article: Strategies for promoting neural regeneration are hindered by the difficulty of manipulating desired neural fates in the brain without complex genetic methods. The subventricular zone (SVZ) is the largest germinal zone of the forebrain and is responsible for the lifelong generation of interneuron subtypes and oligodendrocytes. Here, we have performed a bioinformatics analysis of the transcriptome of dorsal and lateral SVZ in early postnatal mice, including neural stem cells (NSCs) and their immediate progenies, which generate distinct neural lineages. We identified multiple signaling pathways that trigger distinct downstream transcriptional networks to regulate the diversity of neural cells originating from the SVZ. Next, we used a novel in silico genomic analysis, searchable platform-independent expression database/connectivity map (SPIED/CMAP), to generate a catalogue of small molecules that can be used to manipulate SVZ microdomain-specific lineages. Finally, we demonstrate that compounds identified in this analysis promote the generation of specific cell lineages from NSCs in vivo, during postnatal life and adulthood, as well as in regenerative contexts. This study unravels new strategies for using small bioactive molecules to direct germinal activity in the SVZ, which has therapeutic potential in neurodegenerative diseases.
  Controlling the fate of neural stem cells (NSCs) is a key therapeutic strategy in neuroregenerative medicine. The most promising and direct approach would be to use small molecules to promote the generation of a particular neural lineage, without the need to introduce complex genetic methods. A novel strategy consists of identifying drug-like compounds with the ability to induce transcriptional changes that are similar to those observed within neurogenic niches and are associated with acquisition of a specific cell fate [1,2]. Such a strategy is facilitated by the accumulation of publicly available datasets that allows the systematic comparison and identification of similarities between transcriptional signatures of biological and drug-induced samples [3], a principle that lies behind the connectivity map (CMAP) project [4]. In the adult and postnatal brain, neurogenesis is largely restricted to the subventricular zone (SVZ) of the lateral ventricle and the dentate gyrus of the hippocampal formation [5,6]. Within the SVZ, NSCs generate both neuronal precursors (NPs) and oligodendrocyte (OL) precursors (OPs) throughout life in a region-dependent manner [6,7]. Subsequently, NPs and OPs migrate to their final sites in the brain, where they differentiate, respectively, into neurons and OLs. Hence, directing fate of NSCs in the SVZ is a key therapeutic strategy for promoting repair following neurodegeneration or demyelination. The NSCs of the postnatal SVZ are heterogeneous, both in terms of embryonic origins and of the distinct neural subtypes they generate depending on their spatial location [8,9]. This regional NSC heterogeneity is controlled by multiple extrinsic and intrinsic factors that could be exploited for therapeutic manipulation [6,7, 10–12]. In the present study, we have determined links between the signaling pathways and transcriptional networks that define NSC lineages in the SVZ and we have identified small molecules that target them to regulate cell fate in vivo. Our findings facilitate the control of oligodendroglial and neuronal lineages in the postnatal and adult brain and offer new means to fully exploit the regenerative potential of the SVZ.
  The SVZ contains NSCs and their progeny, the transient amplifying progenitors (TAPs), which generate both NPs and OPs. The SVZ can be subdivided into discrete spatial microdomains (or niches) from which distinct neural lineages originate. While subtypes of GABAergic interneurons originate from all SVZ regions, the dorsal SVZ (dSVZ) additionally gives rise to glutamatergic NPs and is the primary source of forebrain OPs (reviewed in [6,7, 13]). To identify the molecular hallmarks that determine cell fate within these microdomains, we previously generated whole transcriptome datasets of NSCs, TAPs, and their respective SVZ niches at postnatal day (P) 4, P8, and P11 [1], which correspond to the postnatal period of greatest germinal activity and lineage diversity [6,14]. Here, we interrogated these datasets to identify signaling and metabolic processes that are unique to NSCs, TAPs, and their respective SVZ niches. Transcripts enriched in dorsal versus lateral datasets were compared using GeneGO Metacore for Process Networks, and the function of individual genes were classified using http: //www. genecards. org. The top ten Metacore categories in each microdomain were ranked (Fig 1A and 1B), and only two categories overlapped, namely “Chemotaxis” and “Notch signaling, ” stressing the importance of these pathways within the neurogenic niche as well as highlighting the existence of discrete signaling processes that are specific to the dorsal and lateral SVZ microdomains (Fig 1A and 1B). Among enriched transcripts generic to the SVZ niche, those coding for secreted signaling factors (grouped here for simplicity as “morphogens”) were prominent in both SVZ microdomains (Fig 1C); many of these were enriched in NSCs/TAPs (Fig 1D). Examination of the dorsal and lateral SVZ revealed a large number of genes differentially enriched within the two microdomains (Fig 1E–1H). Notably, the dSVZ contained the greatest number of genes that were uniquely expressed (Fig 1E), in line with the greater diversity of lineages originating from this microdomain. In particular, the Wnt ligands were specific to the dSVZ, whilst Shh was specific to the lateral SVZ, in accordance with evidence that these signaling pathways have key roles in dorsalization and ventralization of the SVZ, respectively [12,15]. In addition, several members of the TGFβ/Bmp family and their pathway inhibitor Noggin were enriched in the dSVZ (Fig 1E), indicating they may have a specific role in driving cell fate in this microdomain. The lateral SVZ was enriched in the proneural determinants Bmp2 and Tgfa [16], as well as an abundance of chemokines and secreted molecules with undefined roles in neurogenesis (Fig 1G and 1H). A number of Fgf ligands were specific to the dorsal or lateral SVZ, indicative of functional divergence of FGF signaling within the microdomains (Fig 1E and 1G). Together, these results highlight major regionalization of signaling pathways within the SVZ, supporting the possibility they could be directly targeted to instruct lineage commitment of NSCs. We applied a novel pharmacogenomics approach to probe SVZ regionalization. To this end, a meta-analysis was performed to identify relationships between the transcriptional signatures of SVZ niches and/or lineages to those induced by exposure to small bioactive molecules in different contexts. This CMAP approach allows the identification of small bioactive molecules capable of inducing transcriptional changes similar to those observed in the queried samples and therefore to potentially manipulate cell fate in the SVZ (Fig 2A) [3,4, 17]. Small molecules were ranked according to the number of genes that are altered, referred to as “target genes” (See Table 1A–1D), and the protein targets of each small molecule were classified according to Gene Ontology (GO) terms (Fig 2B–2E; see Materials and Methods for further details). Small molecules that may drive NSC/TAP dorsalization or ventralization were identified by comparing the transcriptome of dorsal and lateral NSCs/TAPs (S1 and S2 Tables), as summarized in Table 1A and 1B. Significantly, the GSK3β inhibitor AR-A014418, which we have previously demonstrated dorsalizes the SVZ [10], was identified in the top ten dorsalizing pertubagens (Table 1A). Conversely, 3-nitropropionic acid ranked highly in the ventralizing screen (Table 1B) and is an activator of GSK3β [18], which we have shown represses SVZ dorsalization [15]. Drugs targeting the ventralizing Shh signaling pathway [12] were also identified, such as tolnaftate, which has been described as inhibiting Shh signaling [19]. Altogether, these findings help validate our approach (see below). Among other small molecules, the most prominent category was “Receptor antagonists” (Fig 2B and 2C), consistent with neurotransmitters being major regulators of neurogenesis in the SVZ [20]. Notably, ventralizing perturbagens targeting muscarinic acetylcholine (mACh) receptors were highly ranked, e. g. , terfenadine (Table 1B), suggesting mACh receptors are important determinants of interneuron specification in the lateral SVZ [21]. The two other prominent categories of small molecules were associated with “Signaling” and “Metabolism” (Fig 2B and 2C), which included the most highly ranked dorsalizing perturbagen GW-8510, a potent Cdk inhibitor (Table 1A), and ciclopirox, an inhibitor of prolyl-4-hydroxylase that promotes Notch signaling and NSC activation [22,23]. The most highly ranked ventralizing perturbagen was verteporfin (Table 1B), which alters downstream transcriptional activity of the hippo pathway to regulate cell cycle and neuronal differentiation [24]. Small molecules that may promote oligodendrogenesis were identified by comparing the transcriptome of dorsal NSCs/TAPs (i. e. , the main postnatal forebrain source of OLs) with publicly available transcriptional datasets of oligodendroglial lineage cells [25]. The results are presented in S3 Table and summarized in Table 1C. Most small molecules were related to “Gene regulation” and”Signaling” (Fig 2D). Many from the latter category, including two of the top ten ranked pro-oligodendrogenesis drugs LY-294002 and sirolimus, are inhibitors of PI3K/Akt/mTor signaling (Table 1C), which acts downstream of several ligands enriched in the dSVZ (Fig 1E). Notably, many of the small molecules associated with oligodendrogenesis were within “Epigenetic” and “Cell cycle” categories, which barely featured in the other SPIED analyses (Fig 2B–2E). The highest ranking amongst these were trichostatin-A and vorinostat (Table 1C), which are potent inhibitors of histone deacetylases (HDACs) with broad epigenetic activities, consistent with evidence that HDACs repression is required during NSC differentiation into OPs [26,27]. Similarly, the higher ranking of the Cdk inhibitors GW-8510 and camptothecin (Table 1C) highlights the importance of inhibition of Cdks in regulating cell cycle progression and differentiation of OPs from NSCs, in support of previous studies [28]. Small molecules that may rejuvenate the adult SVZ were identified by comparing the transcriptome of postnatal NSCs [1] and adult NSCs [29] (Fig 2E; S4 Table). The aim of this approach was to identify key transcriptional changes underlying the decline in the activity and loss of competence of NSCs that occurs in adulthood, which is attributed to the combined up-regulation of inhibitory and down-regulation of positive cues [30], and is critical for the response of NSCs to brain injury and degeneration [31,32]. In this manner, key small molecules were identified for “rejuvenating” adult NSC (Fig 2E; Table 1D), approximately 20% of which overlapped with those required in dorsalizing or ventralizing the postnatal SVZ, and were categorized as “Receptor antagonist, ” “Signaling, ” and “Metabolism. ” Some interesting candidates included antagonists for α/β adrenergic receptors, including the highly ranked nadolol (Table 1D), which have been described to promote NSC activation from quiescence in the dentate gyrus [33] and enhance NP survival following exit from the SVZ [34]. A key signaling pertubagen was monensin, which impedes TGFβ processing [35], a major neurogenesis inhibitory factor during aging [36]. Significantly, the GSK3β inhibitor AR-A014418 was one of the top ranking “rejuvenating” small molecules (see below), which we have shown promotes the genesis of glutamatergic NPs in the postnatal SVZ via the canonical Wnt signaling pathway [10,15]. Based on these SPIED/CMAP analyses, LY-294002 and AR-A014418 were identified as promising agents that may specifically regulate oligodendrogenesis (Table 1C) or neurogenesis (Table 1D), respectively, and were selected for further analysis to resolve signaling-to-transcriptional interactions by Genego Metacore network visualization and in vivo validation. LY-294002 is a widely used and highly specific inhibitor of PI3K/Akt. Expression of the target genes of LY-294002 in SVZ cell types/lineages (S5 Table) was compared by hierarchical clustering, highlighting their association with late-stage OLs compared to other cell lineages, including NSCs/TAPS of the dSVZ (Fig 3A). Further target genes analysis provided additional information on the mode of action and predicted effects of LY-294002. Categorizing target genes for GO Pathway Maps and Process Networks revealed up-regulation of genes associated with oligodendrogenesis and myelination and down-regulation of genes related to cell cycle behavior and neurogenesis (Fig 3B). Finally, Genego Metacore network visualization was applied to resolve signaling-to-transcriptional interactions (detailed in Materials and Methods). LY-294002 up-regulated transcriptional nodes were associated with oligodendrogenesis (Fig 3C), while down-regulated nodes included Notch signaling, proneuronal TFs, and astroglial-related genes (Fig 3D). Altogether, LY-294002 appeared as a strong candidate for inducing specifically oligodendrogenesis in the postnatal SVZ. It was infused into the CSF of the lateral ventricle, commencing at P8, and the effects on the SVZ were determined at P11 by immunostaining (Fig 4) and biochemical and quantitative PCR (qPCR) analysis of its target genes (S1A and S1B Fig), as described in our previous studies [10,15]. Intraventricular infusion to achieve a CSF concentration of 3 μM LY-294002 effectively inhibited Akt phosphorylation, the immediate target of PI3K, throughout the SVZ (S1A Fig) and rapidly and specifically promoted oligodendrogenesis in the dSVZ (Fig 4A and 4B). Quantification performed through the rostro-caudal axis of the lateral ventricle revealed a pronounced induction of the OL lineage marker Olig2, particularly in the most dSVZ region (Fig 4A and 4B). NSCs were identified as glial fibrillary acidic protein (GFAP) immunopositive cells in direct contact with the lateral ventricle wall, and their proliferative state was assessed using 5-ethynyl-2 deoxyuridine (EdU; mice received a single intraperitoneal [i. p. ] injection of EdU at P8). Compared to controls, GFAP immunoreactivity and the extent of GFAP+/EdU colocalization were significantly reduced following LY-294002 infusion (Fig 4C and 4F), indicating a general loss of both proliferative and nonproliferative GFAP+ NSCs, consistent with evidence of their precocious differentiation into OPs. Immunolabeling for Dcx and Olig2 in combination with EdU to identify NPs and OPs, respectively, revealed different effects of LY-294002 on these two lineages. Notably, the numbers of Olig2+/EdU+ OPs were increased dramatically (Fig 4D and 4G), and a greater proportion of these cells expressed Ascl1 (Fig 4D and 4H), whereas Dcx+/EdU+ NPs were reduced, as was the overall number of Dcx+ cells in the dSVZ (Fig 4E and 4G). This indicates that LY-294002 promoted the early stages of oligodendrogenesis at the expense of neurogenesis. In addition, OL differentiation was also enhanced as revealed by a doubling of proteolipid protein (PLP) -DsRed+ OLs and a subsequent 30% enhancement in myelination as revealed by myelin index measurements (S1C Fig) by the end of the treatment. Importantly, qPCR of the microdissected SVZ (S1B Fig) confirmed LY-294002 acts via the target genes/nodes identified by the target gene analysis (Fig 3) and provided additional information on its modes of action. Analysis revealed Fgf2 and Igf1 were not increased (S1B Fig), indicating they were not the mechanism of action of LY-294002. Conversely, LY-294002 significantly decreased Shh signaling, which promotes SVZ ventralization, together with Notch signaling (S1B Fig), which stimulates NSCs self-renewal and is a major rate-limiting determinant of OL differentiation [37,38]. Overall, these analyses support that LY-294002-mediated PI3K/Akt inhibition promotes an environment permissive to oligodendrogenesis while inhibiting signaling pathways that promote neuronal cell fates. AR-A014418 is a highly specific inhibitor of GSK3β, which the SPIED/CMAP analysis identified as having one of the highest number of target genes associated with rejuvenation (Table 1D) as well as being positively related to dorsalization of the SVZ (Table 1A) and negatively related to ventralization (Table 1B). Consistent with this, the target genes of AR-A014418 were enriched more prominently in dorsal NSCs/TAPs and OPs, than other cell types (Fig 5A). Furthermore, pathway/network analysis of AR-A014418 target genes revealed an up-regulation of multiple categories related to neurogenesis, oligodendrogenesis, and Wnt signaling (Fig 5B), consistent with our recent evidence that AR-A014418 promotes generation of glutamatergic NPs and OPs from the postnatal dSVZ via canonical Wnt signaling [10,15]. Genego Metacore network visualization identified up-regulated nodes mainly consisting of transcriptional regulators, notably TFs associated with neurogenesis (Fig 5C; e. g. , Tbr2, NeuroD1, Pax6) and oligodendrogenesis (Fig 5C; e. g. , Olig1/2, Sox10). Down-regulated nodes included members of pro-inflammatory cytokines (Fig 5D), such as IL-33, that likely inhibit neurogenesis [39], as well as Id4, which is up-regulated in adulthood and is a potent inhibitor of neurogenesis [40]. These analyses support AR-A014418 as a strong candidate for promoting lineage and signaling pathways that are characteristic of the early postnatal SVZ, whilst down-regulating inhibitory factors associated with the decline in neurogenic capacity in the adult. An age-related decline in SVZ activity was confirmed by qPCR analysis of adult SVZ microdomains, which indicated a parallel decline in neurogenic potential and canonical Wnt/β-catenin signaling in the dSVZ (S2 Fig). This was confirmed by an observed sharp decline in the expression of Lef1 and Axin2 and the dSVZ\glutamatergic NP markers Emx1 and Tbr2 (Fig 6A). Analysis of BAT-gal mice [41] further demonstrated a decline in Wnt/β-catenin activity between P6 and P60, together with a decrease in the densities of Tbr2+ NPs and to a lesser extent Olig2+ OPs in the adult and their apparent loss by P120 (Fig 6B and 6C). These analyses reveal that the neurogenic capacity and lineage diversity of the dSVZ declines in the adult brain [30,42] and, based on the SPIED/CMAP analysis, we predicted GSK3β inhibitors are strong candidates for rejuvenating the adult dSVZ. The effects of the GSK3β inhibitors AR-A014418 and second generation inhibitor CHIR99021 on dorsal lineages in the adult SVZ were examined directly in vivo by infusion into the CSF of the lateral ventricle (Fig 6D and 6E and S3–S5 Figs). First, these agents were tested in postnatal mice to confirm our previous findings that their infusion into the CSF effectively inhibits GSK3β activity and stimulates Wnt/β-catenin signaling in the dSVZ (S3 Fig) [15] and promote the generation of glutamatergic NPs and OPs (S4 Fig). Next, we examined the effects of AR-A014418 or CHIR99021 in the adult (P90) dSVZ; the agents were infusion into the lateral ventricle caudally to ensure no damage to the SVZ by the injection procedure, whilst ensuring effective distribution of agents at a concentration of 3–10 μM at the SVZ, which is rostral to the injection site (S5 Fig). Treatment with AR-A014418 and CHIR99021 dramatically stimulated the germinal activity of the adult dSVZ, increasing proliferation as revealed by Ki67 immunolabeling and EdU incorporation, with profound effects on Mcm2/GFAP+ NSCs and Tbr2+ glutamatergic NPs, which were respectively increased 5-fold and 6-fold (Fig 6D and 6E). There was also an increase in oligodendrogenesis, as evinced by a 3-fold increase in the number of Olig2+ cells (Fig 6D and 6E). Importantly, careful analysis revealed no marker co-expression (i. e. , Olig2/Tbr2 and Dlx2/Tbr2, <50 cells/brain in five animals), supporting appropriate and lineage-specific progenitor specification. Thus, infusion of GSK3β inhibitors was able to rejuvenate the SVZ by promoting the reemergence of lineages associated with early postnatal life, as predicted by the SPIED/CMAP analysis. The results reported above validate the SPIED/CMAP-based approach for lineage-specific manipulation of SVZ germinal activity at various ages. Next, we explored the regenerative potential in a neuropathological context in a model of premature injury that leads to diffuse oligodendroglial and neuronal loss throughout the cortex [43]. To investigate the potential of small bioactive molecules to promote the spontaneous cellular regeneration previously observed in this model [44,45], we selected the GSK3β inhibitor CHIR99021 for its predicted capacity to induce dorsal lineages, i. e. , oligodendrogenesis and neurogenesis [10] (Fig 2), and high activity at low concentrations in inducing dorsal lineages (S4C Fig). Dorsal electroporation of a Cre plasmid in Rosa-YFP Cre reporter mice allowed long-term labeling and fate mapping of dorsal NSCs. Intranasal CHIR99021 delivery in hypoxic animals led to a significant decrease in the number of YFP+ cells in the dSVZ, while their number concomitantly increased in the cortex (Fig 7C and 7D). This efficient cortical cellular recruitment was accompanied by a significant enhancement of migration of YFP+ cells following CHIR99021 treatment (Fig 7B and 7E). Phenotypic characterization of the cells revealed significantly enhanced oligodendrogenesis (YFP+/Olig2+, Fig 7F), regeneration of new myelinating OLs (YFP+/CC1+/MBP+, Fig 7F), as well as increased neurogenesis (YFP+/NeuN+, Fig 7G) following hypoxia and CHIR99021 treatment. Expression of postmitotic markers CC1 and NeuN in OLs and neurons, respectively, support their successful differentiation following CHIR99021 treatment (Fig 7G and 7H). These results illustrate the capacity of small bioactive molecules identified in our bioinformatic approach to promote regeneration following forebrain injury.
  Controlling the fate of NSCs to generate neurons and OLs would represent a key therapeutic strategy in neuroregenerative medicine. In the present study, we used a novel SPIED/CMAP strategy to identify small molecules that are predicted to regulate transcriptional changes associated with neurogenesis in the SVZ neurogenic niche. Importantly, we validate this approach by demonstrating that two of the identified small molecules, which target PI3K/Akt and GSK3β, were able to differentially direct the fate of NSCs in vivo, to promote oligodendrogenesis and neurogenesis, in the postnatal and adult SVZ. Moreover, we show that this approach can be used to promote regeneration in a clinically relevant mouse model of postnatal brain hypoxia, which demonstrates the power of the SPIED/CMAP-based approach for repurposing of clinically approved small molecules for promoting regeneration in neurodegenerative and demyelinating diseases. Transcriptome profiling revealed for the first time the marked heterogeneity of secreted ligands within the dorsal and lateral microdomains of the postnatal SVZ that are likely to contribute towards the regionalized fate of NSCs by regulating downstream transcriptional programs [1,10,12]. These regional differences suggested to us that specific signaling pathways could be probed as a means to manipulate SVZ regionalization as well as the lineages associated with specific SVZ microdomains. To this end, we provide the first interrogation of the SVZ using the CMAP initiative, which has been used extensively in other systems (reviewed in [4]). We identified small molecules that have the potential to manipulate region and/or lineage-specific gene expression signatures. Small molecules were further prioritized by additional analysis of their target genes. First, small molecules can be prioritized by the number of “target genes” (Table 1). Additionally, “target genes” were examined by further by GO analysis, which enabled us to prioritize small molecules predicted to act on select lineages/signaling pathways and exclude those with undesirable side effects (e. g. , cell death). Applying these selection criteria, we selected LY-294002 and AR-A014418 for validation in vivo and showed that they stimulate the genesis of specific lineages during development and in adulthood. To target oligodendrogenesis, we selected the specific PI3K inhibitor LY-294002 on the basis that our SPIED/CMAP analyses predicted an up-regulation of signatures relevant to OL lineage cells. Network analysis revealed that dorsal-derived lineages are promoted by via PI3K/Akt signaling, which was highly ranked in analysis performed in Fig 1A, implying novel fate-specification roles. In addition, in vitro studies indicate an important role for PI3K/Akt signaling in OL development [46,47]. Our findings demonstrate for the first time that intraventricular infusion of LY-294002 specifically targeted oligodendrogenesis in vivo in the dSVZ, which is the primary source of forebrain OLs in the postnatal brain [8]. A further novel finding was that Notch2-signaling is a target of PI3K/Akt in vivo and may be a primary mechanism regulating fate decision of NSC along an oligodendroglial path [37,38], which had not been predicted previously from genetic or in vitro studies. It is significant, therefore, that ciclopirox, an inhibitor of prolyl-4-hydroxylase that promotes Notch signaling and NSC activation [22,23], was the second most highly ranked pertubagen predicted to promote oligodendrogenesis. Other factors identified by our analyses, and worthy of further examination, inhibit HDACs, which is essential for generation of OPs [26]. Indeed, our analyses indicate the key to promoting oligodendrogenesis in the SVZ is to inhibit signaling pathways that promote neuronal cell fate, and future strategies should examine the therapeutic potential of combinations of small molecules. Gene expression profiling of the SVZ demonstrated a marked decline in NSC activity during adulthood, in support of other studies indicating an age-related loss of specific signaling cues that promote NSC proliferation and fate commitment [42,48,49]. Most notable was the decline in canonical Wnt/β-catenin activity, which is consistent with an increase in Wnt inhibitors during adulthood [50]. It is significant, therefore, that our study demonstrates that small molecule inhibitors of GSK3β identified by the SPIED/CMAP analysis act as mimetics of Wnt/β-catenin and successfully reverse the decline in specific neural lineages in the adult SVZ. Significantly, we identified that the GSK3β inhibitors AR-A014418 and CHIR99021 have a profound effect on germinal activity in the dSVZ of 3-mo-old mice. This was primarily illustrated by the pronounced effect on glutamatergic NPs, although OPs were also increased. In summary, our analyses provide a comprehensive catalogue of small molecules that can be used to manipulate SVZ microdomain-specific lineages. The power of this approach is highlighted by our results, demonstrating that two of these compounds, LY-294002 and AR-A014418, stimulate oligodendrogenesis and glutamatergic NPs generation in postnatal and adult contexts. Moreover, experiments performed in a model of postnatal brain injury reveal that this strategy can be applied to promote SVZ germinal activity in a regenerative context. Although further work is required to fully characterize the functional outcome of this repair, these encouraging results will stimulate further studies on repurposing other small molecules and determine their potential for lineage-specific manipulation of SVZ germinal activity and regeneration in multiple neuropathologies. In this regard, it is noteworthy that identified small molecules, for example GSK3β inhibitors, may be useful for reactivating Wnt-signaling, which is down-regulated during impaired cortical development leading to neuropsychiatric disorders in adulthood [51]. Furthermore, our SPIED/CMAP analysis predicts that further specificity could be achieved by combinatorial approaches, most notably promoting adult neurogenesis using AR-A014418 combined with adiphenine, a nACh inhibitor that was the most highly ranked pro-rejuvenating and anti-oligodendrogenesis small molecules. Conversely, specific stimulation of oligodendrogenesis could be achieved by LY-294002 in combination with the HDAC inhibitor trichostatin-A, which was the most highly ranked pro-oligodendrogenesis and anti-neurogenic small molecule. Moreover, our analyses highlighted a number of small molecules that are predicted to have an “anti-dorsalizing” or “ventralizing” effect, hence they would be specifically pro-neurogenic. This is of considerable translational interest, because the more ventral SVZ is more developed in humans [52], and adult neurogenesis has recently been described in the striatum [53], which is more extended than in rodents [54]. Moreover, the ventral SVZ gives rise to specific granule cells, such as those forming the Islands of Calleja [55], and our analyses identifies small molecules that may target the different factors that drive the differentiation of such specific neurons. In conclusion, our study establishes unequivocally the effectiveness of employing pharmacogenomic approaches for generating a robust framework that guides mechanistic experiments to manipulate neurogenic niches in the brain, which has considerable potential for identifying therapeutics for neurodegenerative and demyelinating diseases.
  Whole genome transcriptome datasets of the isolated SVZ microdomains and region-specific NSCs and TAPs are described in detail in a recent study that described transcriptional regulators acting in SVZ regionalization [1]. Briefly, the rostral periventricular regions of postnatal mice of different ages (P4, P8, and P11) of the Ascl1-EGFPBac transgenic reporter mouse line were carefully microdissected under a fluorescent binocular microscope in RNAse-free and sterile conditions. Isolated SVZ microdomains were derived from brain coordinates of +1 and 0 relative to Bregma. The Hes5-EGFP reporter mouse line was used in combination with Prominin-1 immunodetection to isolate NSCs from microdissected dorsal and lateral microdomains by fluorescence-activated cell sorting. Similarly, the Ascl1-EGFPBac transgenic reporter mouse line was used to isolate the 25% brightest cells, i. e. , corresponding to TAPs, from either microdomain. Half a litter of animals were used to pool for each replicate throughout. In the present study, these datasets (recently made publicly available from NCBI Gene Expression Omnibus [http: //www. ncbi. nlm. nih. gov/geo] GEO Series accession number GSE60905) were analyzed using previously applied bioinformatics methods, with only minor modifications [1]. In brief, data were cured (background subtraction, normalization, and summarization) using robust multi-chip analysis (RMA) using the Partek Genomic Suite software package version 6. 6 using stringent false discovery rate (FDR) with p-values where necessary in the analysis. All data were normalized collectively with datasets from previous studies of isolated NSC, NPs, and glia (i. e. , GSE60905, GSE9566, GSE18765) for optimal parameters. Partek was used to assemble affymetrix data and generate hierarchical clustering and gene lists. GO sets were generated using the latest MGI mouse GO datasets via the Broad Institute (http: //www. broadinstitute. org/gsea/index. jsp). The numbers of probes that were differentially expressed across the ten samples analyzed (dNSCs, dTAPs, lNSCs, lTAPs, P4 dSVZ, P4 lateral SVZ, P8 dSVZ, P8 lateral SVZ, P11 dSVZ, and P11 lateral SVZ) represented a total of ~37K probe sets within the 10% FDR range. Genego Metacore (https: //portal. genego. com/) and GSEA (http: //www. broadinstitute. org/gsea/msigdb/index. jsp) were used to filter and select for probes associated as secreted morphogens (tropic factors, growth factors, extracellular signaling molecules, mitogens, and secreted inhibitors of signaling pathways). The numbers of morphogen from this filtered list that were significantly altered amounted to 530 probes, representing approximately 330 individual genes. Identification of spatially enriched signaling ligands, regardless of sample type (Fig 1A and 1B), was done by comparing all dorsal versus all lateral samples. This gene list was uploaded onto Genego Metacore and Process Network option selected using the default parameters. Determination of the spatial expression profiles of secreted signaling factors in SVZ microdomains (Fig 1C–1H) was performed by comparing datasets using appropriate fold changes and FDR cut-offs (Partek, 1. 65-fold change and FDR <5%). For all analyses, raw expression values are provided and Heatmaps are presented in the manuscript. For SPIED identification of small molecules, the “dorsal NSCs/TAPs, ” “lateral NSCs/TAPs, ” “postnatal NSCS/TAPs, ” “oligodendroglial lineage, ” and the “rejuvenating” transcriptional signatures were defined using Partek (1. 8-fold change, FDR < 5%), as follows. Probes significant across multiple normalized datasets in the background (representing ~40K probes) were processed. For identification of “dorsalizing” small molecules, dNSCs/dTAPs datasets (positive range) were compared with probes significantly different in the background. This list was then further refined and compared with other postnatal datasets (negative range), using the advanced tab in list manager followed by criteria configuration in generating lists with merged expression profiles. For identification of “ventralizing” small molecules, lNSCs/lTAPs datasets (positive range) were compared as above against with probes significantly different in the background. This list was further refined and compared with other postnatal datasets from the same study (negative range) [1]. For identification of “pro-oligodendrogenic” small molecules, publicly available datasets of forebrain-derived OL lineage cells (positive range; GSE9566) were compared to dNSCs and dTAPs (negative range) from which they emerge. Finally, for identification of “rejuvenating” small molecules, publicly available datasets of adult NSCs [29] (positive range) were compared with postnatal dNSCs/lNSCs (negative range) [1]. These expression profiles, consisting of gene symbols of “enriched” genes, were next uploaded onto SPIED (http: //www. spied. org. uk/cgi-bin/HGNC-SPIED3. 1. cgi) to interrogate the CMAP initiative in an unbiased way and identify small molecules predicted to promote the positive ranges of gene signatures using default parameters. The Broad CMAP 2. 0 (CMAP2. 0) database consists of the transcriptional profiles corresponding to the effects of small molecules at various concentrations and treatment times using panels of human cell lines. The data are available for download in the form of ranked probe sets for each microarray sample on the [HG-U133A] Affymetrix Human Genome U133A Array platform. Identified small molecules cellular targets were exhaustively characterized using publicly available drug repositories (www. DrugBank. ca/; www. genome. jp/kegg/drug/; http: //insilico. charite. de/supertarget/; www. pharmgkb. org; http: //stitch. embl. de/). Small molecules protein targets identified were cross-checked in http: //www. genecards. org/ for classifying them under general GO terms. All analyses presented in Fig 2 are shown as a percentage. “Target genes” are defined as the genes from the queried expression profiles that are also induced by a given small molecule. Target genes for each analysis were generated as follows. To generate lists of genes perturbed by the small molecules, gene replicates were pooled and the relative expression levels calculated. Changes passing the Student’s t test p-value of ≤0. 05 were processed, and when there were multiple probes for a given gene, the probe with the biggest fold change was assigned to the gene. These were aligned for matching signatures with the transcriptional profiles corresponding to the small molecules repurposed in the CMAP using pattern-matching algorithms that enable identification of functional connections between drugs, genes, and diseases through the transitory feature of common gene-expression changes [4]. The entire database is available for download (http: //www. broadinstitute. org/gsea/msigdb/index. jsp) in the form of ranked probe sets for each microarray sample on the [HG-U133A] Affymetrix Human Genome U133A Array platform. For analysis of “target genes” of select small molecules, we first performed hierarchical clustering of their expression profile in the various cell types and lineage that compose the SVZ using the following datasets: purified postnatal NSCs and TAPs (GSE60905 [1]), for purified glial cells (GSE9566 [25]), and adult NSCs/NPs/ependymal cells (GSE18765 [29]). Target genes were then classified by “Process network and pathway maps” GO categories. Briefly, target gene lists containing the contrasts and fold changes were analyzed via the web platform http: //www. broadinstitute. org/cmap/index. jsp and functionally classified using Genego Metacore (https: //portal. genego. com/) for Process Networks and Pathway Maps. Last, target genes were studied further for obtaining the shortest path between genes associated with highly ranked process networks and pathway maps using standard Dijkstra’s shortest paths algorithm and applying default parameters [7,56]. Background RMA normalized data for all probe sets relevant for the postnatal and adult SVZ derived from postnatal NSCs and TAPs ( (GSE60905) [1]), purified glial cells ( (GSE9566 [25]), and adult NSCs/NPs/ependymal cells ( (GSE18765 [29]) were uploaded onto Genego Metacore (raw data are provided with the manuscript). This allows obtaining, visualizing, aligning, and clustering the most relevant target genes based on small molecule target genes with reference to basally expressed genes in the SVZ. Objects within this network were restricted to 70–80 in accordance with those highly ranked and most significant within the earlier GO analysis, and signaling-to-transcriptional options were selected. Internal clusters (2–4) within the network module were arranged according to the highest ranked GO pathways within the analysis in the pathway selection menu. A full description of the definition of objects and nodes can be found here: https: //portal. genego. com/legends/MetaCoreQuickReferenceGuide. pdf. The SVZ microdomains were microdissected using previously published protocols [1]. In brief, mouse pups were killed humanely by cervical dislocation. In sterile and RNAse-free conditions, brains were rapidly dissected free and placed in ice-cold postnatal-specific coronal brain matrix (Zivic Instruments, US) to obtain tissue segments of 500-μm thickness containing the rostral periventricular tissue as above for whole genome transcriptome analysis. For examination of LY-294002-induced genes by qPCR, pups were treated by intraventricular infusion (see below) at P9 and P10, and 180 min following final infusion, tissue was microdissected. Five pups were used to pool for individual “n” numbers, and RNA was systematically amplified for all “n” numbers as previous [10]. For western blot (S1A Fig), pups aged at P10 were treated with LY-294002 (see below), and 45 min following injection, pups were systematically killed by cervical dislocation and tissue microdissected and flash frozen in lysis buffer in liquid nitrogen for storage at −80°C [10]. One litter of pups was pooled to yield 1 “n” number. For qPCR experiments, relative gene expression was determined using the 2 ΔΔ-CT method versus the housekeeping gene GAPDH (Glyceraldehyde-3-phosphate dehydrogenase). See S7 Table for a list of primers used in the study. Primers were designed by Primer Express 1. 5 software and synthesized by Eurofins (Ebersberg, Germany). Unstated primers in main text were custom designed and obtained from (Qiagen). Protein was extracted with lysis buffer and standard procedures as previous [57]. For SDS-PAGE gels, 15 μg was loaded and transferred to a PVDF membrane (GE Healthcare, Amersham). Blots were preincubated in a blocking solution of 5% BSA in 0. 2% TBST (0. 1 M Tris base, 0. 1% Tween 20, pH 7. 4) for 1 h at RT and incubated with primary antibodies overnight at 4°C and after washing, with a horseradish peroxidase-conjugated anti-rabbit antibody (1: 10,000–1: 25,000; Pierce Biotechnology). Primary antibodies were all obtained from Cell Signaling and used in concentrations of 1: 500 for phosphor-forms and 1: 2000 for total forms of protein. Protein bands were detected by adding SuperSignal West Pico Chemiluminescent Substrate (Pierce) by exposing the blot in a Stella detector (Raytest). Densitometry analysis was performed with NIH software and by normalizing the band intensities to total Akt or total Erk1/2 values. Intensity values for pAkt were combined and pAkt-473 shown only. Gene expression and western blot data are presented as mean + standard deviation of the mean (SD) or standard error of the mean (SEM), respectively, and samples compared for significance using unpaired t test (t test) or (Prism v3. 02 software; GraphPad). Animals were killed humanely by cervical dislocation and brains removed rapidly to ice-cold fixative. Mouse pups of similar size were used throughout. Mice aged P8 were treated by intraventricular infusion into the LV daily for 3 d, and brains sampled at P11, overnight following the final injection. Mice were deeply anesthetized under isofluorane and differing concentrations of LY-294002 (Sigma-Aldrich), dissolved in sterile DMSO, sterile filtered, and co-administered with sterile saline delivered into the CSF of the LV using a Hamilton syringe at point 2 mm from the midline along the Bregma and to a depth of 2 mm. Sterile saline/DMSO vehicle were used as controls throughout this study. Concentrations of small molecules infused into the CSF of the lateral ventricle were based on the known CSF availability of agents, as performed previously by the authors [57] and elsewhere based on CSF volume and turnover in the adult mouse [58]. Methods applied for studying postnatal oligodendrogenesis are based on previous studies using C57/BL6 mice and transgenic mouse line in which fluorescent reporters DsRed are under control of the PLP promoter [57,59]. Methods applied for studying rejuvenation of the adult SVZ were perform by infusing GSK3β inhibitors into the ventricular system of adult P90 mice. Animals were anesthetized with a subcutaneous injection of Ketamin (60 mg/kg body weight), Xylazine (13 mg/kg body weight), and Acepromazine (1. 5 mg/kg body weight) before being fixed in a stereotaxic apparatus. After exposure of the skull surface, a canula (Alzet, Brain infusion kit 3) was stable implanted at the following coordinates (Bregma −0. 5 mm; lateral 1 mm, depth: 2. 5 mm) for intraventricular infusion of the GSK3β inhibitors CHIR99021 and AR-A014418. Delivering the small molecules was achieved over a period of 3 d using an osmotic miniupump (1 μl/h, model 1003D; Alzet Osmotic Pumps) into the CSF. Sham animals received all surgical steps, catheter implantation, and pump insertion. For studying recruitment of SVZ NSCs following hypoxia and small molecule administration, dSVZ NSCs were permanently labeled by dorsal electroporation [60] of a Cre plasmid (Cambridge, MA, www. addgene. org, plasmids 13775) in Cre-reporter mice (ROSA26-Flox-Stop-Flox YFP, Jackson Laboratories). A pCAGs-Cre plasmid under a chicken β-actin promoter was obtained from Addgene (Cambridge, MA, www. addgene. org, plasmids 13775). Mice aged P1 were dorsally electroporated, then placed in a hypoxic rearing chamber maintained at 9. 5%–10. 5% O2 concentration by displacement with N2 as described previously [43]. Hypoxia began at P3 for 8 d until P11. A separate group was maintained in a normal atmosphere (normoxic group). CHIR99021 was administered by intranasal administration as previously described [45]. Mucus was first permeabilized by the use of type IV hyaluronidase, then, 10 μl of CHIR99021 (Sigma) was administrated 4 times (starting at the end of the hypoxic period, then every 12 h), at a concentration of 1. 5 mM in sterile PBS (Vehicle was used as a control). Mice were killed 8 d after cessation of hypoxia at P19. Standard immunofluorescence protocols were applied as previously described [15]. Mice were killed by injection with an intraperitoneal overdose of pentobarbital (Eutha77 in Ringer’s solution) followed by transcardial perfusion with 4% paraformaldehyde (PFA) dissolved in 0. 1 M phosphate buffered saline (PBS; pH 7. 4). Following removal, brains were post-fixed in 4% PFA overnight at 4°C and cut in coronal sections at 30–50 μm thickness for obtaining serial sections. Primary antibodies used were goat anti-Dcx (1: 400 Santa Cruz); anti-Mcm2 (1: 400 Santa Cruz); chicken anti-β-galactosidase (1: 500, Abcam); mouse anti-Ki67 (1: 500, BD Pharmingen); (1: 300, Millipore); rabbit anti-GFAP (1: 300, DAKO); rabbit anti-Olig2 (1: 400, Millipore); chicken anti-GFP (1: 1000; Abcam, AB13970); mouse anti-NeuN (1: 500, Millipore, MAB377); rat anti-MBP (1: 300, Millipore, AB40390); rabbit anti-Tbr2 (1: 500, Abcam). Appropriate secondary antibodies conjugated with Alexafluor 488,568, or 405 (1: 400, Molecular Probes) were applied. Control experiments were performed using appropriate blocking peptides where available or otherwise by omission of the primary antibody. Fluorescent labeling of cells in S-phase by EdU (5-ethynyl-2′-deoxyuridine) detection was performed following manufacturers guidelines using Click-it EdU Alexa Fluor 555 imaging kit (Invitrogen). Tissues were mounted on poly-lysine coated glass slides with Vectashield mounting media (Vector Laboratories) and sealed with coverslips. Imaging and analysis methods are described in detail in our previous methodological study [61]. All quantifications were performed using a homogenous sampling approach that has been optimized for three-dimensional analysis of microdomains in the mouse SVZ [10] and provides an accurate quantification equivalent to exhaustive stereological methods from which it is adapted [61]. In brief, serial coronal sections were processed throughout the entire rostro-caudal extent of the SVZ (series of six sections for adult tissues and at least three for the postnatal SVZ). Quantification was performed on equivalent areas in each experimental group [10]. Images were captured using a Zeiss LSM Meta 5. 1, Zeiss LSM Meta 7. 1, or Leica SPEII confocal microscope and processed with Zeiss LSM Image Examiner (V. 5. 2. 0. 121) or LAS-AF software (V. 2. 7), maintaining the acquisition parameters constant to allow comparison between samples. The number of cells expressing the markers Ki67, EdU, Olig2, Tbr2, Mcm2, Dcx, Ascl1, and GFAP were quantified with at least three fields of view per section on series of equally spaced sections of 40-μm thickness encompassing the entire rostral lateral ventricle as previously described [10,59]. The dSVZ in both ages was defined based on DAPI counterstaining (Invitrogen). Quantifications were performed on confocal z-stacks of 230 μm2 × 230 μm2 in the x-y-plane and 30 μm in the z-plane, with a volume of 1. 6 × 106 μm3. For the case of GFAP+ cells, i. e. , NSCs, only cells directly adjacent to the ependymal layer were analyzed as previous for similar postnatal ages [10]. The myelin index (MI), a means to measure postnatal myelination in the corpus callosum, was done in serial sections from PLP-DsRed mice [59]. The number of myelin sheaths crossing a diagonal transect was counted in each confocal z-section at 1,5, 10,15,20,25, and 30 μm (captured using a 40× objective) so that the MI represents the density of DsRed+ myelin sheaths within a volume of 1. 6 × 106 μm3. Statistical significance was tested using GraphPad Prism v302 for multiple variables, using or one-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test, and for two variables, using unpaired t tests (referred to as t test), where appropriate.
summary: The subventricular zone (SVZ) is the largest germinal zone of the postnatal and adult brain. It contains neural stem cells (NSCs) that give rise to neurons and oligodendrocytes (OLs) in a region-specific manner. Here, we use a bioinformatics approach to identify multiple signaling pathways that regulate the diversity of cell lineages that originate from different subregions of the SVZ. We further use a computational-based drug-discovery strategy to identify a catalogue of small molecules that can be used to manipulate the regionalization of the SVZ. We provide proof that, by administration of small molecules in vivo, it is possible to promote the specific generation of neurons and OLs from NSCs in both the postnatal and adult brain, as well as in regenerative contexts after lesion. This study unravels novel strategies for using small bioactive molecules to direct germinal activity in the SVZ, which has therapeutic potential in neurodegenerative diseases.
keywords: biotechnology
  genetic networks
  chemical compounds
  small molecules
  signaling networks
  organic compounds
  notch signaling
  age groups
  signal inhibition
  adults
  network analysis
  molecular biology techniques
  research and analysis methods
  computer and information sciences
  gene mapping
  chemistry
  molecular biology
  people and places
  signal transduction
  organic chemistry
  cell biology
  gene identification and analysis
  genetics
  biology and life sciences
  population groupings
  physical sciences
  wnt signaling cascade
  cell signaling
  signaling cascades
title: Pharmacogenomic identification of small molecules for lineage specific manipulation of subventricular zone germinal activity