Use of Recombinant Viruses to Manipulate Neural Stem Cell Gene Expression in the Mouse Brain
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Konstantin Khodosevich
Contents
Abstract
Introduction ............................................................
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Neural Stem Cell Biology ......................................
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Recombinant AAVs ................................................
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Recombinant Lentiviruses.....................................
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Recombinant Retroviruses ....................................
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Strategies to Analyze NSC Function in the Postnatal Brain Using Recombinant Viruses .............................................
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Conclusion ..............................................................
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References ...............................................................
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Adult mammalian brain preserves neural stem cells (NSCs) that contribute to postnatal neurogenesis. The last decade has seen a tremendous progress in the identification and analysis of NSCs in the mouse brain. A handful of molecular and cell biology techniques have been applied to confirm the existence and to address the functions of NSCs in the adult brain. Recombinant viruses constitute one of the most powerful and versatile tools for the analysis of NSCs in vivo. This chapter describes major recombinant viruses that are presently available and discusses their applicability to the NSC analysis.
Introduction
K. Khodosevich (*) Department of Clinical Neurobiology/A230, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany e-mail:
[email protected]
Although the majority of NSCs differentiate into neuronal or glial precursors prenatally, the adult mammalian brain preserves a number of NSCs throughout the whole life of the animal (Ming and Song 2011). Functional characteristics of these NSCs meet both criteria of stem cells: the capacity to self-renew and the ability to differentiate into multiple cell types (in neural lineage these are neurons, astrocytes and oligodendrocytes). Since the discovery of NSCs in the adult mammalian brain, several techniques have been employed for NSC analysis. Following the identification of NSC markers, different transgenic mouse lines were used to label NSCs and their progeny in vivo (NSC lineage tracing)
M.A. Hayat (ed.), Stem Cells and Cancer Stem Cells, Volume 7, DOI 10.1007/978-94-007-4285-7_23, © Springer Science+Business Media Dordrecht 2012
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and to analyze NSC proliferation and differentiation. As alternative approaches, in vivo plasmid electroporation as well as infection by recombinant viruses were also applied for the NSC analysis (Fernandez et al. 2011; Khodosevich et al. 2011; Stein et al. 2005). Plasmid electroporation can only be used during a narrow temporal window of perinatal development. In contrast, recombinant viruses can be delivered to the mouse brain at any age. Furthermore, recombinant viruses can be injected into any brain region, whereas electroporation is limited to brain regions that are close to the ventricles. Development of conditional knockout approach and implementation of tamoxifen-inducible Cre-ERT2 system resulted in the creation of mouse lines that can be employed for dynamic and well-controlled studies of NSC functions. However, these methods do not allow for manipulation of a particular subpopulation of NSCs and rather affect all NSCs in the postnatal brain. In contrast, recombinant viruses can be injected to label a small fraction of NSCs in the region of interest. In addition, generation of recombinant viruses cost less time and money than creating a new mouse line. Thus, recombinant viruses are powerful and versatile tools for NSC analysis in vivo in the mouse brain.
Neural Stem Cell Biology In the postnatal/adult mouse brain, NSCs reside in highly organized brain regions called “neurogenic niches”. Besides NSCs, these neurogenic niches contain several other cell types supporting NSC function and controlling their activity. Thus, it is important to know the organization of a given neurogenic niche before proceeding with the experiments aiming at NSC analysis. There are only two regions in the postnatal mouse brain that contain NSCs (Fig. 23.1a–c): the subgranular zone (SGZ) of the dentate gyrus in the hippocampus and the subventricular zone (SVZ) of the lateral ventricles (Lledo et al. 2006; Ming and Song 2011). NSCs (called B cells in both regions) have an astroglial nature and express several glial markers, e.g. glial fibrillary acidic protein (GFAP), brain lipid-binding protein (BLBP) and
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astrocytic glutamate transporter GLAST (Ming and Song 2011). The majority of NSCs in the postnatal neurogenic regions are quiescent (B1 cells) and divide very rarely, once in 2 weeks (Morshead et al. 1998). However, a subset of NSCs, called activated NSCs (B2 cells), divides much faster and their population increases under some pathological conditions, e.g. stroke or seizures (Lugert et al. 2011; Zhang et al. 2004). In addition to NSCs, neurogenic regions contain other proliferating cell types as well as non-proliferating supporting cells (Fig. 23.1b and c for the SVZ and SGZ, respectively). Postnatal/ adult NSCs divide and differentiate into transitamplifying precursors (or C cells), which are fast-dividing cells and in turn give rise to more differentiated neuroblasts (or A cells) (Lledo et al. 2006; Ming and Song 2011). Although the long-held dogma was that postnatal NSCs divided in vivo exclusively asymmetrically, it has recently been challenged by a groundbreaking study showing symmetric NSCs division in vivo (Bonaguidi et al. 2011). Neuroblasts (=immature neurons) in the hippocampal SGZ migrate into the granule cell layer of the dentate gyrus (Fig. 23.1c) and integrate into previously established neural circuits where they have been implied to play a role in certain forms of hippocampal-dependent learning and memory (Lledo et al. 2006; Ming and Song 2011). Neuroblasts originating in the SVZ migrate over long distances via the rostral migratory stream into the olfactory bulb (Fig. 23.1a), where they mature into granule or periglomerular neurons and contribute to olfactory information processing (Khodosevich and Monyer 2011; Lledo et al. 2006). Non-neurogenic or supporting cells of neurogenic niches include endothelial cells of blood vessels, mature astrocytes and neurons, microglia and, in the SVZ, ependymal cells lining the wall of the ventricle (Ming and Song 2011). NSCs communicate with all other cell types within the neurogenic niche either directly via transmembrane receptor–ligand interactions or via secreted ligands. As a result, NSC proliferation and differentiation are tightly controlled by neurogenic niche cells. For instance, ependymal
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Fig. 23.1 Neurogenic niches in the postnatal mouse brain. (a) Sagittal view of the brain showing two neurogenic niches (marked in red): the SVZ of the lateral ventricles (LV) and the SGZ of the hippocampus. Neuroblasts that are generated in the SVZ migrate over long distances via the rostral migratory stream (RMS, depicted as thick grey line) into the olfactory bulb. In contrast, SGZderived neuroblasts migrate a short distance and integrate into the granule cell layer of the dentate gyrus. (b) Schematic representation of the SVZ. There are two populations of NSCs (B cells): quiescent NSCs (B1 cells) and activated NSCs (B2 cells). NSCs divide rarely and produce transit-amplifying precursors (C cells), which are fast-dividing cells. C cells constitute the major neuron-generating unit and give rise to neuroblasts (A
cells) that migrate into the RMS and eventually to the olfactory bulb. Among supporting cells only ependymal cells (E cells) that line ventricle surface are shown on the scheme (other supporting cell types include mature astrocytes and neurons, endothelial cells of blood vessels and microglia). (c) Schematic representation of the SGZ. Quiescent NSCs (B1 cells) divide very rarely and produce activated NSCs (B2 cells) that in turn give rise to transit-amplifying precursors (C cells). C cells generate neuroblasts (A cells) that differentiate into mature neurons (N) and integrate into the granule cell layer (depicted as grey circles symbolizing pre-existing mature granule cell neurons) of the dentate gyrus. Supporting cells for the SGZ are the same as for the SVZ (except for E cells) and are omitted from the scheme
cells release Noggin and potentiate neuronal production in the SVZ (Lim et al. 2000), whereas GABA released by neuroblasts decreases neuronal production (Liu et al. 2005b). Thus, altering gene expression in niche cells might influence NSC functioning, and this should be considered when developing a strategy for NSC analysis. The main recombinant viral types used for in vivo studies in the mouse brain are adeno-associated viruses (AAVs), lentiviruses and retroviruses. Next sections present the advantages and pitfalls of these viral types for the NSC analysis.
viruses. It can accommodate about 1 kb more without severe decrease in viral titers, but above that viral titers drop dramatically (Hermonat et al. 1997). Upon infection of the host cell, typical recombinant AAV plasmid does not integrate into the host genome (although in some cases AAV integration was reported). Recombinant AAVs are classified according to their serotype that is defined by capsid proteins transcribed from the cap gene. There are many AAV serotypes identified so far, including more common AAV1-12 as well as other more rare AAVs, e.g. AAVrh20 or rh43. AAV particles infect host cells via interaction of capsid proteins with particular cell surface receptors. Thus, different AAV serotypes infect only those cells that express serotype-specific receptors resulting in AAV serotype tropism. Since NSCs of the postnatal mouse brain display astrocytic features, AAV serotypes that have astrocytic tropism are more suitable for NSC infection. AAV5 utilizes platelet-derived growth factor receptor (PDGFR) to penetrate into the host cell (Jackson et al. 2006). Because at least a population of NSCs of
Recombinant AAVs AAVs have rather small viral particles (20 nm) and thus are highly diffusible in the brain tissue in comparison to other recombinant viruses that are used for in vivo brain analysis. Thus, AAVs are more suitable for infection of large brain areas. However, AAVs packaging capacity is only 4.7 kb, the lowest among typical recombinant
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the SVZ expresses PDGFR, AAV5 was used to label SVZ B cells following depletion of fastdividing C and A cells (Jackson et al. 2006). Another AAV serotype, AAV4, was shown to infect astrocytes and ependymal cells in the mouse SVZ (Liu et al. 2005a). Recently, a newly isolated AAV serotype, AAVrh43, was found to infect almost exclusively astrocytes (Lawlor et al. 2009). However, its tropism in the neurogenic niches still remains to be studied. New AAV serotypes can be artificially generated via mutagenesis of the cap gene. Using different mutagenesis protocols, most efficient and/or cell type specific AAV variants are selected round by round through a process called “directed evolution”. Eventually, this procedure results in the selection of a few AAV clones tailored to provide potent infection of the cell type of interest. Several AAV variants that efficiently transduce astrocytes or NSCs were engineered so far (Jang et al. 2011; Koerber et al. 2009). However, direct evolution of AAV can be performed only in vitro in NSC/astrocytic cultures and later these novel variants have to be tested in vivo for the efficiency and specificity of NSC infection. AAV serotypes of broad tropism have also been used to transduce NSCs/progenitor cells in the neurogenic niches, e.g., the most common recombinant AAV serotype AAV2 (Khodosevich et al. 2009; Lai et al. 2003). Because AAV2 transduces not only NSCs, but also other proliferating and non-proliferating cells in the neurogenic niche, this precludes AAV2 use for NSC-specific infections. Yet, AAV2 can be employed to study the total neurogenic capacity of NSCs/progenitor cells in the neurogenic niches as well as for the analysis of NSC progeny, e.g. neuroblast migration. AAV2 is the only AAV serotype that can be efficiently produced without ultracentrifugation, but using heparin columns instead (AAV2 capsid proteins bind to heparin). Such procedure of virus preparation results in higher viral titers and higher viral purity. The specificity of AAV2 as well as of other broad tropism AAVs can be enhanced by using cell type-specific promoters that drive gene expression in the AAV vector. For instance, in Khodosevich et al. (2009) expression of red fluorescent protein under the control of the
doublecortin (a marker for immature neurons) promoter restricted fluorescent protein expression to immature neurons in the rostral migratory stream and the olfactory bulb.
Recombinant Lentiviruses Lentiviruses are members of the Retroviridae family. Lentiviral particles are usually around 80–100 nm large and are not as highly diffusible as AAVs. Thus, lentiviruses allow for much more localized infections than do AAVs. Typical recombinant lentiviral vectors are based on the human immunodeficiency (HIV) virus backbone. The major advantage of lentiviral vectors in comparison to AAVs is their increased packaging capacity – recombinant lentiviruses are able to accommodate inserts up to 8–10 kb. However, in contrast to AAVs, upon infection of the host cell, lentiviral DNA integrates into the host genome. Since lentiviral integration might disrupt the function of neighboring genes, this integrative property is a drawback, for example, for clinical applications. As it was described for AAVs, recombinant lentiviruses can also be packed in different envelopes that determine viral tropism. Typical recombinant lentiviruses are pseudotyped (i.e., packed in the envelope of a foreign virus) with the envelope glycoprotein of vesicular stomatitis virus (VSV). Interestingly, VSV belongs to another viral family, Rhabdoviridae. Thus, standard recombinant lentiviruses are packed in an envelope of a foreign virus belonging to a different Viral group. The advantages of VSV envelope are broad tropism, high stability and ability to produce highly concentrated virus (i.e., high titers) (Burns et al. 1993). VSV-pseudotyped lentiviruses were successfully used to infect NSCs/precursor cells and to manipulate gene expression in both neurogenic zones of the mouse brain (Khodosevich et al. 2009; Kuwabara et al. 2004). Importantly, VSV-pseudotyped lentiviruses infect every cell type in the neurogenic niche and specific analysis of NSC population is not possible using these lentiviruses. However, since NSCs in both neurogenic regions produce
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progenitor cells and eventually neuroblasts that migrate some distance to their final destination, it is possible to analyze NSCs using their progeny. Following SVZ injection by VSVpseudotyped lentiviruses, infected neuroblasts appear in the rostral migratory stream already 1 day post-injection. These neuroblasts were infected directly by lentiviruses and migrated out of the SVZ. Another proliferating cell type in the SVZ, transit-amplifying precursors, usually divide 2–3 times and eventually differentiate into neuroblasts that again migrate out of the SVZ. Thus, in 2–3 weeks post-injection all initially infected fast-proliferating cells (precursors and neuroblasts) have migrated out of the SVZ. The only initially infected proliferating cells still residing in the neurogenic niche are NSCs. All infected neuroblasts that enter the rostral migratory stream 4 weeks post-injection are the progeny of initially infected NSCs. This approach was used for the analysis of Eph receptor A4 (EphA4) function in NSCs (Khodosevich et al. 2011). Knockdown of Epha4 in NSCs resulted in premature NSC differentiation and a decline in neuroblast number in the rostral migratory stream starting from 40 days post-injection. Lentiviral particles can be pseudotyped with other foreign envelopes that ensure more specific NSC tropism in comparison to VSV-pseudotyping. Mokola virus-pseudotyped lentiviruses were shown to infect selectively glial cells in the SVZ of mice with depleted pool of transit-amplifying precursors and neuroblasts (Alonso et al. 2008) allowing for the specific analysis of NSCs in the SVZ. In another study, SVZ injections using equine infectious anemia virus (EIAV)pseudotyped lentiviruses resulted in almost exclusive infection of glial cells/NSCs and ependymal cells (Jacquet et al. 2009). One day post-injection, the rostral migratory stream contained only few infected neuroblasts. However, after 1 month, numerous infected neuroblasts were migrating in the rostral migratory stream. Together these data support the notion that EIAV-pseudotyped lentiviruses infect by and large slow-dividing NSCs, but not fast-dividing precursors. Finally, lentiviruses pseudotyped
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with lymphocytic choriomeningitis virus (LCMV) envelope were shown to infect NSCs of the SVZ, but not other proliferating cell types (Stein et al. 2005). Three weeks following LCMVpseudotyped lentiviral injections, the majority of infected neuroblasts in the olfactory bulb were still migrating to their final position. It indicates that these neuroblasts were produced more than 2 weeks post-injection and derived from initially infected NSCs.
Recombinant Retroviruses Although the Retroviridae family includes several viral subfamilies, usually the term “recombinant retroviruses” is applied to only one subgroup of retroviruses – oncoretroviruses. In contrast to lentiviruses, oncoretroviruses cannot infect stationary, non-dividing cells. Mitosis and accompanied nuclear envelope breakdown are necessary for oncoretroviruses to enter the nucleus and to integrate into the host cell genome. Oncoretroviruses have rather large viral particles, around 100 nm, and thus do not diffuse in the brain tissue as easily as AAVs. Typical recombinant oncoretroviruses (hereafter just “retroviruses”) are based on the Moloney murine leukemia virus (MoMLV) backbone and can accommodate rather large inserts, up to 10 kb, between LTRs. Recombinant retroviruses fail to infect the host cell if mitosis occurs more than 6 h after infection (Miller et al. 1990). Since the majority of NSCs in the postnatal brain divide rarely, up to once in 2 weeks (Morshead et al . 1998 ) , retroviruses infect only few NSCs in both SVZ and SGZ. The vast majority of retrovirally infected cells are fast-dividing transit-amplifying precursors and neuroblasts (Khodosevich et al. 2011). Thus, a combination of lentiviral and retroviral injections can be used to distinguish between neurogenic effects derived from slowproliferating NSCs and fast-proliferating precursors. Using this approach, we showed that knockdown of Epha4 by lentiviral injections in the SVZ decreased the number of generated neuroblasts, whereas knockdown by retroviral
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injections did not (Khodosevich et al. 2011), implying EphA4 function in NSCs, but not in precursor cells.
Strategies to Analyze NSC Function in the Postnatal Brain Using Recombinant Viruses Recombinant viruses alone and in combination with other genetic approaches represent a powerful tool to analyze NSC function. Although several examples of NSC analysis using recombinant viruses were discussed above, this section provides a brief overview of different strategies for manipulation of gene expression in NSCs. The most common approach is to interfere with the expression of a gene of interest via its overexpression or knockdown, see e.g. Khodosevich et al. (2011), Lai et al. (2003) and Mao et al. (2009). Knocking down Disc1 (Disrupted in schizophrenia 1) by lentiviral injections into the adult mouse SGZ reduced proliferation of NSCs/progenitor cells, which resulted in changes of mouse behavior (Mao et al. 2009). Conversely, overexpression of Shh (sonic hedgehog) gene by AAV injections in the SGZ enhanced NSC/progenitor cell proliferation (Lai et al. 2003). Recombinant viral injections in conjunction with Cre/loxP recombination system have been successfully applied to obtain gene knockout in specific cell populations/specific regions. For instance, injection of a lentivirus encoding Cre recombinase into the SVZ of Smad4fl/fl mice reduced neurogenesis by knocking out Smad4 in NSCs (Colak et al. 2008). Cre/loxP recombination approach can also be used for cell lineage tracing experiments. Removal of the STOP sequence from the loxP-STOP-loxP-GFP cassette bearing cells via Cre/loxP recombination allows for tracing of the cell progeny. Following the STOP sequence deletion, cells are permanently labeled by GFP, and Gfp coding sequence will be transferred to all descendants of the cell. Injection of recombinant lentiviruses coding for Cre recombinase was used to trace NSC progeny in both SVZ and SGZ (Stein et al. 2005; Suh et al. 2007).
Restriction of gene expression to NSCs can be achieved by the use of specific promoters driving expression of the gene of interest. However, this approach did not receive much attention and there are very few studies employing specific promoters to manipulate NSC gene expression. In one of these, upon SVZ injections, lentiviruses that expressed Cre recombinase under the Gfap promoter induced Cre/loxP recombination only in NSCs (Stein et al. 2005). In the context of AAVrh43 serotype (AAV serotype that has astrocytic tropism), the Gfap promoter was also employed for astrocyte-specific hippocampal labeling. This approach utilized a combination of promoter specificity and viral tropism. As a result, more than 90% of labeled cells were astrocytes (Lawlor et al. 2009). However, NSCs of the hippocampal SGZ were not tested in the study. Such underutilization of NSC specific promoters in recombinant viruses can be explained by the relatively large size of many specific promoters that precludes their use due to the vector size limitations. Additionally, all common NSC markers are also expressed in astrocytes (GFAP, GLAST, BLBP) or in transit-amplifying precursors (nestin, Sox2). Recent transcriptome analysis of SVZ-derived NSCs (Beckervordersandforth et al. 2010) revealed genes that are expressed exclusively in NSCs. Follow-up studies might provide NSC-specific promoters that are short enough to be used in recombinant viruses.
Conclusion In the past decade, recombinant viruses emerged as one of the major approaches to study brain development and neuronal circuitry in vivo. However, only a few of these viruses were used in NSC studies. Furthermore, the majority of NSC studies employed “classical” broad cell type tropism viruses: AAV serotype 2 and VSVpseudotyped lentiviruses. Since NSCs have an astrocytic nature, the use of recombinant viruses with astrocytic tropism might be an advantage to restrict virally-delivered gene expression to NSCs/astrocytes of the neurogenic niches. NSCspecific effect of the virally-delivered gene can
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be analyzed using NSC progeny, precursor cells and neuroblasts. In this case the function of the gene in NSCs can be distinguished from its function in non-proliferating mature astrocytes. Recent NSC transcriptome studies might help to identify genes that have NSC-restricted expression. Thus, a combination of recombinant viruses with other molecular biological techniques, e.g. transgenic animals, will provide an opportunity to manipulate specifically NSC expression sparing all other cells. Furthermore, the implementation of non-classical recombinant viruses for NSC analysis as well as the development of new NSC-specific viral subtypes will improve our toolbox for NSC analysis in vivo. Given the potential of NSCs as a treatment for many brain diseases, it is important to investigate NSC functioning under normal and pathological conditions, and recombinant viruses might prove to be the ideal tool for this.
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