Induced neurogenesis by endogeneous progenitor cells in the adult mammalian brain

M.A. Hofman,G.J. Boer, A.J.G.D.Holtmaat,E.J.W.VanSomeren, J. VerhaagenandD.F. Swaab (Eds.) Progress in Brain Research, Vol. 138 0 2002 Published by Elsevier Science B.V.

CHAPTER 27

Induced neurogenesis by endogenous progenitor cells in the adult mammalian brain Eva Chmielnicki and StevenA. Goldman * Department

of Neurology

and Neuroscience,

Cornell

Introduction

Medical

College,

New York, NY 10021,

Neuronal stem aud progenitor mammalian brain

Neurodegenerative diseases are characterized by the gradual loss of mature functional neuronal populations witbin the nervous system (Kowall et al., 1987; Cummings et al., 1998; Jenner and Olanow, 1998). Mature neurons are postmitotic and hence incapable of self-renewal, and spontaneous replacement of the dying neurons has not been reported in any neurodegenerative disease or experimental model thereof. Nonetheless, the identification of neural progenitor cells in both periventricular and parenchymal regions of the adult mammalian brain suggests that compensatory neuronal replenishment by endogenous progenitor cells should be feasible (reviewed in Weiss et al., 1996; Goldman, 1998, 2001; Gage, 2000). The failure of the adult mammalian brain to utilize these resident progenitor cells to restore depleted neuronal populations has been a fundamental paradox of both evolutionary neurobiology and neurologic therapeutics. This review explores a set of recent studies that have tested the hypothesis that the lack of compensatory neurogenesis to injury and disease might reflect a lack of appropriate postmitotic trophic factors in potentially neurogenic regions of the adult brain. * Correspondence

University

to: S.A. Goldman,

Department

of Neu-

rology and Neuroscience, Cornell University Medical Center, 1300 York Ave. Room E607, New York, NY 10021, USA. Tel.: +l-212-746-6572; Fax: +1-212-746-

8972; E-mail: [email protected]

USA

cells of the adult

A major population of persistent neural progenitor cells resides in the subventricular zone (SVZ) of the lateral ventricle (reviewed in Alvarez-Buylla and Lois, 1995; Goldman and Luskin, 1998; AlvarezBuylla et al., 2000). Following the earlier discovery of VZ progenitor cells in the adult bird brain (Goldman and Nottebohm, 1983; Alvarez-Buylla et al., 1990), mitotically active SVZ progenitor cells were identified in the postnatal mouse brain (Luskin, 1993). These postnatal progenitor cells were found to give rise to both astrocytes and neurons in explant cultures, both in songbirds (Goldman et al., 1992) and mice (Lois and Alvarez-Buylla, 1993). In adult rats, neurogenic progenitors were found to be distributed throughout at least the rostra1 two-thirds of the SVZ, when assessed in explant cultures of the adult ventricular wall (Kirschenbaum and Goldman, 1995). These neuronal progenitors appeared to be lineally related, and either identical to or derived from multipotential neural stem cells that were identified and mapped throughout the adult ventricular neuraxis (Reynolds and Weiss, 1992; Richards et al., 1992). Restrictions on neuronal recruitment normal adult brain

in the

The neuronal progeny of adult SVZ progenitor cells were found to migrate in closely associated chains

452

to the olfactory bulb via the rostra1 migratory stream (RMS). Once in the bulb, these cells differentiate into local interneurons. Remarkably, though, they do not normally invade any other regions of the brain (Luskin, 1993; Lois and Alvarez-Buylla, 1994). Since potentially neurogenic progenitor cells are so widespread in the adult brain, while the regions of actual neuronal recruitment are so few and so segregated, it would appear that compensatory neurogenesis to injury might be limited by regionally specified limitations on either the migration, differentiation, or survival of VZ-derived neurons. To circumvent these restrictions on neuronal recruitment, several strategies have been proposed. First, a redirection of newly generated neurons from the olfactory subependyma has been considered as one strategy to achieve neuronal recruitment to otherwise non-neurogenic regions of the adult brain (NaitOumesmar et al., 1999). Second, since many if not most SVZ progenitor cells appear to give rise to glial cells in the adult, the instruction of SVZ daughter cells away from a glial and towards a neuronal phenotype might provide another strategy for induced neurogenesis (e.g. Shah et al., 1996). Third, since the survival and local recruitment of newly generated neurons may be limited by the availability of postmitotic neurotrophic factors, the provision of these agents might achieve heterotopic or compensatory neuronal addition. This review will focus on recent studies suggesting that targeted ventricular administration of growth factors known to induce neuronal differentiation and survival from SVZ progenitors, as well as the redirected migration of their neuronal progeny, may be viable strategies for inducing neuronal replacement in the adult mammalian brain. Intraventricular infusion of neural mitogens can induce gliogenesis and neurogenesis A variety of studies in tissue culture had demonstrated that neuroepithelial stem cells proliferate in response to EGF, FGF, or TGFcl (Gensburger et al., 1987; Anchan et al., 1991; Reynolds et al., 1992). On the basis of these studies, Craig et al. (1996) examined the effects of intraventricular growth factor infusion on the division and differentiation of adult rodent SVZ cells in vivo. Upon infusion of EGF, cellular proliferation in the SVZ was increased.

Newly generated cells were found within both the SVZ and the brain parenchyma adjacent to the SVZ. Retroviral tagging of the VZ population showed that the newly generated cells in the parenchyma were VZ-derived migrants. However, despite sharp EGF-associated increments in mitotic gliogenesis and parenchymal glial recruitment, little or no neurogenesis was noted in response to EGF. FGF-2 infusion into the lateral ventricle similarly increased cellular proliferation in the SVZ (Kuhn et al., 1997). Furthermore, FGF increased the number of newborn neurons migrating to the olfactory bulb, the usual target of neurons generated in the lateral ventricular SVZ. However, only a very small percentage of the newly generated cells that remained in or near the SVZ differentiated along the neuronal lineage, and no evidence of cortical or striatal neuronal recruitment was noted. Thus, infusion of these factors was insufficient to induce significant neuronal differentiation and recruitment from SVZ progenitor cells into nearby parenchymal regions (Kuhn et al., 1997). While these studies were performed in normal animals, the environment in which a progenitor resides might be drastically different in disease, thereby altering both the exposure and response of progenitor cells to growth factors. Fallon et al. (2000) infused TGFa, a membrane-bound EGF cogener, into the striatum of animals that had unilateral 6-hydroxydopamine (6-OHDA) lesions of the substantia nigra, a model of Parkinson’s disease. In animals that received both the 6-OHDA lesion and the infusion of TGFcl, SVZ cells proliferated and migrated into the striatum, toward the TGFcl infusion site. By immunostaining adjacent sections, the authors demonstrated that the neuronal marker BIBtubulin and the astrocytic marker SlOOg were both expressed within the ‘ridge’ of migrating and proliferating cells, although the relative proportions of dividing cells that differentiated along glial or neuronal lineages was not reported. Furthermore, some of the newly generated cells co-expressed dopamine transporter (DAT) and tyrosine hydroxylase (TH), markers of dopaminergic cells. Therefore, growth factor application in the disease environment resulted in the expansion and local recruitment of new neurons from SVZ progenitor cells (Fallon et al., 2000).

453

Neurotrophins are differentiation and survival factors in the central nervous system The neurotrophin family of proteins, which includes NGF, BDNF, NT3, and NT4/5, are capable of enhancing the differentiation and/or survival of many populations of neurons throughout the nervous system. Specifically, this family of proteins has been shown to provide neurotrophic support to peripheral sensory neurons (Lindsay et al., 1985; Davies et al., 1986), motor neurons (Wong et al., 1993), basal forebrain cholinergic neurons (Morse et al., 1993; Tuszynski, 2000), dopaminergic (Hyman et al., 1991), serotinergic (Mamounas et al., 1995), and GABAergic neurons (Ventimiglia et al., 1995), cerebellar granule neurons (Segal et al., 1992), and retinal ganglion neurons (Johnson et al., 1986). On the basis of these studies, Kirschenbaum and Goldman (1995) examined the role of various neurotrophins on neuronal outgrowth and survival in explant cultures of the adult rat SVZ. In this model, BDNF, but not NGF or NT3, promoted both the maturation and survival of SVZ-derived neurons in a dose-dependent fashion, for as long as 2 months in vitro (Goldman, 1997). Since virtually none of these newly generated neurons incorporated [3H]thymidine in vitro, BDNF appeared to exert its effects directly on either postmitotic neuronal progenitor cells, or on their neuronal progeny. Accordingly, full-length trkl3, the high affinity BDNF receptor, was found to be heavily expressed by the SVZ explant-derived neurons, suggesting their BDNF responsiveness. To distinguish between BDNF’s role in promoting neuronal maturation from that of survival, the authors found that while a 7-day initial pulse of BDNF was sufficient for induction of neuronal maturation of SVZ progenitor cells, it was insufficient to promote long-term SVZ-derived neuronal survival (Goldman, 1997). Rather, continuous BDNF exposure was necessary, confirming the role of BDNF, not only as a neuronal differentiation factor for SVZ progenitor cells but also as a survival factor for SVZ-derived neurons (Fig. 1). In parallel work, Ahmed et al. (1995) also found that BDNF enhanced the neuronal differentiation of EGF-generated spheres from El4 mouse SVZ. However, even with continuous BDNF exposure, this

group found that BDNF did not increase the survival of SVZ-derived neurons. The difference between the results of this group and those of Kirschenbaum and Goldman (1995) likely reflect differences between EGF-expanded neurospheres and SVZ-derived explants. While Kirschenbaum and Goldman (1995) added 2% serum to their explant cultures, Ahmed et al. (1995) raised their neurospheres in serum-free media. As serum contains a variety of agents capable of modulating and supplementing BDNF-dependent signaling events, the more complex humoral environment of a serum-supplemented explant culture might have revealed BDNF-dependent survival effects not apparent in the minimal media utilized by Ahmed et al. (1995). For example, as serum contains insulin, a ligand that signals through the IGF receptor, insulinBDNF signaling pathway interactions might alter the response of SVZ progenitor cells compared with that of BDNF alone (Arsenijevic and Weiss, 1998).

The SW progenitor BDNF in vivo

population

responds to

As further studies corroborated the role of BDNF as a neuronal maturation and survival factor for ‘neural stem and progenitor cells in vitro (Shetty and Turner, 1998), it became apparent that BDNF might be an important factor in SVZ progenitor cell commitment and recruitment in vivo. Indeed, SVZ progenitor cells migrate via the RMS to the olfactory bulb, which may serve as a source of BDNF for the infiltrating neuroblasts by virtue of its BDNF expression (Maisonpierre et al., 1990). In addition, full-length trkB is expressed in the SVZ, as well as by migrating neuroblasts in the RMS (Zigova et al., 1998). Ventricular zone cells and subventricular glia, however, express high amounts of truncated trkl3, which may serve to limit BDNF access to the subventricular zone (Yan et al., 1994). On the basis of this expression pattern, Zigova et al. (1998) hypothesized that intraventricular administration of exogenously applied BDNF protein would be sufficient to induce neuronal differentiation in vivo. They found that chronic intraventricular infusion of BDNF via Alzet mini-pump, with a daily dose of 12 kg BDNF protein (>96 kg/ml CSF/day), resulted in a doubling in the rate of addition of BrdU+/pIII-tubulin+ neurons to the olfactory

Days

in vitro

Days

in vitro

Fig. 1. (A) BDNF promotes the outgrowth and survival of adult rat SVZ explant-derived neurons in vitro. (B) BDNF withdrawal at 7 days in vitro results in the death of new neurons derived from adult rat SVZ explants. (From Goldman, 1997.)

bulb. This, and the Kuhn et al. (1997) report of FGFstimulated olfactory neuronal recruitment, were the first reports of induced neuronal addition in the adult forebrain, but in each case, only olfactory bulb neuronal addition was noted. Intraventricular injection of AdBDNF targets transgene expression to the ventricular wall Taken together, these studies showed that intraventricular delivery of growth factors can affect the

division, differentiation and/or survival of SVZ progenitors and their progeny in the adult forebrain (Craig et al., 1996; Kuhn et al., 1997; Zigova et al., 1998). However, daily infusion of these factors necessitated transcranial intraventricular mini-pump implantations, and the administration of high doses of proteins of variable stability and bioavailability. To obviate the need for daily protein infusions, we therefore chose to use an adenoviral vector to overexpress the gene for BDNF in the ependymal wall (Bajocchi et al., 1993; Yoon et al., 1996). Ade-

455 noviral vectors can carry >7 kb of foreign DNA, can be grown to high titer, and can achieve near-100% infection efficiency in vitro. In addition, adenoviral vectors can infect both mitotic and postmitotic cells, and do not integrate into the host chromosomes, decreasing the potential for insertion-dependent effects unrelated to the viral transgenes. With the goal of inducing high level and long-term BDNF expression in vivo, we therefore constructed a bicistronic adenovirus carrying the gene for BDNF under cytomegalovirus (CMV) promoter control, and humanized green fluorescent protein (hGFP) under internal ribosome entry site (IRES) control. Either this virus, AdCMV : BDNF : IRES : hGFP (AdBDNF), or its control virus AdCMV: hGFP (AdNull), were stereotactically injected into the lateral ventricles of adult rats. In situ hybridization on sagittal brain sections taken 3 weeks after the adenoviral injections showed that BDNF and hGFP mRNAs were restricted to the wall of the lateral ventricle. Expression of the viral transgenes was limited to the ventricular surface; no expression of viral transgenes was noted in either the RMS or olfactory bulb (OB), at any point for over a month after injection, in any of several dozen rats so treated. The selective targeting of viral transgene expression to the ventricular wall was thus accomplished by the spatial restriction of adenoviral infection, which was limited to the ependymal cells. ELISA measurements of CSF protein levels showed that while BDNF was undetectable in saline- and AdNull-injected rats, BDNF levels in the AdBDNF-injected animals averaged 2 rig/ml (Benmiss et al., 2001). This level was within the range of BDNF concentrations that elicit neurotrophic effects in vitro (Lindsay et al., 1994). CSF BDNF levels remained elevated relative to controls for up to 2 months after injection, with a steady decline in the second month. Taken together, these data suggest that a single intraventricular injection of an adenoviral vector can target high level expression of viral transgenes to the ventricular wall for up to 2 months, effectively recruiting the ventricular ependyma as a source of secreted neurotrophin by which to influence the subependymal progenitor population (Fig. 2).

AdBDNF infection of the ventricular wall increased neuronal recruitment to the olfactory bulb To determine if the targeted ventricular overexpression of BDNF could induce neuronal recruitment, mitotic SVZ progenitor cells were labeled by 18 daily intraperitoneal injections of bromodeoxyuridine (BrdU), a thymidine analog. When the animals were then sacrificed on day 21 and their brains examined for the density of BrdU-labeled cells, the olfactory bulbs of AdBDNF-injected animals were found to harbor a 25fold increase in the density of BrdU+ cells compared with AdNull controls (Fig. 3). In addition, over 90% of BrdU+ cells expressed MAP-2 and flIII-tubulin, two neuron-specific markers, indicating that the vast majority of new cells were being recruited to the olfactory bulb as neurons (Lee et al., 1990; Menezes et al., 1995). This percentage was roughly equivalent in both the AdNulland AdBDNF-injected rats, suggesting that BDNF did not alter the fate of SVZ migrants to the olfactory bulb. These observations indicated that the transduction of the adult ependyma to overexpress BDNF resulted in a rapid and substantial increase in neuronal recruitment to the olfactory bulb. Ventricular addition

AdBDNF induced striatal neuronal

Although AdBDNF significantly increased BrdU+ neuronal addition to the olfactory bulb, the BrdU labeling index in the cortex, septum and striatum was approximately the same in AdBDNF- and AdNullinjected animals (Benraiss et al., 2001). While we found that ventricular BDNF overexpression did not result in an increase in the BrdU labeling index in these non-neurogenic brain regions, it remained possible that BDNF might influence either the neuronal maturation of progenitor cells or the survival of their neuronal progeny. BrdU+ cells were therefore examined for their expression of neuron-specific markers. We found that, while almost no BrdU+/fiIIItubulin+ cells were found in the septum or cortex of AdBDNF-injected animals, the striatum of AdBDNF-injected animals harbored 140 newly generated neurons per cubic millimeter. Overall, there was a 7-fold increase in the BrdU+/flIII-tubulin+

456

AdBDNF’

’

AdNull

Fig. 2. Intraventricular AdBDNF injection targets viral transgene expression to the ventricular wall and CSF. (A-C) In situ hybridization on sag&al sections of AdBDNF-injected animals. Hybridization of anti-sense probes for BDNF (A) and GFP (B) show that expression of the viral transgenes is limited to the ventricular wall. Sense probes for BDNF (C) produced no signal. Scale bar: 35 urn. (D) Following cistema magna puncture and CSF withdrawal, ELISA shows that BDNF proteins levels were elevated (to an average of 2 rig/ml) in AdBDNF-injected animals, relative to AdNull- and saline-injected controls, 3 weeks following adenoviral injections. (From Benraiss et al., 2001.)

density compared with the striata of AdNull-injected animals (Benraiss et al., 2001) (Fig. 4). Interestingly, AdNull-injected animals exhibited a small amount of striatal neuronal production, relative to phosphate-buffered saline (PBS)-injected animals that never exhibited neuronal addition to the striaturn. This suggested that adenoviral injection per se might induce a small amount of striatal neuronal recruitment. This might be due to adenoviralinduced release of inflammatory cytokines and/or endothelial-derived BDNF (Leventhal et al., 1999;

Driesse et al., 2000). Regardless of its basis, the adenoviral-associated induction of a small degree of neuronal recruitment was intriguing, and suggests many lines of potentially fruitful investigation. However, to circumvent the limitations of adenoviral vectors, studies will need to be undertaken in which BDNF is overexpressed using viral vectors that contain inducible promoters, and/or using lentiviral vectors that exhibit more prolonged viral transgene expression with less of an immune response (Buchschacher and Wong-Staal, 2000).

457

L1.2mm

L1.7mm

Ll.7mm

L2.2mm

AdBDNF

’

AdNull

-

Treatment Fig. 3. AdBDNF injection increases recruitment to the olfactory bulb. (A,B) Stereological reconstruction of BrdU+ cells, viewed at different mediolateral levels of the olfactory bulb, revealed substantially higher BrdU+ cell density in the olfactory bulb of AdBDNFinjected rats (A) than in their AdNull-injected controls (B). Arrows denote entry to rostral migratory stream (red). (C) The average number of BrdU+ cells/mm3 in the olfactory bulb was 25fold higher in AdBDNF-injected animals compared to AdNull-injected controls 3 weeks following adenoviral injections. (From Benraiss et al., 2001.)

Pencea et al. (2001) have also noted BDNFassociated neuronal addition to the adult striatum, using intraventricular infusion of BDNF protein. However, they also reported that daily BDNF protein infusion into the lateral ventricles increased the BrdU labeling index in the normally non-neurogenic striatum, septum and hypothalamus. They further claimed that in both PBS- and BDNF-infused animals, many of these newly generated cells were

neuronal. The differences between the observation of Benraiss et al. (2001) of AdBDNF-associated striatal neurogenesis and the substantially broader claim of Pencea et al. (2001) of widespread neuronal induction may at least in part be due to the differing bioavailabilities of BDNF in animals infused with protein directly into the ventricle, relative to those transduced by an overexpression vector intended to generate BDNF in situ. For instance,

458

H

T

AdBDNF

Saline

459

the dose used by Pencea et al. (2001), 12 kg/day, should have achieved a minimal CSF concentration of 100 pg/ml, assuming a CSF volume of 125 pl in rats. In our AdBDNF-injected rats, we measured a CSF BDNF concentration of 2 rig/ml, a level 5 x 1O-4 that used by Pencea et al. (2001). This >104-fold greater concentration of BDNF might be expected to trigger any number of trk-dependent cellular responses, rendering comparison between these different approaches difficult. A second difference between these studies was in the dose and location of BrdU administration: while Pencea et al. (2001) infused BrdU intraventricularly, Benraiss et al. (2001) injected BrdU intraperitoneally, possibly resulting in a large difference in the effective BrdU concentration within the brain. Finally, whereas Benraiss et al. (2001) required rigorous confocal analysis of each BrdU-labeled cell as being neuronal (with optical sectioning and orthogonal reconstructions of each cell in order to confirm neuronal identity), Pencea et al. (2001) required only composite imaging by confocal. This approach may be prone to false-positive designation, reiterating the need for rigorous imaging criteria when assessing phenotype by confocal analysis (Kornack and Rakic, 2001). AdBDNF-induced striatal neurons expressed antigens characteristic of medium spiny neurons Because AdBDNF-induced striatal neurons continued to survive and integrate into the striatal parenchyma as late as 8 weeks post-adenoviral injection, it was important to determine the phenotype of these newly generated neurons. While the striatum includes a complex array of neuronal phenotypes, the majority of striatal neurons are GABAergic medium spiny projection neurons containing calbindin, a calcium binding protein, and DARPP-32, a dopamineand CAMP-regulated phosphoprotein (Ouimet et al., 1998). Notably, manygroups h&e shown that BDNF plays an important role in the maturation and survival

of striatal medium spiny neurons. In vitro, Nakao et al. (1995) have shown that BDNF enhances the survival and morphological differentiation of DARPP+ neurons derived from the embryonic striatum. In addition, DARPP-32 expression in the striatum is downregulated in BDNF-/- animals (Ivkovic et al., 1997; Ivkovic and Ehrlich, 1999). These data suggested that AdBDNF-induced striatal neurons might express phenotypic markers characteristic of the medium spiny phenotype. Indeed, at both 3 and 8 weeks postadenoviral injection, most BrdU+ neurons in AdBDNF-treated striata were positive for GAD, calbindin, and/or DARPP-32 (Benraiss et al., 2001) (Fig. 5). AdBDNF-induced striatal neurons could be parenchyma- and/or SVZ-derived Taken together, the above data showed that ventricular BDNF overexpression induced neuronal recruitment not only to normally neurogenic regions like the olfactory bulb, but also to otherwise nonneurogenic regions of the adult rat brain (Benraiss et al., 2001). BDNF-mediated neuronal addition to the neostriatum could occur either by BDNF-induced neuronal differentiation of intraparenchymal progenitor cells, and/or by the induction and parenchymal migration of SVZ progenitor cells. Indeed, we have recently found parenchymal progenitor cells within the subcortical white matter of the adult human brain (Roy et al., 1999). Whether these progenitors are SVZ-derived intraparenchymal migrants is not clear, but Magavi et al. (2000) have suggested that newly generated neurons found in the photolyticallylesioned cortex of mice might be SVZ-derived. Indeed, as targeted cortical apoptosis increases local BDNF expression, BDNF might be necessary for the neuronal differentiation within the cortex of these progenitor cells (Wang et al., 1998). Furthermore, Behar et al. (1997) showed that BDNF stimulates migration of embryonic cortical neurons, suggest-

Fig. 4. AdBDNF injection induces striatal neuronal recruitment (A-G) Confocal images of bIII-tubulin (red)/BrdU (green) doubleimmunolabeled cells (arrows) in the striatum of a rat that, 3 weeks previously, received an intraventricular injection of AdBDNF. (A,D) Composite of six separate confocal images, which are serially displayed in B (for cell in A) and G (for cells in D). (C,E,F) Orthogonal views of double-labeled cells, showing them from the xz and yz axes. Scale bars: 10 urn. (H) Mean density of striatal BIB-tubulin+/BrdU+ cells in AdBDNF-, AdNull- and saline-injected animals at 3 weeks. (From Benraiss et al., 2001.)

Fig. 5. AdBDNF-induced striatal neurons mature to express markers characteristic of medium spiny neurons, and survive for at least 5-8 weeks. (A-F) Confocal images of (A-C) GAD67 (red)/BrdU (green) and (D-F) DARPP32 (red)/BrdU (green) double-immunolabeled cells (arrows) in the striatum of a rat that, 8 weeks previously, received an intraventricular injection of AdBDNF. Scale bars: 10 Wm. (From Benraiss et al., 2001.)

ing that ventricular BDNF overexpression might also mediate the migration of SVZ-derived neurons into the neostriatum. Further studies must be done to elucidate the mechanism by which BDNP induces parenchymal neuronal recruitment from endogenous progenitor cells in the adult mammalian brain. BDNF overexpression as a strategy for restoring neurons in neurodegenerative disease Huntington’s disease is characterized by a progressive motor deficit resulting from the selective degeneration of DARPP+ medium spiny neurons in the neostriatum (Mitchell et al., 1999). The importance of BDNP in the maintenance of the medium spiny population was suggested by Nucifora et al. (2001), who showed that mutant Huntington expression altered the transcriptional activity of CBP, a mediator of BDNF expression. Furthermore, Zuccato et al. (2001) found that wild-type huntingtin protein upregulates BDNF gene transcription. As the tran-

scriptional activity of huntingtin protein is decreased in the disease state, the authors concluded that this would result in insufficient neurotrophic support for medium spiny neurons (Zuccato et al., 2001). As our data showed that BDNF overexpression is a viable strategy for replacement of striatal DARPP-32+ neurons, we are currently studying whether AdBDNFmediated neuronal addition into the striata of mutant huntington transgenic mice is sufficient to restore diminishing DARPP-32+ neuronal numbers in these mice (Mangiarini et al., 1996; Benraiss et al., 2001). Synergistic strategies for inducing neurogenesis in the adult brain These studies showed that induction of neuronal production can be accomplished through BDNFmediated neuronal differentiation of SVZ progenitor cells. By the same token though, inhibition of glial differentiation from SVZ progenitor cells might be another mechanism by which neuronal produc-

tion from endogenous progenitor cells can be enhanced. Several agents have been shown to induce astroglial differentiation from VZ progenitor cells in vitro (Hughes et al., 1988; Lillien et al., 1998). The bone morphogenetic proteins (BMPs) in particular have been found to instruct late fetal VZ progenitors to the glial lineage (Mehler et al., 2000). As BMPs and their receptors are present in high concentrations in the ventricular zone of the adult rat brain, abrogation of BMP signaling in VZ progenitor cells might be a mechanism by which to inhibit astroglial differentiation, and thereby promote differentiation along the neuronal lineage (Gross et al., 1996). Noggin belongs to a class of diffusible BMP antagonists that prevent BMPs from binding to their receptors. Noggin is expressed by the ependymal cells of the ventricular wall, suggesting a mechanism by which a niche is created for neuronal instead of astroglial differentiation of some VZ progenitor cells (Zimmerman et al., 1996; Lim et al., 2000). Indeed, we found that adenoviral ventricular noggin overexpression increased olfactory neuronal recruitment; this effect was accentuated by BDNF expression, such that both the suppression of glial and promotion of neuronal differentiation pathways proved viable means for inducing neuronal addition to the olfactory bulb (Chmielnicki et al., 2001) (Fig. 6). Further studies must be done to determine if co-overexpression of noggin with BDNF, as well as with other factors that induce neuronal differentiation, such as IGF, might further increase in vivo neuronal production, not only to normally neurogenic regions like the olfactory bulb, but also to otherwise non-neurogenic areas of the adult CNS. Conclusion

Neural progenitor cells in the adult mammalian brain are a potential source for neuronal replenishment in neurodegenerative disease. In as much as these progenitor cells can be induced to divide, differentiate as neurons, migrate to appropriate regions of the degenerating brain, survive and make appropriate postsynaptic and presynaptic connections, they may replace the neurons that die in the disease process. As the adult rodent olfactory bulb utilizes these endogenous progenitor cells, recruiting them to replace the neuronal complement that is lost, a state of equilibrium

G

CI

0

AdNoggin

’ AcKflcKfDg#; ’

Fig. 6. Ventricular noggin overexpression results in an increase in neuronal recruitment to the olfactory bulb. Average foldincrease in the number of BrdU+ cells/mm3 in the olfactory bulbs of AdNogginAB2-, and AdBDNF/AdNogginABZ-injected rats compared with AdNull-injected control animals.

between neuronal death and SVZ progenitor-derived neuronal replacement is achieved. However, other regions of the adult mammalian brain do not have this progenitor-mediated homeostatic mechanism for the replacement of dying neurons, often resulting in a gradual loss of neurons with senescence. In the SVZ, the importance of the neurotrophin and BMP families is suggested by their abundant expression, as well as that of their receptors. The studies reviewed here suggest that targeted ventricular administration of these and other factors, rationally designed to capitalize upon the known biologic functions of these molecules, is a viable strategy for inducing neuronal production from endogenous progenitor cells in the adult mammalian brain. Abbreviations

AdBDNF AdNoggin BDNF BMP BrdU CSF CMV

adenoviral BDNF adenoviral noggin brain-derived neurotrophic factor bone morphogenetic protein bromodeoxyuridine cerebrospinal fluid cytomegalovirus

462

DARPP-32 ELISA EGF FGF GAD hGFP IGF IRES NT3 OB 6-OHDA RMS svz TGF TrkB

dopamineand CAMP-regulated phosphoprotein enzyme-linked immunosorbent assay epidermal growth factor fibroblast growth factor glutamic acid decarboxylase humanized green fluorescent protein insulin-like growth factor internal ribosome entry site neurotrophin-3 olfactory bulb 6-hydroxydopamine rostra1 migratory stream subventricular zone transforming growth factor tropomyosin-related kinase B

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