Prion protein (PrP^c) positively regulates neural precursor proliferation during developmental and adult mammalian neurogenesis

Abstract

The misfolding of the prion protein (PrP^c) is a central event in prion diseases, yet the normal function of PrP^c remains unknown. PrP^c has putative roles in many cellular processes including signaling, survival, adhesion, and differentiation. Given the abundance of PrP^c in the developing and mature mammalian CNS, we investigated the role of PrP^c in neural development and in adult neurogenesis, which occurs constitutively in the dentate gyrus (DG) of the hippocampus and in the olfactory bulb from precursors in the subventricular zone (SVZ)/rostral migratory stream. In vivo, we find that PrP^c is expressed immediately adjacent to the proliferative region of the SVZ but not in mitotic cells. In vivo and in vitro studies further find that PrP^c is expressed in multipotent neural precursors and mature neurons but is not detectable in glia. Loss- and gain-of-function experiments demonstrate that PrP^c levels correlate with differentiation of multipotent neural precursors into mature neurons in vitro and that PrP^c levels positively influence neuronal differentiation in a dose-dependent manner. PrP^c also increases cellular proliferation in vivo; in the SVZ, PrP^c overexpresser (OE) mice have more proliferating cells compared with wild-type (WT) or knockout (KO) mice; in the DG, PrP^c OE and WT mice have more proliferating cells compared with KO mice. Our results demonstrate that PrP^c plays an important role in neurogenesis and differentiation. Because the final number of neurons produced in the DG is unchanged by PrP^c expression, other factors must control the ultimate fate of new neurons.

The mammalian prion protein (PrP^c) has been intensively studied for its role in mammalian neurodegenerative disorders such as Creutzfeldt–Jakob disease and the transmissible spongiform encephalopathies (1, 2). Suggested roles for PrP^c, an N-linked glycoprotein tethered to the cell membrane by a glycosylphosphatidylinositol anchor, include cell signaling, survival, adhesion, and differentiation (3). Despite these putative roles for PrP^c, mice that are null for PrP^c exhibit no consistent, overt phenotype other than resistance to infection with prions, the infectious agent in the transmissible spongiform encephalopathies (4). Recent work has shown that PrP^c induces polarization in synapse development as well as in neuritogenesis in embryonic hippocampal neuron cultures (5, 6). PrP^c is also up-regulated after focal cerebral ischemia (7), and PrP^c overexpression reduces the extent of neuronal loss after ischemic insult, suggesting that PrP^c might confer a neuroprotective effect in certain contexts (8). Recent studies have also shown that PrP^c is expressed on the surface of long-term repopulating hematopoietic stem cells and that PrP^c-null mice have limited hematopoietic stem cell self-renewal (9).

Given the role of PrP^c in hematopoietic stem cells and its abundant expression in the developing and adult mammalian CNS, we investigated the role of PrP^c in neural development and in adult neurogenesis, which occurs constitutively in the dentate gyrus (DG) of the hippocampus and in the olfactory bulb from precursors in the subventricular zone (SVZ)/rostral migratory stream (10). We performed analyses of PrP^c knockout (KO), wild-type (WT), and overexpresser (OE) mice to investigate loss- and gain-of-function. By using immunocytochemistry, in vitro analyses of embryonic neural precursor cultures, in vivo cell birth-dating approaches, and morphometry, we find that PrP^c expression is neuronal-specific in differentiated neural cells and that it increases multipotent neural precursor differentiation in vitro and proliferation in neurogenic regions in vivo. Our results describe an important role for the normal prion protein in neurogenesis of the developmental and adult mammalian CNS.

Results

PrP^c Expression Increases as Neurons Mature, but It Is Not Detected in Astroglia or Oligodendroglia.

We investigated PrP^c protein expression in the developing and mature CNS, both in vitro and in vivo. PrP genotypes were confirmed by PCR, and PrP^c protein expression was verified by Western blot analysis (Fig. 1A); the specificity of the PrP^c polyclonal antibody used for immunohistochemistry was confirmed by abundant staining in PrP OE brain and a lack of staining in PrP^c KO brain (Fig. 1 B and C). In vivo, we observed that PrP^c is expressed most strongly immediately adjacent to the proliferative region of the SVZ but not in mitotic cells (Fig. 2 A and B). We also find that PrP^c expression increases in fully differentiated, mature neurons. Both in vivo and in vitro, PrP^c is found in increasing amounts as neuronal differentiation progresses: lowest in a subset of nestin-positive multipotent neural precursors; more in β-III tubulin (TuJ1)- and doublecortin-positive immature neurons and in Hu (RNA-binding protein)-positive early postmitotic neurons; and highest in mature neurons identified by neuron-specific nuclear protein (NeuN) and microtubule-associated protein 2 (MAP-2) expression. In vivo, PrP^c is found at highest levels in mature neurons (Fig. 2C), but it is not detected in astroglia (Fig. 2D). Similarly, PrP^c expression in vitro is highest in mature MAP-2-positive neurons (Fig. 2E), but it is not expressed in recently generated S100β-positive astroglia (Fig. 2F) or O4-positive oligodendroglia (data not shown).

Fig. 1.

Confirmation of PrP protein expression and in vivo antibody specificity. (A) The absence of PrP^c in KO mouse brain and its overexpression in PrP^c OE transgenic mice was confirmed by Western blot analysis of whole-brain homogenate by using a monoclonal antibody against the PrP^c protein. The top, middle, and lower bands of the PrP^c blot (Upper) correspond to di-, mono-, and unglycosylated PrP^c, respectively. The blot was reprobed with an antibody against TuJ1 to demonstrate equal loading (Lower). (B and C) Specificity of the polyclonal antibody against PrP^c was confirmed in 30-μm sections of adult brain from PrP^c OE (B) and PrP^c KO (C) mice at equal exposures. cc, corpus callosum; ctx, cortex; hpc, hippocampus.

Fig. 2.

PrP^c is expressed in neurogenic regions and is strongly expressed in neurons in vivo and in vitro. (A) Proliferation in the SVZ of the adult mouse detected with BrdUrd (green, arrowheads). PrP^c-positive cells (red, arrows) are found adjacent to the neurogenic region. (B) A close-up of the SVZ region shown in (A), indicating that PrP^c (red, arrows) is not expressed in proliferating cells (green, arrowheads) but instead is expressed in cells just lateral to those proliferating in the SVZ. (C and D) 3D confocal reconstructions of the CNS in vivo. (C) PrP^c (red in all micrographs) is strongly expressed in mature NeuN-positive neurons (green, arrows). Note the lack of PrP^c in surrounding DAPI-stained cells with small compact nuclei consistent with glia (arrowheads). (D) PrP^c is not expressed in GFAP-positive astroglia identified by a human GFAP (hGFAP) promoter driving expression of eGFP (arrowhead, green). PrP^c-positive GFAP-negative cells (arrows) surround the astrocyte. (E and F) Embryonic neural precursor cultures. (E) PrP^c (red, arrows) is most strongly expressed in MAP-2-positive neurons (green), whereas it is not detected in S100β-positive astroglia (F) (arrowheads, green). DAPI nuclear counterstain (blue) in B, C, E, and F. cc, corpus callosum; lv, lateral ventricle. (Scale bars: A, 100 μm; B, 10 μm; C and D, 25 μm; E and F, 100 μm.)

PrP^c Levels Directly Correlate with Differentiation of Multipotent Neural Precursors in Vitro.

We isolated multipotent neural precursors from PrP^c KO, WT, and OE embryonic (E13.5) mice to investigate the developmental role of PrP^c in proliferation and subsequent production of differentiated neurons and glia (11). Embryonic neural precursors express the intermediate filament protein nestin, and after withdrawal of basic fibroblast growth factor (11, 12), nestin-positive precursors give rise to immature neurons, followed by astroglia, and then oligodendroglia (12–16).

We find that PrP^c levels are positively correlated with differentiation of nestin-positive multipotent neural precursors. One day after inducing differentiation of neural precursors [termed “1 day in vitro” (1 DIV)], ≈75% of WT cells express nestin (Fig. 3 A and A′). Over the next several days in vitro during which nestin-positive precursors produce neurons, astroglia, and oligodendroglia, the number of such precursors declines so that by 7 DIV only a very low percentage (<5%) remains. In contrast, nestin-positive multipotent precursors derived from PrP^c-null mice remain undifferentiated for much longer, whereas those derived from OE mice leave their multipotent state more rapidly than they do in WT cultures (Fig. 3A). For example, 3 days after inducing differentiation (3 DIV), 48 ± 2% of cells in PrP^c KO cultures are still multipotent precursors compared with 34 ± 2% in PrP^c WT and 17 ± 1% in PrP^c OE cultures (SEM, P < 0.001). By 7 DIV, however, the multipotent precursor populations are depleted in all three conditions. Taken together, these results suggest that PrP^c levels directly increase the rate of multipotent precursor differentiation.

Fig. 3.

PrP^c levels increase differentiation of embryonic neural precursors in vitro. (A) Neural precursors derived from PrP^c KO embryos remain as uncommitted multipotent precursors (A′; nestin-positive, arrow) for a longer period than do precursor derived from WT and, especially, OE embryos. Three days after induction of precursor differentiation via removal of basic fibroblast growth factor (3 DIV), there are significant differences between the percentage of nestin-positive precursors derived from KO, WT, and OE embryos. PrP^c OE precursors differentiate from their multipotent state more rapidly than do those from KO and WT mice, even at 1 DIV. By 7 DIV, there are no significant differences among any of the three groups. (B) Differentiation and maturation into a neuronal phenotype (B′; MAP-2-positive; cell body, arrow; cell process, arrowhead) occurs at a significantly slower rate in PrP^c KO-derived precursors than in WT, and differentiation is significantly more rapid in PrP^c OE-derived precursors. PrP^c KO precursors are still capable of producing neurons, indicating a delay in neuronal production rather than a failure to differentiate. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001. (Scale bars: A′ and B′, 50 μm.)

PrP^c Increases the Rate of Neuronal Differentiation in a Dose-Dependent Manner.

Concomitant with its positive effect on the differentiation of multipotent neural precursors, PrP^c increases the production of mature neurons (Fig. 3 B and B′). For example, 5 days after the induction of differentiation (5 DIV), 26 ± 1% of cells are MAP-2-positive neurons in PrP^c OE cultures compared with 18 ± 2% in PrP^c WT (SEM, P < 0.05) and 14 ± 1% in PrP^c KO cultures (SEM, P < 0.001). The proportion of mature neurons in PrP^c KO animals remains lower than in WT and OE conditions, even after 7 DIV. Nevertheless, PrP^c KO-derived multipotent neural precursors are still capable of generating neurons, suggesting a delay in differentiation rather than a failure to differentiate (Fig. 3B). In marked contrast with its direct effect on neuronal differentiation, PrP^c has no effect on gliogenesis; after 3 DIV, for example, the percentage of S100β-positive astroglia is 27 ± 4%, 24 ± 2%, and 25 ± 3% in PrP^c KO, WT, and OE cultures, respectively.

PrP^c Increases Cellular Proliferation in Vivo.

In the adult CNS, both the SVZ and DG contain multipotent neural precursors. We investigated the effect of PrP^c levels on proliferation in these regions by quantifying the number of mitotic cells 1 h after giving PrP^c KO, WT, and OE mice pulse labels of the thymidine analog BrdUrd.

PrP^c levels increase cellular proliferation in both adult neurogenic regions. In the SVZ, PrP^c OE mice have significantly more proliferating cells compared with WT or KO mice; specifically, PrP^c OE mice have an average of 79 ± 3 cells in each of 101 counted regions of the SVZ (n = 9 mice) compared with PrP^c WT (65 ± 2 cells in 209 counted regions; n = 9) and PrP^c KO mice (60 ± 3 cells in 83 counted regions; n = 11) (P < 0.01) (Fig. 4A). Similarly, in the DG, PrP^c OE and WT mice have significantly more proliferating cells compared with KO mice; PrP^c OE mice have an average of eight mitotic cells (in each of 188 counted regions of the DG; n = 9), as do PrP^c WT mice (in each of 154 counted regions; n = 9), compared with PrP^c KO mice, which have an average of six cells (in each of 160 counted regions; n = 11) (P < 0.001) (Fig. 4B).

Fig. 4.

PrP^c increases proliferation in vivo. (A) One hour after a pulse label of BrdUrd, PrP^c OE mice have significantly more proliferating cells in the SVZ than in PrP^c KO or WT mice. (B) In the DG, PrP^c KO mice have significantly fewer proliferating cells than do WT or OE mice. ∗, P < 0.01; ∗∗, P < 0.001.

PrP^c Levels Do Not Influence the Gross Morphology of the Adult CNS.

Others have reported that there is no notable difference in the mature CNS of mice expressing normal, reduced, or higher levels of PrP^c (4, 17, 18). These reports used methods such as hematoxylin/eosin staining (17). Given the subtle differences in cellular proliferation in PrP^c KO, WT, and OE brains, we reexamined the CNS morphology in greater detail by morphometric studies using the cellular label cresyl violet and fluorescent myelin staining (FluoroMyelin, Molecular Probes). From every sixth thin section from adult, age-matched mouse brains, we performed blinded analyses of the size and overall structure of several selected CNS regions, including the cortex, hippocampus, thalamus, cerebellum, and brainstem. Similarly, we analyzed the size and density of major fiber tracts such as the corpus callosum, medial forebrain bundle, and anterior commissure. We find no morphological differences among any of the experimental groups (data not shown); these observations confirm that, regardless of the PrP^c level during development and adulthood, a morphologically normal CNS is formed.

Neurogenesis in the DG Is Unchanged by PrP^c Level.

Given the positive effect of PrP^c on proliferation in the SVZ and DG and the low level of proliferation in the DG of PrP^c KO mice in particular, we investigated whether deletion of PrP^c leads to a net reduction in adult hippocampal neurogenesis. We injected mice with pulse labels of BrdUrd over a 2-day period and assessed the number of newborn immature and mature neurons 3, 7, and 14 days after the last BrdUrd injection. We found that PrP^c levels do not influence the number of immature (TuJ1-positive) neurons produced in the SVZ or DG nor do they change the net number of new neurons produced in the DG. For example, 14 days after the last BrdUrd injection, there was an average of five BrdUrd/NeuN-positive neurons per DG counted in each of PrP^c KO (n = 4 mice), WT (n = 6), and OE mice (n = 4). Therefore, although PrP^c levels increase proliferation in the DG, they have no detectable effect on the ultimate number of neurons that are produced.

Discussion

We have presented evidence in this article, from multiple modes of analysis, that PrP^c expression positively influences both developmental and adult mammalian neurogenesis. We find that (i) PrP^c is expressed most strongly immediately adjacent to the proliferative region of the SVZ but not in mitotic cells; (ii) PrP^c expression increases in mature neurons but is not detectable in astroglia or oligodendroglia; (iii) PrP^c levels directly correlate with neuronal differentiation from multipotent neural precursors in vitro; (iv) PrP^c significantly increases cellular proliferation in vivo, in both the SVZ and DG; (v) the ultimate number of neurons produced in the DG is unchanged by PrP^c expression level under normal laboratory conditions; and (vi) PrP^c levels do not influence the gross morphology of the murine CNS.

PrP^c Expression.

Cell-type expression of PrP^c in the brain remains controversial, with claims that it is either exclusively neuronal or else is ubiquitously expressed in many glial and neuronal cell types throughout the CNS (19). It has been shown, however, that in disease states PrP^c is strongly expressed in astrocytes, which are also capable of replicating prions after neuronal PrP^c expression is turned off postnatally in transgenic mice (20). Our studies used a PrP^c-specific (goat polyclonal) antibody to label PrP^c-positive cells, which were then analyzed with high-magnification, 3D confocal image reconstructions; our in vivo and in vitro analyses demonstrate that PrP^c is expressed strongly in neurons, but it is not detectable in astroglia or oligodendroglia. Neuronal expression begins with very low levels of PrP^c in nestin-positive multipotent neural precursors, followed by increasing levels from immature neurons on through to mature, integrated neurons. This restricted pattern of neuronal PrP^c expression in the developing and adult CNS strongly suggests that PrP^c has an ongoing and active role in neurogenesis throughout life.

PrP^c Increases Proliferation in Adult Neurogenic Regions in Vivo but Does Not Increase Net Neurogenesis.

We have shown that PrP^c has a positive effect on cellular proliferation in the adult SVZ and DG, indicated by increased proliferation in the SVZ of PrP^c OE mice and by a paucity of proliferation in the DG of PrP^c KO mice. Despite the fact that proliferation in the DG is reduced in PrP^c KO mice, however, there is neither a corresponding reduction in the number of newborn neurons nor a change in the gross morphology of the hippocampus. These results support the concept that cellular proliferation rates alone do not determine the net level of neurogenesis (10). The number of surviving newborn neurons in the adult depends on multiple factors, and PrP^c is probably only one such factor involved in this complex process.

It is possible that the neurogenic niche of the DG produces and maintains only a finite number of new neurons and that cell death acts, in part, to regulate these numbers. The absolute number of proliferating cells in the DG in our studies was low, making it unlikely that significant differences in cell death could be detected by using methods such as TUNEL. The number of new neurons that can be integrated into the adult DG partially depends on local environmental support, including the provision of neurotrophic factors critical for survival of newly generated neurons. Because environmental enrichment increases net neurogenesis in the DG via increased neurotrophic factor production (21), future studies on the role of PrP^c in adult neurogenesis could determine whether such enrichment or even response to injury would increase the possibility that PrP^c OE mice would generate and maintain more neurons than their WT or KO counterparts. It is important to note, for example, that under normal laboratory conditions the hematopoietic compartment of PrP^c KO mice is indistinguishable from that of WT mice; however, under the stress of serial transplantation or myelotoxic injury, hematopoietic stem cells from PrP^c KO mice perform poorly when compared with WT controls (9)

Elucidating the Role of PrP^c in Neural Development.

A role for the normal prion protein in development was proposed in 1992, when its expression at different embryonic stages was described (22). Embryonic multipotent neural precursor cultures are a simple model with which to study cellular proliferation and differentiation. We found that cellular PrP^c levels positively correlate with neuronal differentiation from multipotent neural precursors in that nestin-positive precursors derived from PrP^c-null mice remain multipotent for a longer period, whereas higher PrP^c levels significantly increase neuronal differentiation. Although the rate of neuronal production in PrP^c KO cultures is significantly lower than in the other groups, precursors from KO mice are still capable of generating neurons, indicating a delay in differentiation rather than a failure to differentiate. Whether these differences are related to neuronal survival in vitro is not yet known. Nevertheless, it is evident that PrP^c has no effect on the rate of gliogenesis, indicating that its effects on differentiation are specific to neurons.

Taken together, these results suggest that PrP^c might play a role as a switch from uncommitted, multipotent precursors toward the generation of neurons. Our observations about neural development are further supported by recent studies of neuronal maturation, which demonstrated that folded recombinant PrP^c added to cultured rat hippocampal neurons induces neuronal differentiation and process outgrowth (5). Studies such as these indicate that in vitro neural cell preparations are effective systems with which to study the role of PrP^c in discrete stages of neuronal development and to determine what mechanisms might compensate for the lack of PrP^c in precursors and their neuronal progeny.

Prospects for Determining the Function of PrP^c in Normal and Pathological States.

The downstream signaling pathway of PrP^c is of great interest for studies of the normal and disease-associated isoforms of PrP^c. For example, recent work demonstrated that transgenic mice expressing PrP^c lacking a glycosylphosphatidylinositol anchor are refractory to development of a prion disease phenotype, despite dramatic CNS accumulation of misfolded PrP (23). One intriguing interpretation of this result is that cell surface-bound PrP^c is required to transmit a toxic signal to neurons (24). This phenomenon raises the possibility that PrP^c is normally a signal transducer and that in disease states this signal transduction goes awry. Elucidation of the signaling pathways through which PrP^c influences neurogenesis, along with an understanding of the pathways involved in other key cellular processes (6), will provide insight into how PrP^c misfolding leads to devastating neurodegenerative diseases. Further in vivo and in vitro studies of the role of PrP^c in developmental and adult neurogenesis will contribute to our understanding of the biological function of normal PrP^c and could inform studies aimed toward identifying and counteracting the devastating sequelae of prion diseases.

Materials and Methods

Mouse Strains and Genotyping.

Adult mice were housed and all procedures were performed according to institutional and National Institutes of Health guidelines. PrP^c KO mice (25) were kindly provided by R. Race and B. Chesebro (Rocky Mountain Laboratories, Hamilton, MT) on a mixed 129/Ola and C57BL/10 background and were backcrossed to C57BL/6J for at least 6–10 generations to obtain PrP^c KO and WT control littermates. The PrP^c OE transgenic “Tg20” mice (18) were obtained from the European Mutant Mouse Archive (Munich, Germany) on a mixed C57BL/6 and 129S7/Sv hybrid background and null for PrP^c. These mice were backcrossed to C57BL/6J for five to six generations, and the KO allele was bred out to yield mice that were WT at the endogenous Prnp locus and contained one copy of the PrP^c OE transgene. Genotyping conditions are described in Supporting Materials and Methods, which is published as supporting information on the PNAS web site.

Western Blotting.

PrP deletion and overexpression are confirmed as described in Supporting Materials and Methods.

BrdUrd Administration.

For cell proliferation studies, the thymidine analog BrdUrd (Sigma) was administered as a pulse label (one injection i.p.; 200 mg/kg of body weight, in sterile saline). Animals used for cell proliferation studies were perfused 1 h after BrdUrd administration. For cell differentiation studies, BrdUrd was administered as four pulse labels (four i.p. injections 12 h apart; 100 mg/kg, in sterile saline). Animals used for cell differentiation studies were perfused 3, 7, or 14 days after the last BrdUrd administration.

Tissue Collection and Histology.

Animals were deeply anesthetized with an overdose of Avertin (2,2,2-tribromoethanol, Sigma–Aldrich) and were transcardially perfused with cold 0.1 M PBS followed by cold 4% paraformaldehyde in 0.1 M PBS. Brains were postfixed in 4% paraformaldehyde at 4°C for 16 h. Coronal sections 30 μm thick were cut on a Leica VT 1000S vibrating microtome and stored in 0.1M PBS/0.025% sodium azide.

All immunocytochemical procedures were performed on a minimum of every sixth tissue section. Sections were rinsed in 0.1M PBS and blocked in 0.3% BSA/8% serum (e.g., goat) in 0.3% Triton X-100 in PBS. The following primary antibodies were used: PrP^c (goat polyclonal, 1:500; Abcam, Inc., Cambridge, MA, catalog no. 6664), doublecortin (guinea pig polyclonal, 1:500; Chemicon), GFAP (mouse monoclonal, 1:400; Sigma), GFP (rabbit polyclonal, 1:500; Chemicon), NeuN (mouse monoclonal, 1:500; Chemicon), and TuJ1 (mouse monoclonal, 1:500; Covance, Richmond, CA). For BrdUrd staining, tissue sections were treated for 2 h at room temperature in 2M HCl before application of the primary antibody (rat monoclonal, 1:400; Harlan).

Primary antibodies were applied overnight at 4°C in blocking solution, followed by a series of PBS rinses and incubation in appropriate secondary fluorescent antibodies (1:500, Alexa 488 and 546; Molecular Probes) in blocking solution at room temperature for 2–4 h. In most cases, a nuclear counterstain (DAPI; 1:5,000 in 0.1 M PBS) was used. Specificity of the PrP^c antibody was confirmed with immunocytochemistry on tissue from PrP^c OE and KO mice (Fig. 1 B and C). Cresyl violet and FluoroMyelin staining are described in Supporting Materials and Methods.

Neural Precursor Cell Cultures.

Embryonic neural precursors were cultured following an established procedure (26) and further details are presented in the Supporting Materials and Methods.

Differentiated neural precursor cultures were analyzed immunocytochemically as described above. Primary antibodies used for in vitro analyses were PrP^c (goat polyclonal, 1:1,000; Abcam, Inc.), doublecortin (guinea pig polyclonal, 1:750; Chemicon), MAP-2 (mouse monoclonal, 1:1,000; Sigma), nestin (mouse monoclonal, 1:300; Chemicon), NeuN (mouse monoclonal, 1:500; Chemicon), O4 [mouse monoclonal IgM, 1:400; a gift from P. Follet (Children’s Hospital, Boston)], S100β (mouse monoclonal, 1:1,000; Sigma), followed by PBS rinses and incubation in appropriate secondary fluorescent antibodies (1:500, Alexa 488 and 546; Molecular Probes) in blocking solution at room temperature for 2–4 h. In most cases, a nuclear counterstain (DAPI, 1:5,000 in 0.1 M PBS) was used.

Data and Image Analysis.

Tissue sections and cells were viewed on a Nikon E1000 microscope equipped with an X-Cite 120 fluorescence illuminator unit (EXFO). Images were acquired with a Retiga EX cooled CCD camera (QImaging, Burnaby, BC, Canada) and analyzed with openlab image analysis software (version 3.5). Confocal images were acquired with a Bio-Rad Radiance Rainbow laser scanning confocal microscope equipped for spectral imaging and mounted on a Nikon E800 microscope. 3D image reconstructions were analyzed by using BioRad lasersharp 2000 (version 5.1), laservox 3-d (version 1.0), and imaris 4.1.3 (Bitplane, Saint Paul, MN) rendering software. All confocal images were produced from z stacks, and all cell counts and measurements were made by using National Institutes of Health imagej software.

All cell counts were performed in a blinded fashion, with the counter unaware of the experimental condition being assessed. For in vivo analyses, cells were only counted if a full nucleus was present in the section (e.g., for nuclear labels such as NeuN) or if the cell body and its process could be visualized in the same section (e.g., for cells positive for TuJ1). For in vitro analyses, each coverslip was observed under ×40 magnification, and from each coverslip 10 independent areas were randomly selected for quantification. For each experimental condition, three to five coverslips were analyzed, and each experiment was repeated in two to three separate litters of each genotype.

All statistical analyses were performed with instat software (version 3.0a; GraphPad, San Diego); parametric t tests and nonparametric Mann–Whitney U tests were used where appropriate, with a minimum significance level set at P < 0.05.

Abbreviations

DG

dentate gyrus

SVZ

subventricular zone

KO

knockout

OE

overexpresser

PrP^c

prion protein

TuJ1

β-III tubulin

MAP-2

microtubule-associated protein 2

NeuN

neuron-specific nuclear protein

DIV

days in vitro.