilmington, DE) were maintained at
the Penn State College of Medicine by the Department of Comparative Medicine, an
Association for Assessment and Accreditation of Laboratory Animal Care accredited
facility. Animal experimentation was in accordance with research guidelines set forth by
Pennsylvania State University and the Society for Neuroscience Policy on the use of
animals in Neuroscience research. All animals were fed high fat lab chow (Harlan
Teklad, Madison, WI). After normal delivery, the litter size was adjusted to 10 pups per
litter. Cerebral H/I was produced in 6 d-old rats (day of birth being P0) by a permanent
unilateral common carotid ligation followed by systemic hypoxia (Rice et al., 1981;
Vannucci and Vannucci, 1997). Briefly, pups were lightly anesthetized with isofluorane
(4% induction, 2% maintenance). Once fully anesthetized, a midline neck incision was
made and the right common carotid artery (CCA) was identified. The CCA was separated
from the vagus nerve and then ligated using 3-0 silk. The incision was then sutured, and 50 animals were returned to the dam for 3 h. The pups were prewarmed for 20 minutes in
o jars submerged in a 37 C water bath. They were then exposed to 1.5 h of 8% O2/92% N2.
After this hypoxic interval the pups were returned to their dam for recovery periods of 1,
2, and 3 days, at which time they were either decapitated for neurosphere assays or anesthetized and sacrificed by intracardiac perfusion for immunohistochemistry. Sham operated animals were anesthetized, and the carotid was isolated from the vagus but not ligated. They were not subjected to a hypoxic interval. Control animals were separated from the dam for the same amount of time as experimental animals, but were otherwise not manipulated. In all cases the contralateral and ipsilateral hemispheres from experimental animals were examined separately.
Tissue fixation and BrdU labeling
Animals received 1 i.p. injection of 50 mg/kg BrdU 1 hour prior to sacrifice at 48
h. Animals were anesthetized with a mixture of ketamine (75 mg/kg) and xylazine (5
mg/kg) prior to intracardiac perfusion with 3% paraformaldehyde in PBS. The brain was
then extracted and blocked, with cuts made at about 2 mm from the anterior pole of the
brain and 3 mm from the posterior pole of the cerebrum. The sections were then
incubated in 3% paraformaldehyde for 2 hours and cryoprotected using 20% sucrose with
NaN3 overnight. The 20% sucrose solution was replaced with 30% sucrose for 4-6 hours.
The tissue was removed and frozen in embedding medium (O.C.T., Miles, Inc., Elkart,
IN) on a dry ice/ethanol slush. Frozen coronal sections (12 μm) were cut on a cryostat 51 and mounted on Superfrost Plus microscope slides. Sections were stained with anti-BrdU
(Cappel, 1:10) as described previously (Levison et al., 1996).
Antibodies and Immunohistochemistry
Immunohistochemistry was performed on cryostat brain sections collected at 48 h
of recovery. Primary antibodies were mouse monoclonal IgG2a anti-PCNA (Santa Cruz;
1:50) and mouse monoclonal IgG1 anti-Nestin (Developmental Studies Hybridoma Bank;
1:5). Secondary antibodies were FITC-conjugated goat anti-mouse IgG2a (Southern
Biotech; 1:100) and TRITC-conjugated goat anti-mouse IgG1 (Southern Biotech; 1:100).
Sections were thawed in cold PBS and incubated for 10 min in 1% H2O2 in PBS followed
by an incubation in 0.5% NP-40 in PBS for 10 min. The slides were washed in PBS and
blocked with PGB super block (10% BSA, 0.05% NaN3, 10% normal goat serum in PBS) for 1 h at room temperature. The superblock was aspirated and 1° antibodies were applied in a 1:5 dilution of PGB super block with 0.05% Triton-X-100 and incubated
overnight at 4°C. The slides were rinsed extensively with PBS and then incubated in 2° antibodies for 1h at 37°C. The sections were washed, counterstained with DAPI, and coverslipped with Gel/Mount (Biomeda, Foster City, CA). For Notch1 immunohistochemistry, cryosections were washed in PBS and treated with 5%
BSA/0.2% triton-x-100 for 1 h. Polyclonal antibodies against the intracellular domain of the Notch receptor were diluted (1:50) in the same blocking solution and incubated
overnight at 4°C. Sections were treated with biotin-labeled secondary antibodies and 52 finally in chromogenic solution (DAB/oxygen peroxide). Sections were dehydrated, cleared and mounted in Vectamount.
The dorsolateral SVZ (SVZDL) was assessed at the level of the anterior
commissure (-0.6 mm from Bregma). Single positive PCNA cells and double positive
PCNA/Nestin cells were counted in the ependyma, subependyma and total medial region
of the SVZDL. The total medial region of the SVZDL represented a 80x100 μm field. The ependyma was demarcated as the ciliated pseudostratified layer of cells lining the intact ventricles and the most medial SVZ (SVZm) as those cells within 4 nuclear diameters from the ependymal layer. The Abercrombie correction was applied to all cell counts
(Abercrombie, 1946). The data are presented as the number of PCNA+ or
+ + PCNA /Nestin cells per field in the ependyma, SVZm, and the total medial SVZDL
(Figure 5.1). All counts were conducted on 3 non-adjacent sections per animal from 4
experimental animals. 53
Figure 5.1: Diagram of the brain regions analyzed. (A) View from the top of the brain. Dashed lines represent the region that was blocked for neurosphere assays and RNA isolation. Dotted line indicates approximate level at which sections were taken for Figures 2, 3, and 4. (B) Coronal section at the level of the dotted line in panel A. The region bounded by dashed lines delimits the region dissected for neurosphere assays. The smaller region bounded by the dotted line was removed for RNA isolation. (C) Representation of the medial dorsolateral SVZ (SVZDL) as shown in Figures 2, 3, and 4. LV – lateral ventricle, Ep – ependymal layer, SVZm – 3-4 cell diameters subjacent to Ep, Total – entire medial SVZ including Ep and SVZm (italicized terms are those utilized in figures 3 and 4).
In situ hybridization
Cryosections were thawed and postfixed for 15 min in 4% paraformaldehyde.
After several rinses in phosphate buffer with 0.1% Tween-20 (PBT) they were treated
with Proteinase K (1 ug/ml in phosphate buffer) then rinsed thoroughly in PBT followed
by another postfix in 4% paraformaldehyde, further rinses, and treatment in 0.25% acetic
anhydride in 100mM triethanolamine. Sections were then rinsed and placed in 54 humidified slide chambers and hybridized overnight at 65°C in hybridization solution
(10mM Tris, 100mM EDTA, 600mM NaCl, 0.25% SDS, 10% dextran sulfate, 1X
Denhardt’s solution, 200 ug/mL yeast tRNA, and 50% formamide) with digoxigenin- labeled riboprobe against Hes5 generated according to manufacturer’s instructions
(Roche). Following hybridization, slides were rinsed in 5X SSC, then 1X SSC/50% formamide at 65°C for 30 min, then treated with RNase (20 ug/ml) in TNE (10 mM Tris,
1 mM EDTA, 500 mM NaCl) for 30 min at 37°C. Slides were then washed in 2X SSC and 0.2X SSC for 20 min each at 65°C. Following 2 rinses in MABT (100mM maleic acid, 150 mM NaCl, 0.1% Tween-20) sections were blocked with 1:1 mixture of 10%
Blocking Reagent (Roche) and Tris buffered saline for 15 min. Sections were incubated overnight at 4°C in alkaline phosphatase-conjugated anti-digoxygenin (Promega, 1:500) in blocking buffer. The following day after thorough rinsing in MABT and preincubation with levamisole, sections were developed with NBT/BCIP.
Primary neurosphere propagation
Wistar pups (P7, P8, or P9) were sacrificed by decapitation, and their brains
removed using aseptic techniques. Coronal sections were taken 2 mm from the anterior pole of the brain, excluding the optic tracts, and 3 mm posterior to the previous cut. The sections were then placed in dishes containing PGM solution (PBS with 1 mM MgCl2 and 0.6% dextrose), where the SVZ was dissected out under a microscope. The tissue was mechanically minced and enzymatically dissociated for 5 min at 37°C using a 55 solution of 0.01% Trypsin/EDTA (Invitrogen, Carlsbad, CA) in PGM with 250 ug
DNaseI (Sigma Aldrich, St. Louis, MO), after which an equal volume of 0.02% Trypsin inhibitor (Sigma Aldrich, St. Louis, MO) in ProN media (DMEM/F12 1:1 media containing 10ng/ml d-biotin, 25 μg/ml insulin, 20 nM progesterone, 100 μM putrescine,
5ng/ml selenium, 50 μg/ml apo-transferrin, 50 μg/ml gentamycin) was added. The tissue was triturated in Pro-N media to obtain an even cell suspension. Triturations were done with progressively less media and smaller Eppendorf filter tips with time allowing for the tissue to settle and the supernatant removed and placed in separate tubes before adding new media for the next round of trituration. The cell suspension was then passed through a 40 μm Nitex screen. The number of viable cells was determined with a hemocytometer by exclusion of 0.1% Trypan Blue dye. The cells were plated into plastic 12 well tissue culture plates at a density of 1 x 105 cells/ml in Pro-N media supplemented with 20 ng/mL EGF and 10 ng/ml FGF-2. Cell cultures were fed every 2 days by removing approximately half the media and replacing it with an equal volume of fresh media.
Secondary neurosphere propagation
Primary neurospheres were collected from 12 well plates after 6 days in vitro and
pelleted by centrifugation at 1200 rpm for 5 min. The neurospheres were enzymatically
dissociated for 5 min at 37°C in a 0.01% Trypsin/EDTA solution in GHCKS buffer (11
mM Glucose, 20 mM HEPES, 10 mM Citrate, 4 mM KCl, 110 mM NaCl, 0.002 g/L
Phenol Red). Trypsin was inhibited by adding an equal volume of 0.02% Trypsin 56 inhibitor in ProN. The spheres were dissociated by trituration in ProN media using progressively less media and smaller Eppendorf filter tips. The number of viable cells was determined with a hemocytometer via exclusion of 0.1% Trypan Blue dye. The cells were plated into a plastic 12-well tissue culture plate at a density of 5 x 104 cells/ml in
Pro-N media supplemented with 20 ng/mL EGF and 10 ng/ml FGF-2. Cell cultures were
fed every 2 days by removing approximately half the media and replacing it with an
equal volume of fresh media.
Neurosphere quantitation
A neurosphere was defined as a free-floating, cohesive cluster of at least 8 cells,
although the vast majority of neurospheres (>98%) were substantially larger than this.
Plates were gently shaken before counting each well to ensure an even distribution of spheres. Ten random 10X fields were counted per well. The frequency of sphere-forming cells (i.e. NSPs) was calculated from the average number of spheres per field, the area of the field, and the area of the well. The number of NSPs per hemisphere was then extrapolated by applying the frequency of sphere-forming cells to the total number of cells obtained in the initial dissociation of the tissue.
57 Neurosphere immunohistochemistry
Spheres were collected and resuspended in 5% horse serum in CNM-2 media
(DMEM/F12 1:1 containing 10ng/ml d-biotin, 5 ng/ml insulin, 20 nM progesterone, 100
μM putrescine, 5ng/ml selenium, 50 μg/ml apo-transferrin, 50 μg/ml gentamycin, 150 μl of 0.5M kynurenic acid) at an approximate density of 100-200 spheres/mL. 100 μL of the neurosphere suspension was then plated in plastic 24-well tissue culture plates onto flame-sterilized coverslips precoated with 1% w/v poly-d-lysine and 10 μg/mL laminin.
The spheres were allowed to attach in a 37°C incubator for a minimum of 1.5 hours, after which 400 μl of CNM-2 supplemented to 5% horse serum was added to the well. After
16-20 h, this media was replaced with CNM-2 with 0.03% DMSO. Cultures were allowed to differentiate for five days, with media replenished on day 3. After the differentiation period, the cells were fixed using 2% paraformaldehyde for 15 min, and washed twice with BCH (10% bovine calf serum in Earle’s basal medium with 4.8 mg/mL HEPES). Sections were stained at room temperature for 45 min with O4 culture supernatant diluted 1:3 in BCH supplemented with 10% lamb serum. After thoroughly rinsing, the cells were incubated for 45 min at room temperature in GAM IgM LRSC
(Jackson, Bar Harbor, MN, 1:200). The cells were then permeabilized with BCH-S
(BCH with 0.5 mg/ml Saponin) and stained for 45 min at room temperature with anti-
TuJ1 (Promega, Madison, WI, 1:250) and anti-GFAP (Roche, Indianapolis, IN, 1:200) in
Saponin diluent (BCH-S supplemented with 10% lamb serum). Cells were then incubated for 45 min at room temperature in GAM IgG FITC (Jackson, Bar Harbor, MN,
1:400) and GAR AMCA (Jackson, Bar Harbor, MN, 1:200) in Saponin diluent. The 58 coverslips were rinsed and mounted onto microscope slides with Gel/Mount (Biomeda,
Foster City, CA) and allowed to dry overnight. Colonies were scored according to the types of cells present. Images of stained cells were collected using a SenSys cooled- coupled device camera (CRI, Inc., Woburn, MA) interfaced with IP Lab scientific imaging software (Scanalytics, Fairfax, VA) on an Olympus BX-40 microscope.
RNA isolation
SVZs were dissected out of control and the ipsilateral hemisphere of H/I animals and snap frozen on a dry ice/ethanol slush and immediately stored at –80°C. Dissections
were directed toward the angles of the ventricles to avoid any confounding effects of
ventricular hypertrophy due to striatal degeneration. Tissue samples were then thawed
directly into 0.5 mL Trizol reagent (Molecular Research Center, Cincinnati, OH) and
homogenized using a tissue homogenizer. 100 uL of chloroform was added, and the
samples were centrifuged at 13,000 rpm for 15 min at 4°C. The aqueous phase was then
transferred to a new tube. After adding 250 uL of 70% EtOH, the aqueous phase was
applied to an RNeasy Mini-spin column (Qiagen, Valencia, CA) to remove contaminants
from the RNA, according to manufacturer’s instructions. The concentration of total RNA
was determined by measuring optical density on a spectrophotometer (Becton-Dickson,
Franklin Lakes, NJ). RNA samples were stored at -80°C until needed.
59 Real-time PCR
Total RNA was isolated from 5 individual SVZs per group using TriReagent
(Molecular Research Center, Cincinnati, OH) and RNeasy kits (Qiagen, Valencia, CA)
according to manufacturers’ protocols. 2 μg of was reverse transcribed to cDNA using the Qiagen Omniscript RT kit supplemented with random nonamer primer (Sigma
Aldrich, St. Louis, MO) and RNaseIN (Promega, Madison, WI). Primer pairs specific for
the genes of interest were designed using Lux Primer Design software (Invitrogen,
Carlsbad, CA) or obtained from catalogued TaqMan gene expression assays (Applied
Biosystems, Foster, City, CA) ( Table 5.1 ). Amplification was carried out in 96-well
plates using Platinum-UDP Supermix kit according to the manufacturer’s instructions
(Invitrogen, Carlsbad, CA) and analyzed on an ABI Prism 7700 Sequence Detection
System (Applied Biosystems, Foster City, CA). Fold-changes in gene expression relative to a housekeeping gene were obtained using the Relative Expression Software Tool
(REST) for groupwise comparison and statistical analysis of relative expression results in
real-time PCR (Pfaffl et al., 2002).
Table 5.1: Primers used in qRT-PCR Transcript Primers
EGFR (TaqMan) Applied Biosystems TaqMan Assay ID Rn00580398_m1
5’-CAC GTA CTG CGA GCT GCC CTA CG[FAM]G-3’ Notch1 (Lux) 5’-GGC AGG TGC CTC CGT TCT-3’
18S (TaqMan) Applied Biosystems TaqMan Assay ID Hs99999901_s1
18S (Lux) Invitrogen catalog #115HM-01
60
Statistics
The normality of each data set was verified using the Shapiro-Wilks numerical test for normality. Student’s t-tests for independent samples were conducted to analyze differences between ipsilateral and untouched control hemispheres and between contralateral and untouched control hemispheres. Neurosphere differentiation data were analyzed by two-way ANOVA followed by Bonferroni/Dunn post-hoc tests. Statistical tests were conducted using SPSS software (SPSS Inc., Chicago, IL) or SAS software
(SAS Institute Inc., Cary, NC).
Results
Markers of cell proliferation increase in the medial SVZ following perinatal H/I
The SVZ represents the postnatal remnant of the embryonic germinal zones and continues to produce several cell types after birth (Brazel et al., 2003). The cellular architecture of the SVZ has been highly characterized, revealing that NSPs reside in the most medial region of the SVZ, subjacent to the ependymal layer (Garcia-Verdugo et al.,
1998; Chiasson et al., 1999; Capela and Temple, 2002). To determine how cell proliferation in the SVZ is affected by perinatal H/I, we injected BrdU into rat pups at 48 hours following the insult and perfused them 1 hour later. In the injured brain many 61 BrdU+ cells were found adjacent to or among the ependymal lining at 48 hours; whereas almost all BrdU+ cells in control pups were approximately 5-6 cell diameters removed
from the ependymal lining (Figure 5.2A-C). BrdU-labeled cells in sham-operated
animals with hypoxia showed the same distribution as uninjured control animals after 48
hours of recovery (Snyder, 2001). Restricting counts to the NSP niche, within 4 cell
diameters of the ependymal layer, revealed an increase in the number of BrdU+ cells in
the ipsilateral hemisphere compared to the uninjured contralateral hemisphere (Figure
5.2D). Despite this very selective increase in BrdU labeling within the NSP niche, there
was no difference in the total number of BrdU+ cells within the entire ipsilateral SVZ
compared to controls at this time point (data not shown). 62
Figure 5.2: Hypoxia-ischemia increases the number of BrdU+ cells adjacent to or among the ependymal lining in the ipsilateral SVZ. Control and experimental animals received a single IP injection of BrdU one hour prior to sacrifice at 48 h recovery from hypoxia/ischemia. Cryostat tissue sections were immunostained for BrdU incorporation (brown) and counterstained with hematoxylin (blue). (A) BrdU labeling in a control brain. (B,C) BrdU labeling in the ipsilateral hemisphere. An occasional process apparently extending from a BrdU labeled nucleus contacted the lateral ventricle in the ipsilateral SVZ (arrow in C). (D) Quantitative results for the number of BrdU positive cells per section in from ipsilateral and contralateral hemispheres. Scale bar represents 30 μm in A and B, 10 μm in C.
63 Other sections were processed for PCNA (red) and Nestin (green) immunohistochemistry at 48 hours of recovery (Figure 5.3A-C). PCNA is a protein
expressed during S phase of the cell cycle and is frequently used as another marker of cell
proliferation (Celis et al., 1986). There was approximately a 4-fold increase in the
number of PCNA+ cells in the NSP niche in the ipsilateral hemisphere of injured pups compared to controls (Figure 5.3G “SVZm”; p<0.0003). A smaller increase also
occurred in the contralateral hemisphere (Figure 5.3G, “SVZm”; p<0.002). Again, there
was no significant difference between control and injured pups in the number of PCNA+
cells found in the entire medial SVZ (Figure 5.3G, “Total”). 64
Figure 5.3: Hypoxia-ischemia increases the number of PCNA+ and PCNA+/Nestin+ cells adjacent to or among the ependymal lining in the ipsilateral SVZ. Cryostat tissue sections from control and experimental animals were immunostained for PCNA (red) and Nestin (green). DAPI (blue) is the nuclear stain in D-F. Panels represent control (A,D), H/I contralateral (B,E), and H/I ipsilateral (C,F) hemispheres. The number of PCNA+ cells (G) or PCNA/Nestin double-positive cells (H) per field of view was quantified for the entire medial SVZ (total), the ependymal layer (Ep), and those cells within 4 nuclear diameters of the ependymal layer (SVZm). Counts were conducted on 3 non-adjacent sections per animal. Data represent the mean ± SEM of 4 independent experiments. * = p<0.002 v control; ** = p<0.0003 v control by two-tailed t-test. Scale bar represents 10 μm. 65 To establish which cells were dividing in the SVZ following perinatal H/I we combined immunohistochemistry for PCNA and Nestin, an intermediate neurofilament frequently used to identify NSPs (Figure 5.3D-F). This analysis revealed more than
twice as many double-positive cells, as those depicted in the inset and by the arrows in
panel D, in the most medial region of both the contralateral and ipsilateral SVZs of H/I
pups compared to controls (Figure 5.3H, “SVZm”; p<0.002). This increase persisted into
the ependymal layer in the ipsilateral hemisphere, but not in the contralateral hemisphere
of injured animals (Figure 5.3H, “EP”). Once again, there was no significant difference throughout the medial SVZ as a whole in the number of PCNA/Nestin double-positive cells (Figure 5.3H, “Total”).
More neurospheres can be isolated from the SVZ following perinatal H/I
The clonal neurosphere assay is a critical method for quantifying numbers of
NSPs in the brain. To determine whether the dividing cells observed following perinatal
H/I were NSPs, we microdissected the SVZ from control, contralateral, and ipsilateral
hemispheres, dissociated the cells, and cultured them at clonal density in the presence of
EGF and FGF-2 where they formed primary neurospheres. Quantitative analysis of the
number of neurospheres revealed no difference at 1 or 2 days after the injury; however,
almost twice as many neurospheres were generated from the ipsilateral hemisphere than
from uninjured controls by 3 days of recovery (Figure 5.4A; p<0.05). By contrast there was no significant difference in the number of neurospheres generated from the 66 contralateral hemisphere (Figure 5.4B). Repeated experiments using lower cell-seeding
densities and sham-operated animals rather than normal controls yielded similar results.
After 6 days in vitro, neurospheres from the injured hemisphere after 3 days of recovery
were noticeably larger in diameter than those from contralateral or control hemispheres
(Figure 5.4C-F). This increase in diameter translated to more than a 3-fold increase in
volume compared to sham neurospheres and more than a 2.3-fold increase compared to
contralateral neurospheres. 67
Figure 5.4: Hypoxia-ischemia increases the number of colony-forming NSPs and the self-renewal of NSPs in the ipsilateral SVZ. Control and experimental animals were sterilely decapitated at the designated timepoints and the SVZs were dissociated into single cell suspensions. These suspensions were cultured in the presence of FGF-2 (10 ng/mL) and EGF (20 ng/mL). Each replication consisted of pools of 2-3 animals per group. The number of NSPs per hemisphere for controls was compared to H/I ipsilateral (A) and H/I contralateral (B) hemispheres. Representative neurospheres from ipsilateral H/I (C), contralateral H/I (D), and control (E) SVZs demonstrate the larger size of spheres from the ipsilateral SVZ. Scale bar in C represents 20 μm. The diameter of 10 neurospheres from each of 4 animals per condition was measured (F, error bars represent a 95% confidence interval). Primary neurospheres generated at 3 days of recovery were dissociated and subcultured to determine the relative self-renewal rates of each group (G). Values represent the mean ± SEM of 4 independent experiments. * = p<0.05 v control by two-tailed t-test. 68
NSPs divide symmetrically more often following perinatal H/I
One of the defining characteristics of a stem cell is the ability to self-renew
through asymmetric cell divisions. Under normal circumstances, asymmetric divisions
maintain the population of NSPs at the same size. Alternatively, symmetric cell divisions
can occur in which a NSP can generate 2 NSP progeny, thereby expanding the population
of NSPs (Potten and Loeffler, 1990). Changes in the mode of cell division can be assessed by passaging primary neurospheres. If all NSPs undergo asymmetric cell
divisions, every primary neurosphere would generate a single secondary neurosphere;
however, if some symmetric divisions occur, a single primary neurosphere will give
multiple secondary neurospheres due to the presence of multiple NSPs within that
primary neurosphere. Secondary neurosphere assays, therefore, provide an index for the
frequency of symmetric cell divisions (Reynolds and Weiss, 1996).
Since we observed an increase in the number of neurospheres generated from the
damaged hemisphere we postulated that this was due to an increase in symmetrical
divisions rather than recruitment of quiescent stem cells. To test this hypothesis, we
performed secondary neurosphere assays to determine the frequency of symmetric
divisions following perinatal H/I. We dissociated primary neurospheres, seeded them at
low density, and established the number of 2o spheres formed after 6 days in vitro.
Primary spheres from the ipsilateral H/I hemisphere generated approximately 30% more
secondary neurospheres than contralateral or uninjured controls (Figure 5.4G; p<0.05). 69
A greater proportion of neurospheres are multipotent following perinatal H/I
To verify that the neurospheres obtained following perinatal H/I were derived from multipotential precursors, we examined the progeny generated after differentiating single neurospheres for 5 days. We assessed differentiation by immunostaining for lineage-specific neural cell markers and quantifying the percentage of neurospheres that were unipotential, bipotential, or multipotential (Figure 5.5). Neurospheres generated
from both control brains and the contralateral hemisphere of injured brains demonstrated
a low frequency of tripotency, although approximately 50% produced at least 2 types of
cells (Figure 5.5A,B). Previous experiments have also shown no difference in tripotency
between sham-operated and untouched controls (6.6% and 3.8%, respectively) (Snyder,
2001). By contrast, more than 75% of neurospheres from the ipsilateral hemisphere of
the injured pups produced at least 2 cell types, and nearly 50% exhibited tripotency
(Figure 5.5C,D; p<0.005 for ipsilateral tripotent v. control tripotent). 70
Figure 5.5: Hypoxia-ischemia increases the proportion of multi-potential SVZ-derived neurospheres. NSPs were harvested from animals at 48 hours of recovery and cultured in vitro as neurospheres in the presence of both FGF-2 and EGF. Spheres were plated onto coverslips and differentiated in growth factor free medium for five days. Neurospheres from control (A), contralateral (B), and ipsilateral (C) SVZs were immunostained for neuronal, astrocytic and oligodendrocytic markers and the percentage of glial or neuron/glial producing spheres was quantified. Panel D depicts a representative multipotential clone from the ipsilateral SVZ stained for a neuronal marker (Tuj1, green), an astrocytic marker (GFAP, blue), and an oligodendrocyte marker (O4, red). Data represents the mean of 6 experiments. *=p<0.005 for the % of ipsilateral multipotential spheres vs. control multipotential spheres using ANOVA followed by Bonferroni/Dunn post-hoc test. Scale bar represents 10 μm.
Perinatal H/I induces the expression of Notch1, gp130, and EGFR mRNA
Investigations into the properties of NSPs are beginning to reveal which
molecules regulate neural “stem-cellness” (Gaiano and Fishell, 2002; Cai et al., 2004).
As the increased frequency of symmetric divisions following perinatal H/I suggests an
alteration of the intrinsic properties of the NSPs, we investigated molecular changes
induced by the injury. We isolated total RNA from the SVZ at 48 h of recovery from the 71 insult and performed a microarray analysis with 288 genes related to neural precursors
(SuperArray Stem Cell Array). This analysis revealed induction of 35 transcripts including a number of growth factors known to support the proliferation of NSCs as well as many markers that identify stem-cellness. By contrast, only 4 stem cell-related transcripts from the array were downregulated in the SVZ following perinatal H/I (See
Appendix A). Of the genes induced, we validated the expression of the genes for 3 receptors and one non-receptor regulator of NSP proliferation and maintenance.
Signaling through the epidermal growth factor receptor (EGFR) is a critical mitogenic pathway for NSPs during development and postnatally (Reynolds et al., 1992;
Tropepe et al., 1999; Kornblum et al., 2000; Gritti et al., 2002). Several ligands act through the EGFR to activate multiple signaling cascades including the JAK-STAT and
MAP kinase pathways. Previous studies have shown that perinatal H/I induces the EGFR ligand HB-EGF (Tanaka et al., 1999); therefore, we asked whether perinatal H/I also affects the capacity of SVZ cells to respond to these growth factors. Using qRT-PCR analysis we observed nearly a 3-fold increase in the expression of EGFR mRNA relative to 18S compared to sham-operated controls and about a 2-fold increase compared to the contralateral hemisphere 48 h following the injury (Figure 5.6A; p<0.005). The mild
increase in EGFR expression in the contralateral hemisphere compared to sham controls was not statistically significant. 72
Figure 5.6: Receptors involved in control of NSP fate are induced by perinatal H/I. Ipsilateral H/I and control hemispheres were dissected out after 48 h of recovery. (A) Total RNA was isolated and amplified by qRT-PCR using primers specific for EGFR and Notch1 and normalized to expression of 18S. n=10 for ipsilateral and contralateral conditions, n=4 for sham condition. * = p<0.05 vs. sham; †= p<0.05 vs. contralateral by pairwise fixed reallocation randomiszation. Immunostaining for Notch1 was performed on cryostat sections from H/I (B) and control (C) animals. In situ hybridization was performed on cryostat sections of contralateral (D), ipsilateral (E), and control (F) hemispheres using a digoxygenin-labeled RNA probe for Hes5. Arrows in E delineate region of increased Hes5 expression. Scale bar in F represents 10 μm. 73 Notch1 is a cell surface receptor that regulates cell fate in both the developing and adult brain (Gaiano and Fishell, 2002). It has been demonstrated in vivo and in vitro that
Notch1 is essential for the maintenance of neural precursors (Hitoshi et al., 2002). Using qRT-PCR, we confirmed a 2-fold induction of Notch1 mRNA (Figure 5.6A; p<0.05)
compared to sham-operated and contralateral SVZs at 48 h of recovery from perinatal
H/I. Immunostaining for the Notch1 receptor also revealed intensely stained pockets of
cells in the ipsilateral hemisphere 48h after perinatal H/I (Figure 5.6B,C). In situ
hybridization demonstrated strong expression of Hes5, an effector of Notch1 signaling,
within the NSP niche of the SVZ, although Hes5 expression throughout more lateral aspects of the SVZ was reduced (Figure 5.6D-F). In addition to these results, we have also confirmed by semi-quantitative RT-PCR a 2-fold increase in the expression of gp130
(data not shown), a member of the component of the IL-6 family of cytokine receptors which may influence Notch1 expression (Chojnacki et al., 2003) and an increase in the expression of LIF (personal correspondence with M. Covey), a molecule implicated in
NSP self-renewal that signals through gp130. Furthermore, we have confirmed maintenance of normal expression levels of Numb, another molecule that has been implicated in precursor cell division (Shen et al., 2002) (data not shown).
Discussion
In this paper we provide evidence of a NSP response that temporally precedes the
increase in neurogenesis following a perinatal H/I insult. We demonstrate that: 1) more 74 cells in the medial SVZ incorporate BrdU and express PCNA following perinatal H/I; 2) more Nestin+ cells in the medial SVZ express PCNA; 3) nearly twice as many neurospheres can be isolated from the ipsilateral hemisphere of the damaged brain by 3
days following perinatal H/I; 4) neurospheres from the ipsilateral hemisphere are larger
than those from the contralateral hemisphere or sham-operated animals; 5) expansive
symmetric divisions are maintained in vitro following perinatal H/I to produce more
secondary neurospheres; 6) a greater proportion of neurospheres cultured after perinatal
H/I are tri-potential; and 7) the NSP-related genes EGFR, Notch1, and Hes5 are induced
within the injured NSP niche. These data represent the first evidence that self-renewing,
tri-potential NSPs initiate a regenerative response to perinatal H/I.
Several reports have demonstrated increased neurogenesis following ischemic
injury in adults (Felling et al., 2003), and recent experiments have confirmed the
occurrence and timing of this response in a mouse model of perinatal H/I (Plane et al.,
2004). Proliferation peaks around 1 week following an ischemic insult, but increases
have been observed as early as 2 days following the injury (Jin et al., 2001; Zhang et al.,
2001; Li et al., 2002; Iwai et al., 2003). Some cells proliferating at this timepoint migrate
from the SVZ into the damaged striatum to differentiate into phenotypically mature
striatal neurons (Arvidsson et al., 2002; Parent et al., 2002). Recent studies also suggest
increased oligodendrocyte progenitor proliferation in the peri-infarct region 1 to 2 weeks
following adult stroke, and restoration of myelin basic protein expression 2 weeks
following mild perinatal H/I (Liu et al., 2002; Tanaka et al., 2003; Zaidi et al., 2004). In
this paper we demonstrate a doubling in the number of NSPs in the SVZ between 2 and 3
days following perinatal H/I, a timepoint well situated to supply an increased precursor 75 pool for these restorative observations. Our evidence that tri-potential NSPs are activated following injury is critical to therapeutic interventions, especially for the newborn infant who has sustained brain damage because oligodendrocyte progenitors are extremely vulnerable to such insults (Ness et al., 2001; Back et al., 2002). Consequently, regenerative strategies in the neonatal brain necessitate multipotential cells, not just neuronal precursors, to repair the brain following H/I insults.
We have documented increased proliferation in the medial SVZ after 2 days of recovery using 2 different markers for dividing cells. BrdU, a thymidine analog that is incorporated into DNA during S phase, is a common marker for cell proliferation. Using a single injection of BrdU, we saw more labeled cells in the medial SVZ 48 h after the injury (Figure 5.2). Only cells that were in S phase during the few hours prior to sacrifice
would have incorporated the BrdU; therefore, this is likely an underestimate of the total
number of cells actually dividing following the injury. We observed a similar increase in
cells expressing PCNA, a protein expressed during S phase in proliferating cells (Celis et al., 1986).
The observed increase in proliferating cells was restricted to the most medial cell layers of the SVZ 48 h following the injury (Figure 5.2D and 5.3G,H), a critical point
because the architecture of the SVZ is such that the most immature cells lie in close
proximity to the ventricle, and lineage restriction increases with progression toward the
more lateral part of the region (Garcia-Verdugo et al., 1998). Thus, the increased
proliferation is occurring preferentially in the NSP compartment of the SVZ. Moreover,
using Nestin as a marker of NSPs, we find a significant increase in the number of
PCNA/Nestin double-positive cells, again unique to the most medial region, providing 76 more support for the conclusion that these dividing cells are NSPs. One consideration for the differences in cell proliferation between the medial and lateral portions of the SVZ is the cell death that occurs following injury. Our laboratory has recently demonstrated that cell death is uncommon in the medial region compared to the lateral SVZ (Romanko et al., 2004). Thus, proliferation may be increased within the lateral compartment as well but offset by extensive cell death.
The neurosphere assay is currently the gold standard for quantifying numbers of
NSPs. Using this assay, we find that there are nearly twice as many NSPs present in the brain by 3 days after perinatal H/I (Figure 5.4E). This is substantiated by the observation
that more Nestin+ cells in the NSP niche are proliferating a day earlier. A similar
increase in numbers of neurospheres has been reported 7 days following ischemic injury
in adult rats (Zhang et al., 2004). Here we show in addition to increased numbers of
neurospheres after cerebral injury, that a significantly larger percentage of these
neurospheres are tri-potential. Demonstrating an increase in tripotent cells is critical
considering that oligodendrocyte progenitors are the most vulnerable cell population in
the immature brain and thus need to be replaced (Ness et al., 2001; Back et al., 2002).
Furthermore, we used secondary neurosphere assays to demonstrate that the NSPs
continue to undergo expansive symmetrical divisions in vitro. Assuming that this
expansion reflects the continuation of a mode of cell division that the NSPs adopted in
vivo, these data provide a mechanism for the increased number of NSPs that we have
observed. The observations of more frequent symmetrical expanding cell divisions favor
a mechanism of increased proliferation of active NSPs rather than the recruitment of
dormant NSPs into the cell cycle. 77 An important point to consider is that the contralateral hemisphere also exhibits mildly increased cell proliferation within the medial SVZ according to our markers of cell proliferation. However, although there was a slight increase in the number of neurospheres obtained from the contralateral hemisphere compared to controls, this difference did not reach significance. Furthermore, there was no change in the differentiation potential of the contralateral neurospheres and no increase in the expression of EGFR and Notch1 within the contralateral SVZ (Figure 5.6A). These data
suggest that some of the dividing cells observed in the contralateral hemisphere may
represent more restricted transit-amplifying cells. In the perinatal rat model we employ,
the contralateral hemisphere becomes hypoxic although it does not become ischemic.
While it is possible that hypoxia alone induced these changes, it is more likely that
diffusible factors from the injured hemisphere, communicating via the lateral ventricles,
contributed to changes in the contralateral hemisphere, as there is a noticeable effect on
the contralateral hemisphere that is not observed as a consequence of a sham operation
followed by a similar hypoxic episode (Snyder, 2001).
In this paper we have described an increase in the number of tripotential NSPs
present in the SVZ following perinatal H/I, and initial molecular analyses reveal
mechanisms supporting this observation. We have demonstrated induction of receptors
critical to NSP proliferation and fate determination (Figure 5.6A). EGF receptor
signaling is critical to the proliferation of NSPs, and infusion of EGF into the lateral
ventricles promotes the acquisition of a NSP phenotype by transit-amplifying cells of the
SVZ (Reynolds et al., 1992; Tropepe et al., 1997; Doetsch et al., 2002). Notch1 and
gp130 are important receptors in determining NSP identity and maintaining the NSP 78 population (Gaiano et al., 2000; Chojnacki et al., 2003). Importantly, there is no induction of EGFR or Notch1 in the contralateral SVZ, decreasing the likelihood that the mild increases in proliferation on the contralateral side are a result of true NSP proliferation. Induction of these genes within the injured SVZ could promote NSP proliferation and prevent cells from progressing toward a more differentiated phenotype by increasing the rate of symmetric cell division following brain injury. The cumulative effects of these receptors could support an increase in NSP numbers, and a rigorous analysis of these mechanisms in the context of this injury is the focus of our ongoing studies.
The last decade has seen tremendous advances in our understanding of the brain; however, translation of this knowledge into clinical settings has been comparatively slow.
Many neurological diseases remain uncurable and severely burden our health care system. Perinatal H/I is the leading cause of brain damage resulting from birth complications, and our current capabilities offer no means of restoring normal development after an insult has occurred. Our data indicate that this injury mobilizes endogenous NSPs and that these cells may be a potential source of regeneration.
Furthering our understanding of the mechanisms underlying this response will present targets for the therapeutic use of these vital cells.
79 Acknowledgements and support
The Nestin monoclonal antibody developed by S. Hockfield was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa
City, IA 52242. We would also like to thank Dr. Gord Fishell for his generous donation of the plasmid used for Hes5 in situ hybridization. This work has been presented in part at past meetings (Levison et al., 2001; Snyder, 2001; Felling et al., 2003). The work was supported by MH 59950 and HD 30705 awarded to S. W. Levison and NS0469903-01 awarded to R. J. Felling.
80
Chapter 6
Increased EGF responsiveness following perinatal H/I alters proliferation of neural stem and progenitor cells
Introduction
Neural stem and progenitor cells (NSPs) responsive to EGF appear during late
stages of embryonic development and persist in the subventricular zone (SVZ) of the
postnatal and adult brain, producing neurons, astroocytes, and oligodendrocytes (Temple,
2001). These cells form multipotent, self-renewing colonies, or neurospheres, in the
presence of EGF in vitro (Reynolds et al., 1992). Recently we have demonstrated an
expansion of this population following perinatal hypoxic/ischemic brain injury (H/I)
(Felling et al., in press). The presence of NSPs within the SVZ throughout life has provided hope that regenerative therapies will be realized for neurologic injury and
disease, but this requires us to understand the basic mechanisms underlying their self-
renewal, proliferation and differentiation.
The epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor
expressed within proliferative regions of the late embryonic and postnatal brain (Seroogy
et al., 1995; Weickert et al., 2000). It also is expressed by cells in neurospheres, and
more recently has been used to prospectively identify neurosphere-forming cells
(Reynolds and Weiss, 1996; Kornblum et al., 2000; Ciccolini et al., 2005). Infusion of
exogenous EGFR ligands, EGF or TGFa, into the brain significantly increases
proliferation within the SVZ (Craig et al., 1996; Kuhn et al., 1997). Furthermore, mice 81 deficient in either EGF or transforming growth factor alpha (TGFα) exhibit substantially compromised proliferation within the SVZ and reduced neurosphere forming cells
(Tropepe et al., 1997). More recent evidence suggests that transit-amplifying cells can possess the capacity to behave as stem cells upon EGF stimulation (Doetsch et al., 2002).
We have previously demonstrated an increase in the number of NSPs within the perinatal SVZ following hypoxic/ischemic (H/I) brain injury. This was accompanied by an increase in EGFR expression within the SVZ. Furthermore, neurospheres isolated after injury grew larger in vitro than their control counterparts. Here we show that this effect is specific to EGF. The larger neurosphere size is a direct result of increased proliferation of NSPs in vitro. Finally, we show that this effect requires signals from cells in the surrounding environment. These data suggest that NSPs acquire an increased capacity to respond to EGF following perinatal H/I. This knowledge may provide a means to enhance the expansion of NSPs to provide a regenerative therapy for this class of neurologic injuries.
Methods
Perinatal hypoxia/ischemia model
Timed pregnant Wistar rats (Charles River, Wilmington, DE) were maintained at
the Penn State College of Medicine by the Department of Comparative Medicine, an
Association for Assessment and Accreditation of Laboratory Animal Care accredited 82 facility. Animal experimentation was in accordance with research guidelines set forth by
Pennsylvania State University and the Society for Neuroscience Policy on the use of animals in Neuroscience research. All animals were fed high fat lab chow (Harlan
Teklad, Madison, WI). After normal delivery, the litter size was adjusted to 10 pups per litter. Cerebral H/I was produced in 6 d-old rats (day of birth being P0) by a permanent unilateral common carotid ligation followed by systemic hypoxia (Rice et al., 1981;
Vannucci and Vannucci, 1997). Briefly, pups were lightly anesthetized with isofluorane
(4% induction, 2% maintenance). Once fully anesthetized, a midline neck incision was made and the right common carotid artery (CCA) was identified. The CCA was separated from the vagus nerve and then ligated using 3-0 silk. The incision was then sutured, and animals were returned to the dam for 3 h. The pups were prewarmed for 20 minutes in
o jars submerged in a 37 C water bath. They were then exposed to 1.5 h of 8% O2/92% N2.
After this hypoxic interval the pups were returned to their dam for recovery periods of 1,
2, and 3 days, at which time they were either decapitated for neurosphere assays or anesthetized and sacrificed by intracardiac perfusion for immunohistochemistry. Sham operated animals were anesthetized, and the carotid was isolated from the vagus but not ligated. They were not subjected to a hypoxic interval. Control animals were separated from the dam for the same amount of time as experimental animals, but were otherwise not manipulated. In all cases the contralateral and ipsilateral hemispheres from experimental animals were examined separately.
83 Primary neurosphere propagation
Wistar pups (P7, P8, or P9) were sacrificed by decapitation, and their brains
removed using aseptic techniques. Coronal sections were taken 2 mm from the anterior pole of the brain, excluding the optic tracts, and 3 mm posterior to the previous cut. The sections were then placed in dishes containing PGM solution (PBS with 1 mM MgCl2 and 0.6% dextrose), where the SVZ was dissected out under a microscope. The tissue was mechanically minced and enzymatically dissociated for 5 min at 37°C using a solution of 0.01% Trypsin/EDTA (Invitrogen, Carlsbad, CA) in PGM with 250 ug
DNaseI (Sigma Aldrich, St. Louis, MO), after which an equal volume of 0.02% Trypsin inhibitor (Sigma Aldrich, St. Louis, MO) in ProN media (DMEM/F12 1:1 media containing 10ng/ml d-biotin, 25 μg/ml insulin, 20 nM progesterone, 100 μM putrescine,
5ng/ml selenium, 50 μg/ml apo-transferrin, 50 μg/ml gentamycin) was added. The tissue was triturated in Pro-N media to obtain an even cell suspension. Triturations were done with progressively less media and smaller Eppendorf filter tips with time allowing for the tissue to settle and the supernatant removed and placed in separate tubes before adding new media for the next round of trituration. The cell suspension was then passed through a 40 μm Nitex screen. The number of viable cells was determined with a hemocytometer by exclusion of 0.1% Trypan Blue dye. The cells were plated into plastic 12 well tissue culture plates at a density of 1 x 105 cells/ml in Pro-N media supplemented with various combinations and concentrations of EGF and FGF-2 as described in the Results. Cell
cultures were fed every 2 days by removing approximately half the media and replacing it
with an equal volume of fresh media. 84
3H-thymidine Assay
Following 72 hours of recovery from perinatal H/I, the SVZs were dissociated as
described previously. Neurospheres were cultured in ProN media supplemented with 20
ng/mL EGF. Cultures were established overnight before being spiked with 8 uCi thymidine per well in 12-well plates. Neurospheres were allowed to grow in the presence of thymidine for 4-24 hours, after which cells were collected on Whatman GF/C filters on a vacuum manifold. Following a 5 min rinse with PBS, the DNA was precipitated with ice cold 5% TCA for 5 minutes, and then the filters were washed 2x5 min with PBS.
Filters were dried, placed in plastic scintillation bottles with 5 mL scintillation fluid, and counted on a scintillation counter (Becton-Dickson).
In vitro H/I insult
Primary neurospheres were prepared as described above in plastic 6 well tissue
culture plates. After 3 days in vitro (DIV) cells in the H/I group were resuspended in
DMEM with 0.15 mM glucose and cultured in an incubator under 3% O2/5%
CO2/balance N2 for 3 hours. After this time period, cells were returned to their normal
culture conditions in ProN supplemented with 20 ng/mL EGF and 10 ng/mL FGF-2.
Control cells were collected and resuspended in parallel with the H/I cells, but were 85 always kept in ProN supplemented with 20 ng/mL EGF and 10 ng/mL. At designated recovery intervals or 1, 2, or 3 days, 8 μCi 3H-thymidine was added to each well for 24
hours, after which the cells were collected and incorporation determined as described
above.
Co-culture experiments
Neurosphere and neonatal mixed brain cultures were prepared as described above
in plastic 6 well tissue culture plates and cultured in parallel for 3 DIV. After 3 DIV
mixed brain cells in the H/I group were resuspended in DMEM with 0.15 mM glucose and cultured in an incubator under 3% O2/5% CO2/balance N2 for 3 hours (neurospheres were not manipulated during this interval). Control cells were maintained in MEM-C under normal culture conditions during this time. After the H/I interval, the mixed brain cells were placed in ProN supplemented with 20 ng/mL EGF and 10 ng/mL. The neurospheres transferred to transwells suspended in the media in the wells of mixed brain cells. After 48 hours, 8 μCi 3H-thymidine was added to each well for 24 hours, and the
neurospheres were collected and incorporation determined as described above.
86 Results
Larger neurospheres are observed following perinatal H/I in the presence of EGF, but not in the presence of FGF-2 alone
Our previous studies which demonstrated an increase in the size of neurospheres
following perinatal H/I were performed using both EGF and FGF-2 in the culture media.
As our microarray and real-time PCR experiments revealed that the EGF receptor is
induced by this insult, we hypothesized that the SVZ cells in the injured hemisphere would be more sensitive to EGF and possibliy FGFs. Therefore, we performed pilot experiments in which we lowered the concentration of EGF and FGF in the media from
20 ng/ml and 10 ng/ml respectively to 2 ng/nl and 1 ng/ml. With both EGF and FGF-2 present in the medium at these concentrations, the spheres from the ipsilateral hemisphere
were indeed larger (data not shown). To determine whether the cells were more
responsive to EGF or FGF-2 or both, we performed the neurosphere assay 48 hours after
H/I but used either EGF alone at 2 ng/mL or FGF-2 alone at 1 ng/mL. In cultures grown
in EGF alone the neurospheres generated from the ipsilateral SVZ grew larger compared
to neurospheres from either contralateral or control SVZs (Figure 6.1A-C). In the
presence of FGF-2 alone, neurospheres grew to a similar size regardless of the source
(Figure 6.1D-F). 87
Figure 6.1: Neurospheres grow larger in the presence of EGF, but not FGF-2. SVZ cells were dissociated from ipsilateral (A,D), contralateral (B,E), and control (C,D) hemispheres and cultured for 6 DIV in ProN media supplemented with either 2 ng/mL EGF (A-C) or 1 ng/mL FGF-2 (D-F). Phase contrast images of representative neurospheres were captured for comparison.
Proliferation is increased in ipsilateral neurosphere cultures
The larger size of neurospheres following perinatal H/I suggested more cell divisions within the culture period. To address this hypothesis, primary neurosphere cultures were established from animals at 3 days of recovery from perinatal H/I and grown overnight in 20 ng/mL EGF alone. The medium was then spiked with 8 µCi/ML
3H-thymidine and incorporation was assessed at 4 hour intervals for up to 20 hours. All 88 cultures exhibited a similar degree of incorporation during the initial 4 hours, but the cultures prepared from the ipsilateral hemisphere rapidly increased their rate of incorporation and exhibited higher counts at all subsequent timepoints (Figure 6.2). The contralateral cultures also showed a steeper growth curve than sham cultures, but not as steep as the ipsilateral curve.
4500 4000 3500 3000 2500 2000 1500 1000 DPM (% of 4h Control) 4h of (% DPM 500 0 4 8 12 16 20 Hours
Figure 6.2: NSPs proliferate more rapidly in the presence of EGF following perinatal H/I. Animals were sacrificed 3 days after H/I and neurospheres were cultured from ipsilateral (♦), contralateral (■), and control (●) SVZs in 20 ng/mL EGF overnight. The following morning 3H-thymidine was added to the culture medium (8 µCi/mL). At 4 h intervals, cells were collected on Whatman filter discs by vacuum, the DNA was precipitated with TCA and 3H-thymidine incorporation was quantified. This figure depicts one representative experiment from 3 repetitions, normalized to the 4 h control. Diamonds represent ipsilateral, squares represent contralateral hemisphere and triangles represent controls. Values are averages ± SEM.
89 Neurospheres require external signals to exhibit increased proliferation following H/I
To test whether the proliferative changes observed in neurosphere cultures following perinatal H/I is due to a direct effect of H/I on the NSPs, we developed an in vitro model. Neurospheres were established in EGF-supplemented medium as described previously for 3 DIV, after which they were resuspended in media with low glucose (0.15 mM) and no growth factors and cultured for 3 hours in 3% O2. After this H/I interval,
they were returned to normal neurosphere medium supplemented with EGF. A 24-hour
pulse of 3H-thymidine was given on days 1, 2, or 3 following the H/I exposure. At days 1
and 2 of recovery, the cultures exposed to in vitro H/I only incorporated ~50% as much
3H-thymidine as control cultures. By 3 days of recovery there was no significant
difference in the level of thymidine incorporation (Figure 6.3A). 90
Figure 6.3: NSPs respond to signals from injured tissue following perinatal H/I. Normal neurospheres were subjected to in vitro H/I alone (A), or neurospheres were cultured in transwells exposed to media from mixed brain cells that had been subjected to in vitro H/I (B). In both cases, EGF and FGF was present in the medium. At the designated time points, 3H-thymidine was added to the culture medium (8 µCi/mL) for 24 hours after which cells were collected on Whatman filter discs by vacuum, the DNA was precipitated with TCA and 3H-thymidine incorporation was quantified. White bars=control, black bars=in vitro H/I; *=p<0.05 vs. control by Student’s t-test. 91 A second strategy was implemented to determine whether signals derived from more mature cells surrounding the NSP niche would alter the responsiveness to EGF.
Mixed brain cell cultures were prepared from neonatal rat cerebral cortices and cultured in parallel with neurosphere cultures for 3 DIV, after which they were exposed to the same in vitro H/I interval described above, after which they were returned to normal culture conditions. The neurospheres were then cultured suspended above the mixed brain cells using transwells for 2 days and 3H-thymidine was added for 24 hours. Under
these conditions where the neurospheres were exposed to factors secreted by the mixed
brain cells, the NSPs incorporated more 3H-thymidine when the brain cells had been
exposed to H/I compared to control brain cell cultures. (Figure 6.3B).
Discussion
Here we have demonstrated that the previously described size differences in
neurospheres following perinatal H/I are the specific result of increased responsiveness to
EGF. Furthermore, this increased responsiveness cannot be directly induced within NSPs
by H/I, but occurs after exposure to signals secreted from more mature neural cells
exposed to H/I. As we have previously observed increased EGFR expression in the SVZ
following perinatal H/I, the current data suggests that this increased expression is
responsible for the increased sensitivity of the NSP population, possibly contributing to
their expansion in response to injury in vivo. 92 Previously we demonstrated that larger neurospheres could be generated from
NSPs isolated from the injured SVZ when cultured in the combined presence of EGF and
FGF-2 (Felling et al., in press). Our present data show that this effect is predominantly due to increased sensitivity to EGF because no size differences were observed in the presence of FGF-2 alone. Interestingly, studies have shown that FGF-2 stimulation can induce EGFR expression within NSPs (Ciccolini and Svendsen, 1998). Many studies have shown that FGF-2 is strongly induced following brain injury, primarily within glial cells (Alzheimer and Werner, 2002). Moreover, our microarray analysis revealed increased expression of multiple FGF isoforms 48 hours after perinatal H/I. These data suggest a link between increased levels of FGFs and the induction of EGFR expression in this model, which is supported in part by our demonstration that the change in proliferative behavior of NSPs following H/I requires soluble factors released from more mature cells recovering from exposure to H/I.
It is well established that EGFR+ cells can give rise to multipotent neurospheres in vitro (Reynolds and Weiss, 1996; Kornblum et al., 2000). Recent data suggests that the majority of neurospheres in vitro are actually generated by rapidly dividing transit- amplifying cells, or Type C cells, rather than their more quiescent Type B NSCs. EGF infusion stimulated proliferation of Type C cells in vivo, but it also “activated” a subset of
GFAP+ Type B cells and promoted their contact with the ventricle (Doetsch et al., 2002).
The transit-amplifying compartment may represent an additional cellular reserve that can revert to a NSC-like phenotype under situations of increased EGF stimulation, a condition that we have demonstrated is present following perinatal H/I; however, the 93 observed recruitment of quiescent cells into the cell cycle suggests that H/I also mobilizes the NSCs.
The data presented in this paper show that NSPs exhibit intrinsic changes in growth-factor sensitivity and proliferative potential following perinatal H/I. Here we show that these cells maintain an enhanced sensitivity to EGF after being removed from the brain and cultured in vitro, indicating a strong intrinsic change in the properties and behavior of the NSPs. This is promising for therapeutic efforts because it promises that the enhanced sensitivity to EGF stimulation is not short lived. These results may represent a mobilization of this population for tissue regeneration, similar to what is observed in other tissues following injury. In adult stroke, EGF infusion has been successfully used to enhance repair following the injury (Nakatomi et al., 2002). Our data validate the EGF signaling pathway as a promising target for future therapeutic strategies attempting to promote functional recovery after stroke from endogenous NSPs. 94
Chapter 7
Induction of Notch1 promotes increased neural stem/progenitors following perintatal H/I
Introduction
The Notch pathway is an evolutionarily conserved mechanism for regulating cell
fate decisions (Artavanis-Tsakonas et al., 1999). In mammals there are 4 homologues of the Notch receptor (Notch 1-4). Signaling is mediated through cell-cell contact through the Delta-Serrate-Lag2 (DSL) family of Type 1 transmembrane receptors. Activation of the Notch receptor prompts a series of intramembranous cleavage events, ultimately
releasing the effector, Notch Intracellular Domain (NICD). The mature Notch1 receptor
consists of a furin-generated transmembrane protein with a relative molecular weight of
~120 kDa (Blaumueller et al., 1997). Upon ligand binding, Notch1 is ultimately cleaved
at the transmembrane portion by γ-secretase to release an ~80 kDa intracellular domain
(Schroeter et al., 1998; De Strooper et al., 1999). NICD translocates to the nucleus and
binds directly to CSL proteins to regulate target genes including the Hairy/Enhancer of
Split (Hes) family of basic helix-loop-helix (bHLH) transcription repressors, particularly
Hes1 and Hes5 (Mumm and Kopan, 2000).
Significant evidence confirms that Notch is crucial to the function of neural stem and progenitors (NSPs). The pathway is essential for the maintenance of the NSP population both in vivo and in vitro, but interestingly it is not essential for the initial establishment of the NSC population. The presinilin gene encodes the γ-secretase 95 necessary for release of the NICD. Presinilin-deficient mice are born with normal numbers of NSPs, indicated by the numbers of neurospheres that can be obtained from the brain, but they show a rapid decline in this population compared to wild-type mice
(Hitoshi et al., 2002). Inhibition of Notch signaling within neurospheres in vitro, either by pharmacologically inhibiting γ-secretase or by knocking down Notch1 mRNA, also decreases self-renewal as indicated by the formation of fewer secondary neurospheres upon subcloning (Chojnacki et al., 2003). Overexpression of an active form of Notch in the brain has revealed the context dependent nature of its action. In cortical precursors just prior to embryonic neurogenesis active Notch overexpression promotes a radial glial phenotype, while overexpression in early postnatal SVZ cells inhibits any differentiation and delays emergence from the SVZ (Chambers et al., 2001; Gaiano and Fishell, 2002).
Accumulating data indicates that radial glia serve as NSCs; therefore, these results suggest that Notch can promote either the acquisition or the maintenance of a precursor state.
Many studies have revealed the inherent plasticity of the brain and increased neurogenesis frequently occurs following brain injury (Romanko et al., 2004). Ischemic injury, in particular, incites a strong proliferative response in the subventricular zone
(SVZ), a region of the brain known to harbor NSPs throughout life (Felling et al., 2003).
We have recently shown that perinatal hypoxic-ischemic (H/I) brain injury stimulates proliferation of precursors within the SVZ prior to the peak of neurogenesis described by others (Felling et al., in press). These precursors form more multipotent, self-renewing neurospheres in vitro, indicating that the increase is relatively specific for the most 96 undifferentiated precursors. Accompanying this increase in NSPs is an induction of
Notch1 expression in the injured SVZ (Felling et al., in press).
Here we further characterize the role of Notch1 in the increase in NSPs following perinatal H/I. We present evidence of selective increases in Notch1 as well as one of its ligands, Delta-like 1 (Dll1), and its downstream effectors, Hes1 and Hes5. We further show that pharmacologically decreasing Notch1 activity during the acute recovery period in vivo reduces the response of the NSPs. These results indicate that increased Notch1 signaling is important for the expansion of NSPs in this injury model. Thus enhancing
Notch signaling may be therapeutically beneficial in promoting regeneration from endogenous NSPs.
Methods
Perinatal hypoxia/ischemia model
Timed pregnant Wistar rats (Charles River, Wilmington, DE) were maintained at
the Penn State College of Medicine by the Department of Comparative Medicine, an
Association for Assessment and Accreditation of Laboratory Animal Care accredited
facility. Animal experimentation was in accordance with research guidelines set forth by
Pennsylvania State University and the Society for Neuroscience Policy on the use of
animals in Neuroscience research. All animals were fed high fat lab chow (Harlan
Teklad, Madison, WI). After normal delivery, the litter size was adjusted to 10 pups per 97 litter. Cerebral H/I was produced in 6 d-old rats (day of birth being P0) by a permanent unilateral common carotid ligation followed by systemic hypoxia (Rice et al., 1981;
Vannucci and Vannucci, 1997). Briefly, pups were lightly anesthetized with isofluorane
(4% induction, 2% maintenance). Once fully anesthetized, a midline neck incision was made and the right common carotid artery (CCA) was identified. The CCA was separated from the vagus nerve and then ligated using 3-0 silk. The incision was then sutured, and animals were returned to the dam for 3 h. The pups were prewarmed for 20 minutes in
o jars submerged in a 37 C water bath. They were then exposed to 1.5 h of 8% O2/92% N2.
After this hypoxic interval the pups were returned to their dam for recovery periods of 1,
2, and 3 days, at which time they were either decapitated for neurosphere assays or anesthetized and sacrificed by intracardiac perfusion for immunohistochemistry. Sham operated animals were anesthetized, and the carotid was isolated from the vagus but not ligated. They were not subjected to a hypoxic interval. Control animals were separated from the dam for the same amount of time as experimental animals, but were otherwise not manipulated. In all cases the contralateral and ipsilateral hemispheres from experimental animals were examined separately.
DAPT injections
P7 Wistar pups underwent common carotid ligation followed by systemic hypoxia
o with 8% O2 at 37 C. Animals were given subcutaneous injections with the compound at a dose of 10 mg/kg in a volume of 0.1 mL of 5% EtOH/0.3% DMSO in corn oil 98 administered subcutaneously. Controls were injected with the vehicle. Animals were given 3 injections, 1 every 18 hours beginning 18 hours after the surgery. At 72 hours of recovery from surgery, pups were decapitated under sterile conditions, and the SVZs were isolated and dissociated as described previously below.
In situ hybridization
Cryosections were thawed and postfixed for 15 min in 4% paraformaldehyde.
After several rinses in phosphate buffer with 0.1% Tween-20 (PBT) they were treated
with Proteinase K (1 ug/ml in phosphate buffer) then rinsed thoroughly in PBT followed
by another postfix in 4% paraformaldehyde, further rinses, and treatment in 0.25% acetic
anhydride in 100mM triethanolamine. Sections were then rinsed and placed in
humidified slide chambers and hybridized overnight at 65°C in hybridization solution
(10mM Tris, 100mM EDTA, 600mM NaCl, 0.25% SDS, 10% dextran sulfate, 1X
Denhardt’s solution, 200 ug/mL yeast tRNA, and 50% formamide) with digoxigenin-
labeled riboprobe against Hes5 generated according to manufacturer’s instructions
(Roche). Following hybridization, slides were rinsed in 5X SSC, then 1X SSC/50%
formamide at 65°C for 30 min, then treated with RNase (20 ug/ml) in TNE (10 mM Tris,
1 mM EDTA, 500 mM NaCl) for 30 min at 37°C. Slides were then washed in 2X SSC
and 0.2X SSC for 20 min each at 65°C. Following 2 rinses in MABT (100mM maleic
acid, 150 mM NaCl, 0.1% Tween-20) sections were blocked with 1:1 mixture of 10%
Blocking Reagent (Roche) and Tris buffered saline for 15 min. Sections were incubated 99 overnight at 4°C in alkaline phosphatase-conjugated anti-digoxygenin (Promega, 1:500) in blocking buffer. The following day after thorough rinsing in MABT and preincubation with levamisole, sections were developed with NBT/BCIP.
Primary neurosphere propagation
Wistar pups (P7, P8, or P9) were sacrificed by decapitation, and their brains
removed using aseptic techniques. Coronal sections were taken 2 mm from the anterior pole of the brain, excluding the optic tracts, and 3 mm posterior to the previous cut. The sections were then placed in dishes containing PGM solution (PBS with 1 mM MgCl2 and 0.6% dextrose), where the SVZ was dissected out under a microscope. The tissue was mechanically minced and enzymatically dissociated for 5 min at 37°C using a solution of 0.01% Trypsin/EDTA (Invitrogen, Carlsbad, CA) in PGM with 250 ug
DNaseI (Sigma Aldrich, St. Louis, MO), after which an equal volume of 0.02% Trypsin inhibitor (Sigma Aldrich, St. Louis, MO) in ProN media (DMEM/F12 1:1 media containing 10ng/ml d-biotin, 25 μg/ml insulin, 20 nM progesterone, 100 μM putrescine,
5ng/ml selenium, 50 μg/ml apo-transferrin, 50 μg/ml gentamycin) was added. The tissue was triturated in Pro-N media to obtain an even cell suspension. Triturations were done with progressively less media and smaller Eppendorf filter tips with time allowing for the tissue to settle and the supernatant removed and placed in separate tubes before adding new media for the next round of trituration. The cell suspension was then passed through a 40 μm Nitex screen. The number of viable cells was determined with a hemocytometer 100 by exclusion of 0.1% Trypan Blue dye. The cells were plated into plastic 12 well tissue culture plates at a density of 1 x 105 cells/ml in Pro-N media supplemented with 20 ng/mL EGF and 10 ng/ml FGF-2. Cell cultures were fed every 2 days by removing approximately half the media and replacing it with an equal volume of fresh media.
Neurosphere quantitation
A neurosphere was defined as a free-floating, cohesive cluster of at least 8 cells,
although the vast majority of neurospheres (>98%) were substantially larger than this.
Plates were gently shaken before counting each well to ensure an even distribution of spheres. Ten random 10X fields were counted per well. The frequency of sphere-forming cells (i.e. NSPs) was calculated from the average number of spheres per field, the area of the field, and the area of the well. The number of NSPs per hemisphere was then extrapolated by applying the frequency of sphere-forming cells to the total number of cells obtained in the initial dissociation of the tissue.
Neurosphere immunohistochemistry
Spheres were collected and resuspended in 5% horse serum in CNM-2 media
(DMEM/F12 1:1 containing 10ng/ml d-biotin, 5 ng/ml insulin, 20 nM progesterone, 100
μM putrescine, 5ng/ml selenium, 50 μg/ml apo-transferrin, 50 μg/ml gentamycin, 150 μl of 0.5M kynurenic acid) at an approximate density of 100-200 spheres/mL. 100 μL of 101 the neurosphere suspension was then plated in plastic 24-well tissue culture plates onto flame-sterilized coverslips precoated with 1% w/v poly-d-lysine and 10 μg/mL laminin.
The spheres were allowed to attach in a 37°C incubator for a minimum of 1.5 hours, after which 400 μl of CNM-2 supplemented to 5% horse serum was added to the well. After
16-20 h, this media was replaced with CNM-2 with 0.03% DMSO. Cultures were allowed to differentiate for five days, with media replenished on day 3. After the differentiation period, the cells were fixed using 2% paraformaldehyde for 15 min, and washed twice with BCH (10% bovine calf serum in Earle’s basal medium with 4.8 mg/mL HEPES). Sections were stained at room temperature for 45 min with O4 culture supernatant diluted 1:3 in BCH supplemented with 10% lamb serum. After thoroughly rinsing, the cells were incubated for 45 min at room temperature in GAM IgM LRSC
(Jackson, Bar Harbor, MN, 1:200). The cells were then permeabilized with BCH-S
(BCH with 0.5 mg/ml Saponin) and stained for 45 min at room temperature with anti-
TuJ1 (Promega, Madison, WI, 1:250) and anti-GFAP (Roche, Indianapolis, IN, 1:200) in
Saponin diluent (BCH-S supplemented with 10% lamb serum). Cells were then incubated for 45 min at room temperature in GAM IgG FITC (Jackson, Bar Harbor, MN,
1:400) and GAR AMCA (Jackson, Bar Harbor, MN, 1:200) in Saponin diluent. The coverslips were rinsed and mounted onto microscope slides with Gel/Mount (Biomeda,
Foster City, CA) and allowed to dry overnight. Colonies were scored according to the types of cells present. Images of stained cells were collected using a SenSys cooled- coupled device camera (CRI, Inc., Woburn, MA) interfaced with IP Lab scientific imaging software (Scanalytics, Fairfax, VA) on an Olympus BX-40 microscope.
102 In vitro H/I
P1 Wistar pups were sacrificed, and the brain was extracted under sterile culture
conditions. The SVZ was isolated, and neurospheres were cultured as described above.
The cortex and striatum were mechanically dissociated and subjected to enzymatic dissociation with trypsin at 37oC for 30 min with gentle shaking. The enzymatic
digestion was stopped with the addition of MEM-C (Minimal Essential Medium,
dextrose, fetal bovine serum). Tissue was triturated by passage through 10mL pipette.
After settling, the supernatant was removed and and 10mL fresh media was added. This
was repeated 4 times, and the supernatant collection was passed through a 130 μm filter.
After another round of trituration the suspension was passed through a 40 μm filter. Cells
were plated in MEM-C at a density of 2x106 cells/well in 6-well culture plates to
establish confluent beds of mixed brain cells. Media was changed every other day. After
4 DIV, experimental cultures were changed to DMEM-F12 without serum and placed in
2% O2 for 3 hours. Control cultures received fresh and were left in normal culture
conditions for this interval. After the H/I interval, cells were rinsed once with DMEM-
F12, and the media was changed to Pro-N+EGF/FGF-2 (see Primary neurosphere propagation for media components). At this time, neurospheres which had been
established simultaneously at P1 were transferred to Transwells (Corning, Corning, NY)
and cultured in the same wells with either experimental or control mixed brain cells for
an additional 3 days in ProN+EGF/FGF-2. Separate neurosphere cultures were also
subjected to the in vitro H/I paradigm at the same time as the mixed brain cells. These 103 neurosphere cultures were returned to their normal culture conditions following the H/I interval for an additional 3 days.
Western Blot
The cortex and striatum was isolated from control and experimental animals
homogenized in lysis buffer (20mM Tris, 150mM NaCl, 1mM EDTA, 1mM EGTA, 1%
Triton X-100, 2.5mM sodium pyrophosphate, 1mM glycerolphosphate, 1mM sodium
orthovanadate) with protease inhibitor cocktail (Sigma, St. Louis, MO) by mechanical
shearing through a 25-guage syringe followed by sonication. Protein concentrations were
calculated using a standard BCA assay according to the manufacturer instructions
(Pierce, Rockford, IL) prior to western blot analysis. For western blotting, 30ug of
protein sample was combined with 4X NuPage LDS sample buffer and 10X NuPage
reducing agent, heated at 70oC for 10 min and loaded onto a NuPage 7% Tris-Acetate
pre-cast gel (Invitrogen, Carlsbad, CA). Standard lanes were loaded with 5uL
MagicMark XP (Invitrogen) and Kaleidoscope (Bio-Rad, Hercules, CA) molecular
weight standards. After separation of proteins by gel electropohresis, proteins were
transferred to nitrocellulose and stained with Ponceau-S to determine total protein
present. The blots were rinsed and incubated overnight at 4oC with gentle shaking with
polyclonal antibody to Notch1 C-terminus (Santa Cruz, sc-6014, 1:100). After 3 rinses in
PBS-Tween, they were incubated with HRP-conjugated secondary antibody for 2 hours at
room temperature with gentle shaking. The blots were exposed with Western Lightning chemiluminescence reagent according to manufacturer’s instructions (PerkinElmer, 104 Wellesley, MA). Images were captured in a UVP EpiChem3 darkroom and processed
using Labworks 4.0 digital quantification software (UVP, Upland, CA).
RNA isolation
SVZs were dissected out of control and the ipsilateral hemisphere of H/I animals and snap frozen on a dry ice/ethanol slush and immediately stored at –80°C. Dissections
were directed toward the angles of the ventricles to avoid any confounding effects of
ventricular hypertrophy due to striatal degeneration. Tissue samples were then thawed
directly into 0.5 mL Trizol reagent (Molecular Research Center, Cincinnati, OH) and
homogenized using a tissue homogenizer. 100 uL of chloroform was added, and the
samples were centrifuged at 13,000 rpm for 15 min at 4°C. The aqueous phase was then
transferred to a new tube. After adding 250 uL of 70% EtOH, the aqueous phase was
applied to an RNeasy Mini-spin column (Qiagen, Valencia, CA) to remove contaminants
from the RNA, according to manufacturer’s instructions. The concentration of total RNA
was determined by measuring optical density on a spectrophotometer (Becton-Dickson,
Franklin Lakes, NJ). RNA samples were stored at -80°C until needed.
Real-time PCR
Total RNA was isolated from 5 individual SVZs per group using TriReagent
(Molecular Research Center, Cincinnati, OH) and RNeasy kits (Qiagen, Valencia, CA) 105 according to manufacturers’ protocols. 2 μg of was reverse transcribed to cDNA using the Qiagen Omniscript RT kit supplemented with random nonamer primer (Sigma
Aldrich, St. Louis, MO) and RNaseIN (Promega, Madison, WI). Primer pairs specific for the genes of interest were designed using Lux Primer Design software (Invitrogen,
Carlsbad, CA) or obtained from catalogued TaqMan gene expression assays (Applied
Biosystems, Foster, City, CA) ( Table 7.1 ). Amplification was carried out in 96-well
plates using Platinum-UDP Supermix kit according to the manufacturer’s instructions
(Invitrogen, Carlsbad, CA) and analyzed on an ABI Prism 7700 Sequence Detection
System (Applied Biosystems, Foster City, CA). Fold-changes in gene expression relative to a housekeeping gene were obtained using the Relative Expression Software Tool
(REST) for groupwise comparison and statistical analysis of relative expression results in
real-time PCR (Pfaffl et al., 2002). 106
Table 7.1: Primers used in qRT-PCR Transcript Primers
Hes1 Applied Biosystems TaqMan Assay ID Rn00577566_m1
Hes5 Applied Biosystems TaqMan Assay ID Rn00821207_g1
5’-GAA CCA ACA GAC TCG TCT CTG CAT GG[FAM]TC-3’ Nov/CCN-3 5’-AGG TGG ATG GAT TTC AGG GAC T-3’
5’-GAA CTG GCT CCG CCG CAA CAG [FAM]TC-3’ Jagged1 5’-CAT GCA GAA CGT GAA CGG AGA-3’
5’-CAC GGA AGG AGT GCA AAG AAG CCG [FAM]G-3’ Jagged2 5’-CCC GTG GAG CAA ATT ACA TCC T-3’
5’-GTA CCT CAG GGC TGC TGG CAG G[FAM]AC-3’ Delta-like 1 5’-GGC CGC TAC TGC GAT GAA T-3’
5’-CAC GTA CTG CGA GCT GCC CTA CG[FAM]G-3’ Notch1 (Lux) 5’-GGC AGG TGC CTC CGT TCT-3’
18S (TaqMan) Applied Biosystems TaqMan Assay ID Hs99999901_s1
18S (Lux) Invitrogen catalog #115HM-01
Results
Notch1 and Delta expression increase within the first 2 days of recovery from perinatal H/I
Previously we have shown increased expression of Notch1 within the SVZ 2 days following perinatal H/I. To determine the time course of Notch 1 induction, we 107 microdissected the SVZ from the ipsilateral and contralateral hemispheres of H/I animals as well as from sham-operated controls and performed real time PCR to assess the expression of Notch1 mRNA as well as several Notch ligands. This analysis revealed that
Notch1 expression peaks at 48 h after H/I at approximately 2-fold compared to the contralateral hemisphere followed by a decrease over the next 48 hours towards levels observed in the contralateral hemisphere ( Figure 7.1 A). Expression in the contralateral
hemisphere at 48 h of recovery is comparable to expression in sham-operated controls.
An important Notch ligand, Dll1, shows a similar increase in expression in the ipsilateral hemisphere at 48 hours, but its expression is almost equally increased in the contralateral 108
Figure 7.1: Components of the Notch signaling pathway are induced following perinatal H/I. (A) RNA was isolated from ipsilateral (circles) and contralateral (baseline) SVZs at designated timepoints and amplified by qRT-PCR. (B) RNA was isolated from ipsilateral (solid bars), contralateral (white bars), and sham (line) SVZs 48 h following perinatal H/I and amplified by qRT-PCR. *=p<0.05 vs sham by REST; †=p<0.05 vs. contralateral by REST. 109 hemisphere. Other Notch ligands, Jagged-1 (Jgd) and Nov/CCN3, remain unchanged in either hemisphere at 48 hours of recovery. Similarly, Hes1, a downstream target of
Notch signaling, is unchanged compared to a sham-operated animal, but Hes5, another downstream target, is actually reduced in the ipsilateral hemisphere compared to the contralateral hemisphere or a sham-operated control.
In situ hybridization revealed regional differences in the expression of these
Notch pathway components within the SVZ. Notch1 mRNA is particularly induced within the most medial aspect of the SVZ, just subjacent to the ependymal layer (Figure
2A). Both control and contralateral hemispheres exhibit comparably homogeneous expression throughout the SVZ ( Figure 7.2B,C). This pattern of higher expression in the
medial SVZ with normal or decreased expression throughout the rest of the SVZ also
occurs for the ligands Dll1 and Jgd (Figure 8.2). 110
Figure 7.2: Notch1 receptor and its ligands are induced within the NSP niche following perinatal H/I. In situ hybridization was performed on cryostat sections of contralateral (A,D,G), ipsilateral (B,E,H), and control (C,F,I) hemispheres using a digoxygenin- labeled RNA probe for Notch1 (A-C), Dll1 (D-F), or Jgd1 (G-I). Scale bar in I represents 10 μm. 111 Pharmacologically inhibiting Notch1 in vivo reduces the increase in NSPs following perinatal H/I
To determine whether Notch signaling is essential for the increase in NSPs after
perinatal H/I we administered a commercial γ-secretase inhibitor (DAPT) by subcutaneous injection and then analyzed the numbers of NSP using the neurosphere assay and also assessed their differentiation potential. We used a γ-secretase inhibitor as this is the enzyme believed to be responsible for the final cleavage event that releases the active NICD (De Strooper et al., 1999). We administered 3 doses of the inhibitor every
18 hours beginning 18 hours after perinatal H/I. Previous experiments have demonstrated persistent levels of compound in excess of the in vitro LD50 within brain tissue up to 18 hours following this route of administration (Dovey et al., 2001). Western blotting for Notch1 protein revealed a decrease in the ratio of the NICD fragment (80 kDa) to its immediate precursor (100 kDa) following injection of the inhibitor compared to vehicle (Figure 7.3A, 0.13 DAPT vs. 0.24 Vehicle, p=0.01). Following administration
of vehicle alone, the ipsilateral hemisphere yielded greater than 50% more neurospheres
than the contralateral hemisphere, as we have shown previously. However, following
administration of DAPT, equivalent numbers of neurospheres were obtained from the
contralateral and ipsilateral hemispheres 3 days following perinatal H/I (Figure 7.3B). 112
Figure 7.3: Inhibition of Notch activity reduces the expansion of NSPs following perinatal H/I. A γ-secretase inhibitor was administered subcutaneously during the first 3 days of recovery from perinatal H/I. (A) The active NICD is reduced in animals injected with inhibitor compared to animals injected with vehicle. (B) There was less than a 30% increase in NSPs following injection of DAPT compared to a 60% increase following injection of vehicle, n=10 DAPT, 9 Vehicle, *=p<0.05 by Student’s t-test, DAPT vs. Vehicle.
To further examine the effect of Notch inhibition on cell fate following perinatal
H/I, we differentiated 6 DIV primary neurospheres from the ipsilateral hemisphere of vehicle and DAPT injected pups. After staining for neural cell markers (GFAP, O4, and
Tuj1) we scored the colonies according to the combinations of cells present. In the 113 vehicle-injected animals, ~80% of the neurospheres produced neurons, oligodendrocytes, and astrocytes compared to ~30% from the DAPT-injected animals (Figure 7.4)
Figure 7.4: Inhibition of Notch activity after perinatal H/I reduces the frequency of tripotent neurospheres. A γ-secretase inhibitor was administered subcutaneously during the first 3 days of recovery from perinatal H/I. After 6 DIV, spheres were plated on sterile coverslips and allowed to differentiate for 4 DIV in the presence of serum after which they were stained for neural cell markers (GFAP-astrocytes, O4-oligodendrocytes, Tuj1-neurons). Colonies were scored according to which cell types they possessed. Fewer neurospheres contained all 3 cell types after injection of DAPT vs. vehicle, n=3 animals per group, p<0.03 by Student’s t-test for tripotential groups.
Mixed brain cells subjected to H/I in vitro stimulate Notch1 expression in neurospheres
We designed an in vitro H/I insult to determine whether Notch1 induction
following perinatal H/I in vivo is a direct effect of injury on the cells or the response of
NSPs to injury cues from surrounding cells. Neurospheres were established as usual, but
after 3 days in vitro (DIV), they were resuspended in low glucose media to simulate 114 ischemia and cultured at 3% O2 for 3 hours. After this H/I exposure, the neurospheres were resuspended in normal media supplemented with EGF and FGF-2. Notch1 expression was not induced within neurospheres after subjecting them to this in vitro H/I paradigm (Figure 7.5A). In a separate set of experiments we established confluent beds
of mixed brain cells grown in the presence of serum and exposed these cells to the same
in vitro H/I paradigm. After the H/I interval, the media was replaced with our normal
neurosphere media supplemented with EGF/FGF-2, and neurospheres that had been
cultured under normal conditions for 4 days were placed in transwells with these mixed
brain cells. This allowed the neurospheres, which had never been exposed to H/I
themselves, to be exposed to any of the factors secreted by the H/I-stimulated mixed
brain cells without direct contact. This co-culture paradigm induced Notch1 expression
1.5 fold in the neurospheres 48 hours after exposing them to the H/I mixed brain cultures
(Figure 7.5B). 115
Figure 7.5: Signals from NSP niche triggers Notch1 induction following exposure to H/I. Neurospheres were exposed to in vitro H/I (neurospheres alone) or cultured with mixed brain cells that had been exposed to in vitro H/I (co-culture). RNA was isolated 48 h after the in vitro insult and amplified by qRT-PCR. n=6 per group.
Discussion
Notch-DSL signaling is essential for regulating stem and progenitor cell populations. Here we present evidence for a role for Notch signaling in a regenerative 116 response to perinatal H/I. This response consists of an expansion of the NSP population in the SVZ, in part caused by increased proliferation and possibly a shift in the mode of cell division. These data demonstrate that this expansion is preceded by an upregulation of Notch1 receptor and Dll1 expression. Additionally, selective increases in the expression of other components of the pathway occur in the most medial cell layers of the
SVZ. Pharmacological inhibition of Notch activation in vivo during the acute recovery period reduces Notch1 activation and diminishes the NSP expansion.
Notch1 expression peaks in the ipsilateral SVZ 48 hours following perinatal H/I.
At this timepoint, the contralateral hemisphere exhibits no difference in Notch1 expression relative to a sham-operated control. In our animal model, the contralateral hemisphere is uninjured, but it is exposed to systemic hypoxia. This indicates that brief hypoxia alone is insufficient to induce Notch1 expression, suggesting that the induction observed following perinatal H/I is the result of injury signals produced from cells either surrounding the NSC niche or within the cells themselves. This is supported by observation that mixed brain cells exposed to in vitro H/I can induce Notch1 in normal neurospheres, but Notch1 is not induced in neurospheres exposed to in vitro H/I. Dll1, on the other hand, is equally induced both in the ipsilateral and contralateral hemispheres, suggesting that a brief exposure to hypoxia induces this Notch ligand.
Other components of the Notch signaling pathway did not exhibit the robust upregulation seen with Notch1 and Dll1. Rather, they appeared to be reduced throughout most of the SVZ with maintained or increased expression in the most medial cells of the region. Importantly, the medial SVZ provides the niche for NSPs (Garcia-Verdugo, et 117 al., 1998). This region remains relatively undamaged following perinatal H/I, allowing it to remain capable of responding to injury signals from the surrounding damaged tissue.
Notch receptors play important regulatory roles in cell fate decisions in many systems both during development and in adulthood. In the brain, Notch1 is essential in maintaining the NSC population by promoting self-renewal. Mice genetically deficient in Notch1 or other pathway components, including presenilin (γ-secretase) or RBP-Jκ, exhibit a progressive loss of NSPs as reflected by declining ability to generate neurospheres, an defect that can be rescued with the introduction of activated Notch1
(Hitoshi et al., 2002). Inhibition of Notch activity in neurospheres in vitro, either pharmacologically or genetically, also reduces the self-renewal capacity of NSPs to generate secondary neurospheres (Chojnacki et al., 2003).
Our data demonstrate a similar role for Notch in the acute expansion of the NSP population following perinatal H/I because inhibitions of Notch signaling eliminates this expansion. Interestingly, the level of Notch inhibition achieved in these studies is insufficient to appreciably reduce the number of NSPs in the normal brain, but it produces a significant reduction in the number of new NSPs generated in response to the injury. One reason for this could be the length of treatment. In the normal adult SVZ,
NSCs are a slowly dividing population with a cell cycle time estimated at 15 days
(Morshead et al., 1994). In these experiments, the inhibitor compound was only present in the brain for 48 hours prior to sacrifice, making it unlikely that it would have an appreciable effect on the numbers of existing NSCs. Following perinatal H/I, however, we have shown a doubling in their numbers; therefore, our data suggest that Notch 1 is 118 required for the expansion in the numbers of NSCs which would normally occur during the treatment period.
The effects of Notch signaling on NSPs are principally mediated by the bHLH transcription factors, Hes1 and Hes5 (Ohtsuka et al., 1999). While deficiency in either of these genes alone leads to compensatory expression of the other, deficiency in both of these genes leads to disorganized neural tube formation and premature neuronal differentiation of neuroepithelial precursors at the expense of astrocytes and oligodendrocytes (Kageyama et al., 2005). Overexpression of Hes1, on the other hand actually promotes astrogliogenesis (Wu et al., 2003). Furthermore Hes1-/-;Hes5-/- mutants exhibit reduced ability to generate neurospheres, although the few spheres that they do form are multipotential (Ohtsuka et al., 2001). Consistent with these data, we reduced the expansion of NSPs following perinatal H/I, a process likely related to the self-renewal of these cells, by pharmacologically inhibiting Notch. In contrast, however, inhibition of Notch in our studies compromised the multipotentiality of neurospheres.
This difference is likely a distinction between the embryonic NSPs analyzed in the Hes1-/-
;Hes5-/- mice and the postnatal NSPs present in our studies, although it could indicate a
Hes-independent function of Notch.
A concern with the use of DAPT is that γ secretases are involved in other functions in the brain besides Notch cleavage, most notably the processing of amyloid precursor protein (APP). This leaves the possibility that the effect of the inhibitor that we have seen is due not to decreased Notch activity, but rather to altered APP processing. In fact, a recent study has shown that the soluble form of APP (sAPP), generated mostly through cleavage by an α-secretase, actually stimulates proliferation of cells within the 119 SVZ (Caille et al., 2004). By inhibiting γ-secretase, we would expect a larger amount of
APP to be processed via the alpha cleavage, thus generating more sAPP and stimulating proliferation within the SVZ. Rather we see a decrease in these precursors with the inhibition of γ-secretase, suggesting that our results are not due to altered processing of
APP.
Using the in vitro H/I insult paradigm, we were able to demonstrate that NSPs required signals from the surrounding environment to upregulate their expression of
Notch1. In transwells, the neurospheres were never in contact with the mixed brain cells, indicating that a diffusible factor is responsible for this effect. One candidate molecule is leukemia inhibitory factor (LIF). LIF signals through a receptor complex that includes gp130, a protein that appears to regulate the expression of Notch (Chojnacki et al., 2003).
Our laboratory has shown increased levels of LIF following perinatal H/I that coincides with the increase in Notch1 induction (M. Covey, unpublished results), suggesting that
LIF could be responsible for the induction of Notch receptor expression in NSPs following perinatal H/I. Since astrocytes are the major source of LIF in the CNS, it is likely that H/I stimulates the astrocytes surrounding the NSPs to produce LIF. Our data suggest that though there may be precursors within neurospheres that share the properties of astrocytes, these precursors are evidently not at a sufficient stage of maturation to render them competent to produce LIF.
These data indicate that Notch1 is an important contributor to the expansion of
NSPs following perinatal H/I. This class of neurologic injury is devastating to children and those with the responsibility of caring for them. Current therapies do not provide sufficient improvement in neurologic function after such insults, and it is incumbent upon 120 science to identify new and better strategies. Understanding how increased Notch signaling in the context of perinatal H/I alters NSP proliferation will provide important clues as to how these valuable precursors can be further mobilized to provide regenerative therapy. 121
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Appendix A
Altered Expression of NSC-Related Genes After Perinatal H/I
Table 1. Result from the SuperArray stem cell array Total RNA pooled from at least 4 animals at 48 h of recovery was labeled and hybridized to the SuperArray Nylon membrane. Fold differences vs control are indicated for genes exhibiting a similar in expression in each of 2 separate experiments. Values represent averages from 2 experiments with 2 independent sets of mRNAs. Only averaged values greater than 2 fold are shown. GENE DESCRIPTION CHANGE FAMILY
INDUCED
Integrin αL Strain DBA/2J integrin alpha L (Itgal) 5.92 ECM Molecules
Integrin α5 fibronectin receptor alpha (Itga5) 5.03 ECM Molecules
Integrin ß6 Integrin beta 6 (Itgb6) 4.18 ECM Molecules
Cadherin 5 cadherin 5 3.54 ECM Molecules
Integrin α6 Integrin alpha 6 (Itga6) 2.93 ECM Molecules
BMPR2 Bone morphogenic protein receptor, type II 20.37 Growth Factor/Cytokine
Notch1 Notch1 7.3 Growth Factor/Cytokine
TGFb1 Transforming growth factor, beta 1 9.14 Growth Factor/Cytokine
Fzd3 Frizzled homolog 3 (Drosophila) 7.80 Growth Factor/Cytokine
ALK-3 BMP receptor, type 1A 6.41 Growth Factor/Cytokine
FGF6 Fibroblast growth factor 6 5.43 Growth Factor/Cytokine
Ntrk2 Neurotrophic tyrosine kinase receptor type 2 5.20 Growth Factor/Cytokine
FGF23 fibroblast growth factor 23 5.20 Growth Factor/Cytokine
Wnt7b Wingless-related MMTV integration site 7B 4.84 Growth Factor/Cytokine
FGF4 Fibroblast growth factor 4 3.47 Growth Factor/Cytokine
Wnt3a Wingless-related MMTV integration site 3A 3.33 Growth Factor/Cytokine 147 FGF3/int-2 Fibroblast growth factor 3 2.84 Growth Factor/Cytokine
GENE DESCRIPTION CHANGE FAMILY
FGF22 Fibroblast growth factor 22 2.31 Growth Factor/Cytokine
Krt1-17 Keratin complex 1, acidic, gene 17 35.99 Molecular Markers
Igf1r Insulin-like growth factor 1 receptor 2.65 Growth Factor/Cytokine
Glut 1 facilitated glucose transporter, member 1 12.35 Molecular Markers
Prox1 Prospero-related homeobox 1 11.69 Molecular Markers
Gp130 IL-6 signal transducer 8.41 Molecular Markers
MHR a-1 keratin complex 1, acidic, gene 5 8.00 Molecular Markers k14 Keratin complex 1, acidic, gene 14 5.73 Molecular Markers
Erbb2ip Erbb2 interacting protein 4.90 Molecular Markers
Neurotrophin neurotrophin 3 4.50 Molecular Markers
3
Fabp7 Fatty acid binding protein 7, brain 4.41 Molecular Markers
Pou6f1(Brn5) POU domain, class 6, transcription factor 1 4.28 Molecular Markers
LIF Leukaemia inhibitory factor (LIF) 3.67 Molecular Markers
Olig1 Oligodendrocyte transcription factor 1 2.76 Molecular Markers
Pou3f3 (brn-1) POU domain, class 3, transcription factor 3 2.68 Molecular Markers
EGFR Epidermal Growth Factor Receptor 2.65 Molecular Markers
Brn2 POU domain, class 3, transcription factor 2 2.11 Molecular Markers
Tep1 Telomerase associated protein 1 5.74 Others
REPRESSED
Fgf1 Fibroblast growth factor 1 0.17 Growth Factor/Cytokine
Scgn10 Superior cervical ganglia, neural specific 10 0.38 Molecular Marker
Nfl Mouse neurofilament-L mRNA 0.46 Molecular Marker
Ncam2 Neural cell adhesion molecule 2 0.46 Molecular Marker
RYAN J. FELLING
Education
M.D. The Pennsylvania State University College of Medicine August, 2000 – May, 2007
Hershey, PA 17033
Ph.D. The Pennsylvania State University College of Medicine August, 2000 – May, 2006
Hershey, PA 17033
B.S. The Pennsylvania State University August, 1996 – May, 2000
University Park, PA 16801
Honors/Awards
Medical School: Medical Scientist Training Program Fellowship (2000-2002)
Graduate School: NIH Ruth Kirchstein Predoctoral Fellowship
1st Place, Life Sciences, Penn State Graduate Student Exhibition (2003)
Selected for oral presentation, National MD/PhD Conference (2005)
Undergraduate: Schreyer Honors College (Honors in Biology)
Ridge Riley Alumni Scholarship
Phi Kappa Phi Honor Society
Research
Doctoral Dissertation
A Regenerative Response of Endogenous Neural Stem Cells to Perinatal Hypoxic/Ischemic Brain
Damage; PI – Dr. Steve Levison
Undergraduate Honors Thesis
Genetic Basis of Synaptic Transmission in Drosophila; PI – Dr. Richard Ordway
Activities
Physician Scientist Student Association, Dance Marathon – student run philanthropy, Student Pediatric
Society, Bethesda Mission – medical care for homeless, Brain Awareness Week
Professional Memberships
Society for Neuroscience, American Medical Association, American Academy of Pediatrics