Adenoviral brain-derived neurotrophic factor induces both neostriatal and olfactory neuronal recruitment from endogenous progenitor cells in the adult forebrain

Abstract

Neural progenitor cells persist throughout the adult forebrain subependyma, and neurons generated from them respond to brain-derived neurotrophic factor (BDNF) with enhanced maturation and survival. To induce neurogenesis from endogenous progenitors, we overexpressed BDNF in the adult ventricular zone by transducing the forebrain ependyma to constitutively express BDNF. We constructed a bicistronic adenovirus bearing BDNF under cytomegalovirus (CMV) control, and humanized green fluorescent protein (hGFP) under internal ribosomal entry site (IRES) control. This AdCMV:BDNF:IRES:hGFP (AdBDNF) was injected into the lateral ventricles of adult rats, who were treated for 18 d thereafter with the mitotic marker bromodeoxyuridine (BrdU). Three weeks after injection, BDNF averaged 1 microg/gm in the CSF of AdBDNF-injected animals but was undetectable in control CSF. In situ hybridization demonstrated BDNF and GFP mRNA expression restricted to the ventricular wall. In AdBDNF-injected rats, the olfactory bulb exhibited a >2.4-fold increase in the number of BrdU(+)-betaIII-tubulin(+) neurons, confirmed by confocal imaging, relative to AdNull (AdCMV:hGFP) controls. Importantly, AdBDNF-associated neuronal recruitment to the neostriatum was also noted, with the treatment-induced addition of BrdU(+)-NeuN(+)-betaIII-tubulin(+) neurons to the caudate putamen. Many of these cells also expressed glutamic acid decarboxylase, cabindin-D28, and DARPP-32 (dopamine and cAMP-regulated phosphoprotein of 32 kDa), markers of medium spiny neurons of the neostriatum. These newly generated neurons survived at least 5-8 weeks after viral induction. Thus, a single injection of adenoviral BDNF substantially augmented the recruitment of new neurons into both neurogenic and non-neurogenic sites in the adult rat brain. The intraventricular delivery of, and ependymal infection by, viral vectors encoding neurotrophic agents may be a feasible strategy for inducing neurogenesis from resident progenitor cells in the adult brain.

Figures

Fig. 1.

Ependymal restriction of intraventricular adenoviral infection. A, A single intraventricular injection of an adenoviral vector bearing GFP, expressed under the control of the constitutive CMV promoter, shows the widespread infection of the ventricular ependyma, bilaterally and throughout the ventricular system 1 week after viral injection. B, Along the striatal and septal walls, GFP expression was primarily limited to the ventricular surface, with little subependymal and no parenchymal extension. A, B, Sagittal sections. C, A coronal section taken at the level of the main body of the lateral ventricles again reveals GFP expression by the infected striatal and callosal ventricular surfaces. Unlike the striatal and septal walls, the callosal wall shows subependymal and some parenchymal extension of labeled cells. Str, Striatum; LV, lateral ventricle; CC, corpus callosum; D, dorsal; V, ventral;A, anterior; P, posterior.

Fig. 2.

Adenoviral BDNF infection yielded high-level BDNF expression in vitro and in vivo. A,B, HeLa cells transduced with AdBDNF secreted BDNF in a viral dose-dependent manner (n = 3).C, D, AdBDNF-injected animals showed sustained expression of high levels of BDNF in CSF, as measured on day 20 (n = 5). A and Cshow results in picograms per milliliter, and B andD are given in picograms per microgram of protein.

Fig. 3.

AdBDNF transduced expression of BDNF and hGFP mRNAin vivo. Serial sections of AdBDNF–GFP-injected brain were treated with antisense probes for BDNF (A,D) or GFP (B, E). mRNA expression was restricted to the wall of the lateral ventricle.C, Sense probe for BDNF, as control. D , dorsal; V , ventral; R , rostral;C , caudal. Scale bar, 35 μm.

Fig. 4.

Strategy used to induce adult neuronal recruitment. A, Delivery: schematic coronal section showing site of injection of adenovirus into the lateral ventricle.B, Vector: E1-deleted (ΔE1) adenoviral type 5 constructs used to express a bicistronic transcript of BDNF and hGFP (or hGFP alone, as a control vector) under the control of the constitutive CMV early promoter. C, Experimental protocol: adenovirus was injected on day 1, followed by intraperitoneal injections of 100 mg/kg BrdU for the next 18 d. On day 20, CSF was extracted for BDNF ELISA, and the brains were processed for BrdU immunohistochemistry in tandem with phenotype-specific immunolabeling.

Fig. 5.

AdBDNF injection increased recruitment to the olfactory bulb. A, B, BrdU+ cells in the olfactory bulbs of AdBDNF:IRES:hGFP (A) AdNull:GFP (B) injected brains, at day 20. C, Stereological reconstruction of BrdU+ cells, viewed here at different mediolateral levels of the olfactory bulb, revealed substantially higher BrdU+ cell densities in the olfactory subependyma and granular layers of AdBDNF-treated rats (C) than in their AdGFP-injected controls (D). Arrows denote entry to rostral migratory stream in red. E, The average number of BrdU+ cells/mm3in the olfactory bulb (n = 4 per group), plotted as a function of treatment, again revealed significantly higher numbers of newly generated BrdU+ cells in AdBDNF-treated rats than their controls.

Fig. 6.

AdBDNF-associated newly generated olfactory cells were neurons. Confocal imaging confirmed that BrdU+cells added to the olfactory bulb were almost entirely neurons in rats injected with virus 3 weeks before being killed and given BrdU daily until the day before death. A–C, Mergedz-dimension stacks of confocal images of BrdU (green) colabeling with βIII-tubulin+ (A, B; in red) and MAP-2+ (C;red) neurons. This suggested that the AdBDNF-associated increase in the olfactory bulb BrdU labeling index reflected enhanced neurogenesis and/or recruitment to the bulb. Scale bars, 25 μm.

Fig. 7.

AdBDNF stimulation of BrdU+cell addition was pronounced in the olfactory bulb but not appreciable elsewhere. AdBDNF treatment promoted net BrdU+ cell addition to the olfactory bulb but not to the septum, striatum, or cortex. The difference between AdBDNF (red)- and AdNull (blue)-treated olfactory bulb BrdU labeling indices was significant to p < 0.001. No other comparisons based on total BrdU+ cell counts were significant. However, whereas BrdU+ cell addition to non-olfactory regions was almost entirely non-neuronal in AdNull control rats, the BrdU+ cell population included newly generated neurons in several regions of the AdBDNF-injected brains. Thus, when BrdU+–βIII-tubulin+ neurons were specifically compared between AdBDNF and AdNull treatment groups, a significant effect of AdBDNF on neuronal recruitment to the striatum was noted (see below). Red, AdBDNF treated;blue, AdNull treated. VZ, Ventricular zone; RMS, rostral migratory stream; OB, olfactory bulb; Sep, septum; Str, neostriatum; Ctx, neocortex.

Fig. 8.

AdBDNF treatment was associated with neuronal addition to the neostriatum. A shows sagittal (left) and coronal (right) schematics of the neostriatal region assessed for neuronal addition (indicated ingreen) in AdBDNF-injected rats and their AdNull-injected controls. B plots the mean density of βIII-tubulin+–BrdU+ cells in AdBDNF- and AdNull-injected striata and in PBS-injected controls at day 20 (n = 4).

Fig. 9.

AdBDNF induced the heterotopic addition of BrdU+–βIII-tubulin+ neurons to the striatum. Confocal images of BrdU-labeled neurons found in the neostriata of AdBDNF-treated rats 3 weeks after virus injection. These cells were identified by immunostaining for both BrdU (green) and βIII-tubulin (red).A–C, A representative βIII-tubulin+–BrdU+ cell (arrow). A shows az-dimension composite of serial 0.9 μm images, showing βIII-tubulin+ (red) and BrdU (green) immunoreactivities. B, Az-dimension series of six separate 0.9 μm confocal images taken 0.6 μm apart, displaying the concurrence of BrdU and βIII-tubulin in the new neuron. C, A single optical section with reconstructed orthogonal images, as viewed from the sides in both the x–z and y–z planes.D–F, Another newly generated, βIII-tubulin+–BrdU+neostriatal neuron (arrow), similarly viewed as az-stack composite (D). By way of contrast, this field also includes both a non-neuronal BrdU+ cell and a βIII-tubulin+but BrdU-unlabeled resident neuron. Like A–C, this field is also viewed as a series of optical sections (E) and in orthogonal side views (F). G–J, A pair of βIII-tubulin+–BrdU+ striatal neurons (arrows), composited in G with split red and green images separately indicating βIII-tubulin+ and BrdU, respectively.H, I, An optical section with orthogonal images taken at two different points to allow individual assessment of the βIII-tubulin staining of each of these BrdU+cells. Both BrdU+ nuclei are completely surrounded by βIII-tubulin. J, A series ofz-dimension optical sections through these cells, again confirming the coincident expression of BrdU and βIII-tubulin.K, L, Low-power views of the fields shown in A–C and G–J, respectively, to visualize the range of morphologies of both resident (examples asarrowheads) and newly added (arrows) neurons. * in L shows a myelinated bundle passing through the striatal matrix. Scale bars, 10 μm.

Fig. 10.

Newly recruited striatal neurons included medium spiny neurons. The BrdU+ neurons found in AdBDNF-treated striata expressed neuronal markers other than βIII-tubulin, which included NeuN. They also expressed characteristic antigenic markers of medium spiny neurons of the adult caudate putamen, including calbindin-D28k, GAD67, and DARPP-32. A–C, A typical NeuN+–BrdU+ striatal neuron (arrow); local resident neurons (NeuN+–BrdU−) shown byarrowheads. A shows az-dimension composite of serial 1 μm images, with split red (NeuN) and green (BrdU) images on the right. B shows a single optical section with reconstructed orthogonal images, as viewed from the sides in both the x–z (top) andy–z (right) planes. Cshows a z-dimension series, viewed as four separate 0.9 μm optical sections taken 0.6 μm apart. D, A BrdU+(green)–calbindin+(red) neuron, with the split red andgreen images of each to show calbindin (arrow) and BrdU staining separately. Eand F show confocal images of a GAD67+(red)–BrdU+(green) neuron in an AdBDNF-treated striatum.E shows a confocal section with reconstructed orthogonal side views. In the orthogonal side views, the greenBrdU+ nuclei remain completely surrounded by thered GAD67 antigen. F shows az-dimension series of four separate 0.9 μm confocal images taken 0.6 μm apart, displayed to reveal the correspondence of BrdU and GAD67 in the same cell (arrow;arrowheads indicate resident GAD67+ cells) at multiple z-levels. G–I show analogous images of a DARPP-32 (red)–BrdU+(green) neuron in the same striatum (arrowheads, show examples of BrdU-unlabeled resident neurons). G shows the z-dimension composite of serial 0.9 μm images, again with splitred and green images to show DARPP-32 (arrow) and BrdU staining individually. Hshows an optical section with reconstructed orthogonal side views, as described. In both the x–z and y–zplanes, the BrdU+ nucleus is completely surrounded by DARPP-32 signal. I shows a z-dimension series through this cell. All images were taken of striatal sections sampled from AdBDNF-treated rats killed 3 weeks after virus administration. Scale bars, 10 μm.

Fig. 11.

AdBDNF-induced striatal neurons matured and survived for at least 5–8 weeks. βIII-tubulin+–BrdU+, GAD67+–BrdU+, and DARPP32+–BrdU+ striatal neurons persisted in AdBDNF-injected rats. A–C, A typical βIII-tubulin+–BrdU+ neuron found in an AdBDNF-treated striatum 8 weeks after virus injection.A, The z-dimension composite of serial 1 μm images, with split red and greenimages to show βIII-tubulin+(arrow) and BrdU, respectively. B, A confocal section with reconstructed orthogonal images, as viewed from the sides in both x–z (top) andy–z (right) planes. C, Az-dimension series of four separate 0.9 μm confocal images taken 0.6 μm apart, confirming the βIII-tubulin immunoreactivity of the BrdU+ cell.D–F, Analogous images of a GAD67+–BrdU+ neuron viewed in an AdBDNF-treated striatum at 8 weeks. Only one of the two adjacent GAD67+ neurons (arrow) is BrdU-labeled; its neighbor is unlabeled (arrowhead).G–I, A representative DARPP-32+–BrdU+ neuron, again found in an AdBDNF-treated striatum 8 weeks after virus injection.G, A z-dimension composite of serial optical sections, showing DARPP-32 (red) and BrdU (green) immunoreactivities. DARPP-32+–BrdU+ neurons are indicated by arrows; BrdU-unlabeled resident neurons are indicated by arrowheads. H, Orthogonal views of the DARPP-32+–BrdU+striatal neuron. I, Serial 0.9 μm optical sections taken 0.6 μm apart confirm the coincidence of BrdU and DARPP-32 in the same cell. Scale bars, 10 μm.