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. Author manuscript; available in PMC: 2015 Jan 1.
Published in final edited form as: Stem Cells. 2014 Jan;32(1):313–326. doi: 10.1002/stem.1590

Pten Loss in Olig2 Expressing Neural Progenitor Cells and Oligodendrocytes Leads to Interneuron Dysplasia and Leukodystrophy

Cécile L Maire 1,*, Shakti Ramkissoon 1,2,*, Marika Hayashi 1, Sam Haidar 1, Lori Ramkissoon 1, Emmanuelle DiTomaso 3, Keith L Ligon 1,2,4
PMCID: PMC4096461  NIHMSID: NIHMS554999  PMID: 24395742

Abstract

Therapeutic modulation of PI3K/PTEN signaling is currently being explored for multiple neurological indications including brain tumors and seizure disorders associated with cortical malformations. The effects of PI3K/PTEN signaling are highly cell context dependent but the function of this pathway in specific subsets of neural stem/progenitor cells generating oligodendroglial lineage cells has not been fully studied. To address this we created Olig2-cre:Ptenfl/fl mice which showed a unique pattern of Pten loss and PI3K activation in Olig2-lineage cells. Olig2-cre:Ptenfl/fl animals progressively developed CNS white matter hypermyelination by 3 weeks of age leading to later onset leukodystrophy, chronic neurodegeneration and death by nine months. In contrast, during immediate post-natal development oligodendroglia were unaffected but abnormal and accelerated differentiation of lateral subventricular zone stem cells produced calretinin-positive interneuron dysplasia. Neural stem cells isolated from Olig2-cre:Ptenfl/fl mice also exhibited accelerated differentiation and proliferation into calretinin-positive interneurons and oligodendrocytes indicating such effects are cell autonomous. Opposition of the pathway by treatment of human primary neural progenitor cells (NPCs) with the PI3K inhibitor, NVP-BKM120, blocked in vitro differentiation of neurons and oligodendroglia indicating PI3K/PTEN effects on NPCs can be bidirectional. In summary, our results suggest Pten is a developmental rheostat regulating interneuron and oligodendroglial differentiation and support testing of PI3K modulating drugs as treatment for developmental and myelination disorders. However, such agents may need to be administered at ages that minimize potential effects on early stem/progenitor cell development.

Keywords: hypermyelination, Olig2, Pten, interneurons, leukodystrophy

Introduction

The phosphatidylinositol 3-kinase (PI3K) signaling pathway has tissue specific functions, which are regulated by multiple mechanisms depending on the cellular context driving pathway activity. Indeed in the central nervous system (CNS), PI3K signaling plays critical roles in regulating cell growth, proliferation, survival, metabolism and migration [1, 2]. Mouse models, wherein components of the PI3K pathway are genetically mutated or deleted, lead to constitutive activation or downregulation of the signaling pathway. Loss of phosphatase and tensin homolog (Pten) function results in activation of PI3K signaling, which significantly affects brain development and neural function. Loss of Pten in GFAP expressing neural stem cells from the subventricular zone (SVZ), results in premature death of mice, due to global enlargement of the brain (megalencephaly) and increased neuronal size in the cortex, cerebellum and hippocampus in association with ectopic neurons [3–5]. Moreover, in GFAP+ or nestin+ neural stem/progenitor cells (NPCs), neurogenesis and NPC self-renewal are increased but not at the expense of glial differentiation [6–10].

The status of PI3K pathway activation and its potential involvement in oligodendrocyte specification and maturation has yet to be fully deciphered. While GFAP and Nestin-cre conditional Pten deletion revealed obvious roles for Pten in neuronal development, the targeting and functional effects in oligodendrocytes was limited. Other PI3K pathway studies targeting mature oligodendrocyte or Schwann cells demonstrate excessive myelin production [11–15] as well as an increase of astrocyte proliferation and cell size [16]. However, these studies did not fully address the role of PI3K pathway in earlier stages of progenitor cells and brain development.

During normal brain development, Olig2 a bHLH transcription factor shows restricted expression in NPCs that give rise to all oligodendrocyte progenitor cells (OPC) and specific subsets of GABAergic inhibitory interneurons [17–19]. In the adult CNS, Olig2 expression is retained in 20% of the SVZ-NPCs [20, 21] as well as all proliferating glial progenitors diffusely present in white and gray matter [22]. These Olig2+ cells give rise to mature myelinating oligodendrocytes and to mature GFAP+ astrocytes in gray matter [22–24]. As a reservoir of adult glial progenitors, these Olig2+ cells play crucial roles in response and repair of brain injury. However, the role of PI3K pathway activation in these cellular compartments remains largely unexplored. To this end, we investigated the role of Pten in these two critical cellular compartments in adult and developing brains using methods for activation or suppression of PI3K signaling in glial and neuronal progenitors expressing Olig2.

Material and Methods

Animal studies

All animal experiments were performed in accordance with Dana-Farber Cancer Institute animal facility regulations and policies. Ptenfl/fl (JAX #006068), RosaYFP (JAX #006148), CAGCATGFP (gift from Dr. Miyasaka UCSF) and hGFAP-cre mice (JAX #004600) were genotyped as reported previously [25] and Olig2-cre mice (JAX #011103) were genotyped with Cre and Olig2 primers designed by Transnetyx (Cordova, TN, USA). To verify CNS-specific Pten deletion, genomic DNA was extracted from tails and brains of Ptenwt, Ptenfl/fl and Olig2-cre:Ptenfl/fl mice and amplified using the following primers: P1 5′-ACTCAAGGCAGGGATGAC-3′, P2 5′-AATCTAGGGCCTCTTGTGCC-3′, P3 5′-GCTTGATATCGAATTCCTGCAGC-3′.

NVP-BKM120 (Novartis) was administrated by oral gavage (30mg/kg in corn oil) to C57Bl6 wild type mice (n=6), every other day for 1 month. Control mice (n=5) were given corn oil. Mice were euthanized after the last treatment. 500 microliter of insulin (4mg/ml) was injected into 2 control and 2 BKM-treated mice 30 min prior to euthanasia for pharmacodynamic assessment.

Tissue processing

Post natal mice were subjected to intracardiac perfusion with 4% paraformaldheyde (PFA) in 0.1M phosphate buffer saline (PBS) solution under terminal anesthesia. Brains were extracted, stored overnight in a solution of 20% sucrose and frozen in OCT (tissueteck) or dehydrated prior to paraffin embedding. Frozen sections were analyzed at twelve micrometer and paraffin sections at five micrometers. For protein extraction, brain and liver tissues were snap frozen in liquid nitrogen immediately after dissection and stored at −80C.

Electron microscopy

Half a centimeter square of tissue extracted from cortex, corpus callosum and striatal tissue from Olig2-cre:Ptenfl/fl mice and Olig2-cre:Ptenfl/+ control were fixed using 2.5% PFA 2.0% glutaraldheyde in 0.2M cacodylate buffer, pH 7.2. Toluidine blue stained semi-thin sections were generated according to standard protocol. Electron microscopy was performed at the Brigham and Women Hospital EM core facility. Axonal myelin wrapping was measured using g-ratio methodology (ratio of axon diameter to diameter of axon plus surrounding myelin).

Immunostaining

Paraffin or frozen sections were subjected to antigen retrieval in 10mM citrate buffer for 20min. After cooling, slides were incubated overnight at 4°C with primary antibody then washed in TBST. For immunofluorescence studies, slides were incubated for 1h with the following secondary antibodies: Alexa 488- or 568-conjugated anti mouse, rat or rabbit, anti mouse IgM-FITC conjugated. Sections were counterstained with bisbenzimide 33342 (sigma-aldrich) and mounted in fluoromount medium (Southern technology). For DAB-based immunostaining, Dako EnVision+ kit was used according to manufacturer’s instructions. Primary antibodies used were directed against Olig2 (1:10000, gift from Dr C. Stiles lab, DFCI, Boston, MA); phosphor-S6 ribosomal protein S235/236 (1:1000, 2211, Cell Signaling Technology); GFP (1:500, A-11122, Invitrogen); NG2 (1:100, Chondroitin sulfate proteoglycan, AB5320, EMD Millipore); Calretinin (1:200, 18-0211, Zymed); myelin basic protein (1:1000, MBP, SMI-94R, Covance); NeuN (1:1000, MAB377, EMD Millipore); SMI31 (1:100, Covance); Mac-2 (1:20000, CL8942AP, Cedarlane); 5-bromo-2-deoxyuridine (1:100, BrdU, BD pharmingen); Ki67 (1:100, clone MM1, Novocastra); adenomatous polyposis coli (APC, clone CC1, 1:100, Calbiochem); Iba1 (1:500, Wako); nestin for mouse (clone RAT401, 1:1000, Milipore); nestin for human cells (1:2000, AB5922, Chemicon); Gfap (1:1000, G3893, Sigma-aldrich); O4 (1:50, MAB345, Millipore); Galactocerbroside C (GalC, 1:1000, MAB342, Milipore); Map2 (1:1000, M9942, Sigma-Aldrich); doublecortin (Dcx, 1:100, sc-28939, SantaCruz technology); beta III tubulin (1:500, Tuj1, BABCO); pAKT S473 (1:50, D9E, Cell Signaling Technology); CD3 (1:200, Abcam); B220 (1:200, BD pharmingen). Co-staining of Olig2 and pS6 were performed using the dual link system-HRP (Dako #K4063). Hematoxylin & Eosin (H&E), Bodian and luxol-fast-blue (LFB) stains were performed on paraffin sections according to standard protocols. All the quantifications were performed on 3 Olig2-cre:Ptenfl/fl and 3 controls. Formalized quantification of staining was performed for the following antibodies: Ki67, GFP, Gfap, NG2, Calretinin. Such quantification was performed from multiple levels of the dorsal neocortex and corpus callosum as 12um paraformaldehyde fixed frozen serial sections spaced at a distance of 120um apart for all experiments to prevent counting of single nuclei multiple times.

Western blot

Protein lysates were prepared from fresh mouse brain tissue and homogenized in RIPA buffer with protease (complete tablets, Roche diagnostics) and phosphatase inhibitors (PhosSTOP, Roche diagnostics). Twenty micrograms of protein were separated on 4–12% Tris Glycine gels (Invitrogen), transferred onto nitrocellulose membranes, blocked in 5% BSA and incubated with the following primary antibodies: pS6 S235/236, pAKT S473, pAKT T308, AKT, PTEN, GAPDH all from Cell Signaling Technology. The secondary antibodies were anti rabbit HRP (GE Healthcare) and anti mouse HRP (Dako). After washing in PBST, membranes were revealed by ECL reaction and images were acquired using the AlphaInnotech FluorChem HD2 imagery system.

Neural stem cell isolation

Embryonic neural stem cells (NSC) were isolated from either E14.5 embryos or P6 pups according to previous protocol [26]. Briefly, the meninges were removed and lateral and medial ganglionic eminences were harvested, mechanically dissociated in mouse NeuroCult media (Stem cell Technology). Cells were seeded in Neurocult media enriched by FGF 10 ng/ml and EGF 20 ng/ml on low adhesion plates (Corning) at 100,000 cells/ml. After 3d, established neurospheres were passaged every 10d by mechanical dissociation. Adult NSCs were isolated from 5-month-old mice by precise dissection of the SVZ from a coronal section under a dissecting microscope. Adult NSCs were extracted, plated and passaged as described above.

Proliferation and differentiation assay

In vitro proliferation of NSC was assessed using CellTiter-Glo (Promega) luminescent cell viability assays. Two thousand cells from 2 independent Ptenwt or Olig2-cre:Ptenfl/fl cell lines were plated in sixplicates and subsequently lysed to quantify ATP content every 2 days, according to the manufacturer’s protocol. Standard curves were established for each cell line. Cells were incubated with BrdU (10mg/ml) for 3h and analyzed by IHC to determine the number of cells that entered the cell cycle.

To assess cellular differentiation, Ptenwt or Olig2-cre:Ptenfl/fl neural progenitors were plated on laminin-coated chamber slides with 2% FBS and allowed to differentiate for 8d before analysis.

Human neural progenitor cell culture

Tissues were obtained from temporal lobectomy specimens (white and gray matter) of patients undergoing seizure surgery (BWH IRB approved protocol) and dissociated using a combination of mechanical and papain treatment (Neural tissue dissociation kit, Miltenyi). Cells were plated on laminin-coated plates in human NeuroCult media (Stem cell technology) supplemented with FGF 10ng/ml and PDGFBB 5ng/ml. A mixed culture of glial and neuronal human progenitors were allowed to grow for 1 month then plated on laminin chamber slides with 2% FBS supplemented either with DMSO (control) or 1μM NVP-BKM120 (Novartis) to assess differentiation. Cells were harvested after 9d for immunofluorescence analysis.

mRNA expression profile analysis

Total RNA was extracted from Olig2-cre:Ptenfl/fl (n=3) and Ptenfl/+ (n=3) brains with Trizol (Invitrogen) followed by purification with RNeasy cleanup kit (Qiagen). mRNA expression profiles were obtained using mouse 430 2.0 microarrays (Affimetrix) in collaboration with the DFCI microarray core facility. Microarray data were analyzed using Genepattern [27] [28] [29, 30] and IPA software (Ingenuity® Systems, www.ingenuity.com).

Results

Pten loss in oligodendroglia leads to ectopic activation of PI3K/Akt signaling

To determine unique effects of PI3K activation on the oligodendroglial lineage and unique subpopulations of Olig2-expressing neural stem/progenitor cells (NPCs), we performed targeted deletion of Pten using the previously described Olig2tm2(TVA,cre)Rth mice (hereafter referred to as Olig2-cre mice) [31]. In order to provide detailed fate mapping in the forebrain, we crossed Olig2-cre mice with animals containing two independent reporter alleles, CAG-CAT-EGFP and B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J (hereafter referred to as GFP-Reporter line), which when combined give complete fate mapping results compared to either reporter line alone. Olig2-cre:GFP-Reporter mice had strong GFP signal in the corpus callosum and SVZ with reduced staining in neuron containing regions of the cortex and striatum (Fig. 1A). In comparison to hGfap-cre:GFP-Reporter mice, frequently used in prior Pten deletion studies, the Olig2-cre driver fate mapped more cells in the white matter and less in the stem cell niches, while the number of GFP+ cells in the gray matter were comparable between the two lines. Double immunofluorescent staining with GFP and a specific marker to designate cell types, shows that 70% of NG2+ oligodendrocyte progenitors fate mapped to the corpus callosum of Olig2-cre mice compared to only 25% in hGfap-cre mice. Post-mitotic GABAergic inhibitory interneurons that stain positive for Calretinin were equally fate mapped to cortex in both lines, while more GFAP+ astrocytes colocalized with GFP in the cortex of hGfap-cre mice (Fig. 1A and Supplementary Figure 1A).

Figure 1.

Figure 1

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PI3K signaling is activated by Pten deletion in Olig2+ cells.

(A) Fate mapping analysis in Olig2-cre:GFP-Reporter and GFAP-cre:GFP-Reporter mice by immunofluorescence staining of adult dorsal neocortex. Olig2-cre more effectively targets oligodendrocytes (NG2+) and interneurons (Calretinin+) compared to GFAP-cre which more effectively targets astrocytes (Gfap+). White arrows indicate co-localization of GFP (green) with lineage specific markers (red). Right panel shows quantification of GFP labeling in specific cell lineages within the neocortex and corpus callosum. Scale bar = 50μm. (B) Schematic illustrating engineered alleles for Olig2-cre and Ptenfl/fl mouse lines subsequent cross to produce conditional deletion of exon 5 of Pten. (C) PCR confirmation of exon 5 deletion (849 bp band) as well as Ptenfl and Ptenwt primers detect appropriate bands at 335 and 225 bp, respectively. (D) Olig2-cre:Ptenfl/fl mouse brain is notably expanded on gross images. Scale bar = 1 cm. (E) Western blot analysis confirming PI3K pathway activation following Pten deletion in 9-month-old brain by increased pAkt and pS6. (F) Immunohistochemistry for pAkt shows ectopic pathway activation in subpopulations of cells (arrows) within the SVZ and striatum of Olig2-cre:Ptenfl/fl mice at 3 weeks of age. Scale bar = 50 μm. (G) Double immunohistochemistry in 9 month old corpus callosum shows increased co-localization of Olig2 (red) and pS6 (brown) in oligodendroglial cells of Olig2-cre:Ptenfl/fl mice. Scale bar = 50μm.

Having established that Olig2-cre mice target oligodendroglial cell populations more effectively than hGfap-cre, we crossed Olig2-cre mice to the previously described conditional Ptenfl/fl line [25] (Fig. 1B, 1C). Olig2-cre:Ptenfl/fl mice were generated at expected Mendelian frequencies. Previous studies of Pten deletion in Gfap-cre and Nestin-cre mice resulted in death by 3 weeks of age [3, 4, 6, 9]; however Olig2-cre:Ptenfl/fl mice were viable, fertile, and grossly normal until early adulthood. By 6 months they developed progressive ataxia, megalencephaly and decreased motor function progressing to bilateral hind leg paralysis culminating in premature death by age 9 months.

In contrast to the normal low baseline activity, western blot analysis on protein isolated from coronal sections at the level of the anterior commissure of Olig2-cre:Ptenfl/fl brains showed strong ectopic activation of the PI3K pathway demonstrated by increased pAkt (S473), pAkt (T308) and pS6 (S235/6) (Fig. 1D). Immunohistochemical staining with pAkt (S473) on Olig2-cre:Ptenfl/fl brain sections highlighted a greater number of positive cells in the cortex and stem cell niche (SVZ) compared to littermate controls (Fig. 1E, arrows). Additionally, pS6 (S235/6) protein was highly expressed and co-localized with Olig2 protein following Pten deletion (Fig. 1E). This pattern of co-expression was not seen in controls suggesting that Pten deletion in the oligodendroglial compartment results in ectopic PI3K signaling.

Olig2-cre:Ptenfl/fl mice show early megalencephalic and leukomegalic features with later progression to leukodystrophy

Histological analysis of Olig2-cre:Ptenfl/fl brains at 3 weeks showed enlarged neocortex with striking expansion of the SVZ (Fig. 2A). Interestingly, the severe gross developmental anomalies reported in the hGfap-cre:Ptenfl/fl mice [3, 4, 9] including enlarged cerebellum and neuronal dysplasia were not seen in Olig2-cre:Ptenfl/fl animals. However, we noted that 100% of Olig2-cre:Ptenfl/fl animals became moribund by 9 months of age (n=20 median survival 306 days). Necropsy and neuroanatomic examination revealed megalencephaly and massive hypermyelination without evidence of neoplasia. Quantification of the corpus callosum and cortex width on H&E sections demonstrates that the gross enlargement observed (Fig. 1D) is mostly due to the increase of size of corpus callosum (103%), while the cortex was enlarged by only 13% (Fig. 2B). Examination of cerebellar compartments at embryonic, juvenile and adult time points revealed normal development without evidence of tissue patterning defects. The progressive white matter vacuolization noted in the forebrain was also evident in deep cerebellar and folial white matter by 9 months of age (Supplementary Fig. 1C).

Figure 2.

Figure 2

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Pten deletion in Olig2+ cells leads to hypermyelination and oligodendrocyte loss.

(A) Histological analysis of 3 week old Olig2-cre:Ptenfl/fl brains shows normal cortex (Cx) and white matter development but an expanded SVZ compared to controls. (B) By 9 months of age Olig2-cre:Ptenfl/fl brains are larger, secondary to white matter expansion best demonstrated by the thickened corpus callosum (CC). (C) Hematoxylin and eosin (H&E) stains highlight white matter vacuolization in the CC of 9 month old Olig2-cre:Ptenfl/fl brains (arrows), while Olig2 immunolabeling shows a 40% reduction (graph) of oligodendrocytes compared to controls. Luxol fast blue (LFB) staining and myelin basic protein (MBP) immunolabeling illustrate hypermyelination, which contributes to the macrocephaly phenotype in Olig2-cre:Ptenfl/fl animals. Scale bar = 100 μm (D) mRNA extracted from 9 month old Olig2:Ptenfl/fl brains show upregulation of myelin associated genes (Table) compared to controls, which highly correlates with the oligodendrocyte lineage signature by Gene Set Enrichment Analysis (GSEA, enrichment plot). (E) Electron microscopy (magnification: 10000x) of CC from 9 month old Olig2:Ptenfl/fl mice reveals excessive myelin wrapping and increased axonal diameters (bar graph), which are associated with decreased g-ratios (scatter plot).

To determine the temporal etiology of megalencephaly in Olig2-cre:Ptenfl/fl mice, we performed histopathological analysis. Although we found no difference in the ventricular zone thickness in neonates or number of Olig2+ oligodendrocytes or NG2+ OPC (Supplementary Fig. 1B), starting at 3 weeks of age we observed an enlargement of the white matter with hypermyelination highlighted by LFB and MBP staining (Fig. 2C). Interestingly we also noted that the cells present in the expanded SVZ in the Olig2-cre:Ptenfl/fl mice appear less densely packed, probably because of the increase number of hypermyelinating axons crossing through the SVZ/RMS in the mutant mice (Fig. 2B). mRNA expression profiling and gene set enrichment analysis (GSEA) supported these histological findings by identifying significant up regulation of key myelin genes and associated pathways (Fig. 2D, supplementary Fig. 2 and 3). Electron microscopy of thickened corpus callosum white matter from 9-month-old mice demonstrated excess myelin wrapping by oligodendrocytes with a significant decrease in g-ratios (Fig. 2E). Over time, myelin accumulation resulted in oligodendrocyte toxicity (40% loss of Olig2+ cells) leading to vacuolization of white matter and leukodystrophy (Fig. 2C, graph).

Hypermyelination precedes inflammation and neurodegeneration

As leukodystrophies often elicit or are associated with strong immune responses, e.g metachromatic and adrenoleukodystrophy (ALD), we investigated the nature of the inflammatory response in areas of white matter degeneration observed in Olig2-cre:Ptenfl/fl mice. Starting at 3 months, we noted white matter invasion by reactive macrophages (Mac-2+) followed by reactive microglia (Iba1+) at 9 months, which subsequently provoked a diffuse, prominent cerebral white matter gliosis highlighted by GFAP (Fig. 3A) and expression profiling analysis (Supplementary Fig. 3C). Given the delayed onset and progressive increase of inflammatory cells, their recruitment appears to be secondary to ongoing white matter degeneration, rather than a primary CNS autoimmune disorder. Additional examination of the inflammatory response showed rare lymphocytes scattered diffusely in the white matter. No perivascular collections were noted. Such lymphocytes were determined to be CD3+ T-lymphocytes by immunohistochemistry while no B220+ B-lymphocytes were detected (Supplementary Fig. 4). In addition to the inflammatory response, we also noticed that neuronal cell bodies in the cortex as well as axons in the spinal cord showed signs of progressive degeneration and vacuolization but only after hypermyelination was identified (Fig. 3B). Immunohistochemistry for SMI-31, a neurofilament protein, showed axonal depletion in the lateral white matter of the spinal cord, while Bodian staining highlighted axonal thickening and swelling in the striatum (Fig. 3B). Collectively these findings demonstrate that directed Pten loss in Olig2+ cells provide a novel model of leukodystrophy with the combination of hypermyelination, inflammation and neuronal degeneration.

Figure 3.

Figure 3

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Hypermyelination leads to massive inflammatory response and axonal death.

(A) Immunohistochemical analysis for Mac2 highlights macrophage invasion of white matter at 3 months in Olig2:Ptenfl/fl animals. Nine-month-old animals show marked gliosis (Gfap) and increased microglial activation (IBA1). Scale bar = 200 μm. (B) Massive spinal cord degeneration observed at 9 months (H&E), with axonal loss in the spinal cord (SMI31) and axonal swelling (Bodian staining) in Olig2:Ptenfl/fl mice. Left panel scale bar = 200 μm, right panel scale bar = 25 μm. DC: dorsal cord; CX: cortex; CC: corpus callosum; LWMT: lateral white matter track.

Increased specification and abnormal migration of neuroblasts is associated with loss of Pten in Olig2+ stem/progenitor cells

We next sought to determine the effect of ectopic PI3K pathway activation in Olig2+ SVZ NPCs. We characterized the expanded SVZ at 3 weeks and found no significant increase in proliferation (Ki67), or Olig2+ cells in the SVZ (data not shown). Surprisingly, the number of doublecortin positive (Dcx+) migrating neurons was markedly increased in Olig2-cre:Ptenfl/fl animals (Fig. 4A). Furthermore, accumulation of post-mitotic calretinin positive GABAergic interneurons were identified in the expanded SVZ. These ectopic neurons aberrantly migrated in chains away from the rostral migratory stream (RMS) and SVZ through the corpus callosum, terminating in the cingulum and deep cortical layers (V–VI) (Fig. 4B). Ectopic neurons (Map2+/pAKT(S473)+), continued to proliferate throughout their migration (Fig. 4B, graph) ultimately differentiating into Calretinin positive, GABAergic interneurons colonizing in the deep cortical layers (Supplementary Fig. 5). This pool of ectopic proliferating neuroblasts represents a distinct population from those in the SVZ, which are mature, non-proliferating neurons. Surprisingly by 3 months these ectopic calretinin+ neurons were no longer present in the cortex, suggesting cell death mechanisms are intact in the Pten deleted cells and that the wave of aberrant neurogenesis occurs only during a brief early developmental period. All the ectopic postmitotic calretinin positive neurons in the cortex were dead in the older animals. Taken together we demonstrate that activation of PI3K signaling in Olig2+ NPCs is sufficient to drive postnatal neurogenesis but cannot maintain mature neuron survival.

Figure 4.

Figure 4

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Pten deletion in Olig2+ cells leads to expanded SVZ and ectopic neuronal migration.

(A) Schematic of coronal and sagittal sections of mouse brains to indicate the location of micrographs presented in panels B, C and D. (B) Immunohistochemical analysis on coronal sections from 3 week old brains reveals increased doublecortin (Dcx) and Calretinin positive neurons in the expanded SVZ of Olig2-cre:Ptenfl/fl animals compared to controls, consistent with ectopic neuron maturation in the SVZ. Map2 (red) colocalizes with pAkt (green) by immunofluorescence indicating that abnormal neuron maturation in the SVZ occurs in Akt activated cells. (C) Sagittal sections of 3 week old brains highlight ectopic, Dcx positive neurons that form abnormal chains as they migrate away from the RMS through the corpus callosum (black arrows). Immunofluorescence images in the middle panel confirm increased PI3K signaling (pAkt, green) in ectopic neurons (Map2, red) of Olig2:Ptenfl/fl animals compared to control. These ectopic neurons continue to proliferate (Ki67, red) while in the CC (white arrows) and then differentiate into post-mitotic CalR (green) GABAergic interneurons (white arrowheads) in the deep cortex (right panel). Quantification of Ki67+ migrating neuroblasts was achieved by counting the entire corpus callosum on a sagittal section of three different animals at P18 (graph). (D) The SVZ of 9-month-old Olig2:Ptenfl/fl mice show retention of ectopic Dcx+ neuroblasts and modest increases in proliferating (Ki67+) cells. Gfap illustrates prominent gliosis while Olig2 shows loss of oligodendrocytes in Olig2:Ptenfl/fl animals compared to controls. Scale bars = 50 μm.

Examination of aged adult Olig2-cre:Ptenfl/fl brains revealed no significant difference in the number of Olig2+ or proliferating cells in the SVZ, whereas GFAP staining showed prominent gliosis and reactive astrocytes (Fig. 4C). However, within the SVZ, increased PI3K activity enhanced adult neurogenesis as evidenced by increased Dcx staining suggested a role for Pten in Olig2+ NPCs and neuronal specification.

Pten deletion increases proliferation and differentiation of NPCs in a cell autonomous manner

To determine whether the neural differentiation findings seen in vivo were cell autonomous we isolated and examined the growth and differentiation potential of embryonic (E14.5) NPCs from Olig2-cre:Ptenfl/fl mice. The sphere-initiating potential of Olig2-cre:Ptenfl/fl NPCs was significantly increased compared to controls under standard stem cell growth conditions (Fig. 5A). Additionally Pten null NPCs showed a marked increase proliferation (Fig. 5A, graph). Moreover, analysis of sectioned neurospheres demonstrated approximately 2-fold increase in the fraction of cells in S-phase (3 hr BrdU pulse) and non-G0 stages (Ki67) of the cell cycle (Fig. 5B). Such increases appeared to result predominantly by affecting the proliferation of Olig2+ cells as measured by co-localization of Olig2 and Ki67 (Supplementary Fig. 6A).

Figure 5.

Figure 5

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In vitro E14.5 Olig2-cre:Ptenfl/fl NSCs show precocious differentiation.

(A) Olig2-Pten null NSCs show increased self-renewal (sphere images, magnification 10x) and proliferation (graph). (B) Increased proliferation in Olig2-cre:Ptenfl/fl NSCs compared to controls demonstrated by BrdU and Ki67 immunohistochemistry and quantification. Scale bar = 50 μm. (C) Differentiation assays of E14.5 NSCs or P6 NPCs show accelerated and precocious differentiation into multiple lineages: O4+ OPCs, Galactocerebroside+ (GalC) mature oligodendrocytes, Map2+ neuroblasts and Calretinin+ mature neurons. Scale bar = 50 μm.

Examination of adherent NPC cultures under differentiation conditions showed that Olig2-cre:Ptenfl/fl NPCs at both embryonic (E14.5) and early postnatal (P6) stages advanced differentiation along oligodendroglial and neuronal lineages, without altering the overall ratio of glia to neurons (Fig. 5C, supplementary Fig. 6B). Oligodendrocytes from Pten null cultures appeared to mature earlier with more processes and galactocerebroside (GalC) pseudo-myelin sheaths compared to controls (Fig. 5C). Pten loss at both developmental stages led to a robust increase in the number of neuronal progenitors (Map2) as well as cells expressing postmitotic neuronal markers (Calretinin) suggesting accelerated differentiation (Fig. 5C, supplementary Fig. 7). E14.5 Pten null NPCs gave rise to roughly 2-fold more O4 and 5-fold more GalC positive oligodendrocytes, 1.7-fold more Map2 positive neuroblasts, and a reduced proportion of undifferentiated, nestin-expressing cells after 7d in culture. In contrast, Gfap expressing cells were not significantly altered after Pten deletion in Olig2+ cells (Supplementary Fig. 6B).

Inhibition of PI3K signaling in human NPCs suppresses neuronal and glial differentiation in vitro but has minimal effects on NPC development in vivo

To further establish the level at which PI3K signaling regulates neural differentiation and determine the potential for bidirectional pathway activity in human cells, we treated human primary NPCs with NVP-BKM120 (Novartis Inc.), a pan-PI3K small molecule inhibitor. This agent was selected because of its reported effects on CNS function and current use in clinical trials for glioblastoma and other cancers [32, 33]. Differentiation was assessed using two independent primary mixed NPC cultures that were plated in 2% FBS on laminin supplemented with either DMSO (control) or BKM120 (1μM). After 9 days of treatment we observed notable delays in neuronal (Tuj1), oligodendroglial (O4) and astroglial (GFAP) differentiation (Fig. 6).

Figure 6.

Figure 6

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Human neural progenitor in vitro differentiation is compromised following PI3K pathway inhibition.

Immunofluorescence staining for Nestin (red, neural stem cells), O4 (green, oligodendrocyte progenitors), Tuj1 (red, neuroblasts) and GFAP (red, astrocytes) in human neural progenitor cells treated with BKM120 (1μM) shows inhibition of cellular differentiation without increased cell death. Scale bar = 50 μm.

Given our in vitro findings and the mood alteration side effects reported in the phase I clinical trial for BKM120 [33] we performed in vivo administration of BKM120 for 1 month on juvenile animals (3 weeks, incomplete myelination) to analyze the effects on brain development and acquire knowledge as to the connection between myelination and PI3K pathway in pediatric population. Analysis of treated mice demonstrated marked pharmacodynamic changes on pAKT308, pAKT473, and pS6 in all brain regions (Fig. 7A). BKM120 was particularly effective at inhibiting pAKT in neuronal and SVZ compartments (Fig. 7B); however, examination of multiple other cell types with lineage and proliferation markers (DCX, NeuN, MBP, IBA1, NG2, Ki67, GFAP, Calretinin) demonstrated no significant alterations in brain structure or development in postnatal stages even after 1 month of treatment (Fig. 7C). In particular, oligodendroglia and myelin appeared normal by histological and EM analysis indicating that postnatal inhibition of PI3K signaling has few effects on formed brain and cellular structures. These findings suggest that clinical use of PI3K inhibitors is not likely to disrupt neuronal and glial homeostasis in the adult CNS.

Figure 7.

Figure 7

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In vivo Inhibition of PI3K pathway by BKM120 does not affect myelination or neurogenesis.

(A) Western blot analysis from brain and liver demonstrating PI3K pathway suppression by decreased pAKT levels in treated animals. (B) Immunohistochemistry for pS6 and pAKT illustrates decreased PI3K signaling in multiple CNS compartments of 3-week-old BKM-treated mice compared to vehicle-treated controls. Scale bar = 50 μm. (C) BKM-treated with BKM120 showed no differences in neuroblast migration (Dcx), cortical and hippocampal neuronal density (NeuN), myelin (MBP), migroglia (Iba1), OPCs (NG2), astrocytes (GFAP) or GABAergic neurons (Calretinin). Otherwise specified scale bar = 50 μm.

Discussion

Our results suggest several new perspectives on cell-context specific Pten function in cortical interneurons and myelinating oligodendrocytes. Pten loss at early stages of development is associated with an increase in NPC proliferation within the SVZ together with an accelerated differentiation of glial and neuronal progenitors in vitro and in vivo. At later stages the disruption of Pten signaling in oligodendroglia leads to hypermyelination, brain enlargement, eventual leukodystrophy, and death at an advanced age. These findings have implications for understanding developmental disorders as well as cancer and its treatment in several ways.

During neural development, Olig2 is responsible for oligodendroglial and neuronal specification of neural stem cells residing in the germinal zone of the medial, lateral and caudal ganglionic eminences (GE). By E18.5 GABAergic interneurons derived from Olig2+ NPCs that originated from the medial and caudal GE, radially migrate to restricted cortical layers (II–III/IV) [34, 35]. In the adult CNS, neuroblasts derived from Olig2+ NPCs in the SVZ migrate in chains along the RMS to populate the olfactory bulb. Surprisingly loss of Pten in Olig2+ cells resulted in early post-natal waves of increased neurogenesis in the SVZ producing Calretinin+ GABAergic interneurons that also aberrantly migrated homophilically to the deep cortical layer V. We did not observe any abnormalities of embryonic (E14.5 and E18.5) neurogenesis or migration and therefore the presence of these ectopic post-mitotic neurons in the SVZ of 3 week old Olig2-cre:Ptenfl/fl animals appears to result from Pten effects within a specific developmental window in the life of these cells. The ectopic calretinin+ neurons did not seem to be a result of failure of apoptosis or pruning of such cells given that at later stages such cells are no longer present in the brain. Furthermore no alterations in the level of apoptosis as measured by cleaved caspase 3 IHC were noted (data not shown). Therefore while excess PI3K signaling promoted transient ectopic neurogenesis, it ultimately failed to maintain the survival of ectopic neurons possibly due to the absence of essential micro-environmental cues in the post-natal cortex or axonal targets present during embryogenesis.

Adult neuroblast migration along the RMS was not affected in Olig2-cre:Ptenfl/fl mice contrary to previous studies wherein Pten was deleted in Nestin+ progenitors [10]. This difference highlights the need for complementary use of additional cell-type restricted Cre-deletion lines in investigating the effects of critical pathways. With recent evidence suggesting a role for GABAergic interneurons in neurologic disease, including schizophrenia, autism, epilepsy and Rett syndrome, our studies in Olig2+ NPCs indicate that PI3K signaling functions to fine-tune the balance of neuronal specification and migration in post-natal stages [36]. Further studies investigating the role of Olig2+ cells in interneuron disease are warranted.

Within the oligodendroglial compartment, targeted deletion of Pten in Olig2 expressing cells resulted in hypermyelination due to excessive myelin wrapping leading to leukodystrophy. In the peripheral nervous system (PNS) this phenomena of increased axonal wrapping has been well described in subtypes of Charcot-Marie-Tooth disease and in patients with hereditary neuropathy with liability to pressure palsy. Although these disorders typically arise as a result of defects in myelin specific genes including PMP22, P0 and MAG, our studies show that ectopic PI3K signaling leading to excess of apparently normal myelin produce a similar phenotype in the CNS. Interestingly our mRNA expression profiling analysis demonstrated upregulation of PNS associated myelinating genes in the CNS of Olig2-cre:Ptenfl/fl mice which may also contribute to the development of leukodystrophy and inflammatory response. Previous reports in the PNS identified a role for Pten in directly regulating peripheral myelin production, wherein Pten complexes with DLG1 to create a switch that prevents excessive myelin wrapping [15]. Olig2-Cre:Ptenfl/fl mice showed no changes in DLG1 levels compared to controls (data not shown) suggesting alternate mechanisms likely exist for PI3K signaling in balancing CNS myelin production.

The hypermyelination phenotype due to Pten loss in the oligodendrocytes leads to a late onset inflammatory leukodystrophy. The severity and timing of the leukodystrophy and axonopathy suggest the findings are not due to loss of Pten in rare neurons targeted by Olig2-cre (~10%). The inflammatory response in our mice appears most likely secondary to the presence of myelin and/or early neuronal degeneration based on the delayed appearance of the inflammatory response. That altered myelin formation can lead to significant inflammatory responses is suggested by their presence in transgenic mice overexpressing PLP protein within the brain [37] as well as the fact that inflammation is a common feature in several types of leukodystrophies. In Pten mice we noted only rare CD3+ T cells in severely affected areas and no B cells, which contrasts with the usual prominence of T cells in human adrenoleukodystrophy. Pten mice may have a more similar inflammatory response to that seen in metachromatic leukodystrophy, where significant macrophage and microglial are the predominant component. Pten mice may serve as a useful tool therefore to help differentiate how the inflammatory responses and the excess myelin may uniquely contribute degenerative phenotypes commonly encountered in leukodystrophies.

A clear role for aberrant PI3K signaling has been identified in multiple neurodevelopmental disorders. For example, patients with Cowden syndrome (CS), an autosomal dominant inherited disorder due to inactivating mutations in Pten, often present with megalencephaly and gangliocytomas of the cerebellum (Lhermitte-Duclos Disease). Neuroimaging analysis of CS patients with known Pten mutations demonstrated no detectable white matter abnormalities [38] with only extremely rare cases of neuropathy. The features revealed in Olig2-cre:Ptenfl/fl mice therefore are unlike those features characteristic of CS. This suggests that abnormal PI3K activation in restricted CNS cellular compartments generates unique neuroanatomic findings unlike those reported in other “pan-CNS” Pten null animal models [3, 4].

While Olig2-cre:Ptenfl/fl mice did not develop tumors, our findings have implications for the mechanisms by which Pten may produce tumor suppression. Previous studies of Pten loss in human GFAP-cre expressing NSC promoted proliferation but blocked differentiation of cells, however this did not lead to tumor formation in vivo [7, 9]. In contrast, our findings suggest a more complex role whereby the rate of proliferation and differentiation were both increased, and apparently balanced in degree, such that neoplasia did not develop in any animals in our study. Our findings perhaps explain more simply a mechanism for the observation that Pten loss alone is insufficient to generate neoplasia in the brain or several other cellular contexts. However, that such an effect can promote tumor formation is provided by the fact that additional gain of function oncogenic events or loss of additional tumor suppressors (e.g. Tp53) which increase proliferation or reduce cell cycle exit readily produce gliomas in the hGFAP-cre mice [7, 39].

Abnormal PI3K signaling in a number of CNS diseases has sparked pharmacologic and clinical interest in developing therapies that target this pathway. For example, BKM120 is currently in clinical trials for glioblastoma patients with additional compounds in the pipeline directed against downstream pathway components. Our findings are the first demonstration of the in vivo pharmacodynamic effects of BKM120 on the normal brain and, combined with prior results in orthotopic glioblastoma models, strongly support exceptional brain penetration of this drug [40]. Given that in vivo adult neurogenesis and myelination were not affected despite robust pathway suppression, these findings suggest that the use of this agent in patient care is unlikely to adversely alter adult brain cytoarchitecture.

However, further studies will likely be needed to determine the degree to which neuronal or possibly astrocyte physiological function and communication may be affected by the demonstrated reductions in PI3K signaling within these cells. That BKM120 has such effects is suggested by the acute and rapidly reversible effects on mood and other CNS cognitive functions observed in cancer patients treated with the drug [32]. Our results further suggest that PI3K inhibitors may therefore have potential uses in psychiatric disorders. Moreover, abnormal PI3K signaling has been linked to neurodevelopmental disorders, such as hemimegalencephaly secondary to activating AKT3 mutations, which raises the possibility of using PI3K pathway inhibitors as early therapeutic interventions to reduce activity of other AKT isoforms [41]. However, since our studies with human NPCs treated with BKM120 showed altered differentiation profiles, further investigations are necessary to determine the effects in vivo on the developing CNS. This is of particular interest in the context of pediatric glioma patients who, with the recent discovery of activating PIK3CA mutations [42, 43] are now being considered for clinical trials of PI3K inhibitors, which might have deleterious effect in the younger patients.

Supplementary Material

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Acknowledgments

The authors wish to thank Tom Roberts and Jean Zhao for technical advice and comments on the manuscript. We thank Marian Slaney, Mei Zheng, Colleen Ford and Brant Douglas in the BWH Pathology Department for specialized histopathology staining and expertise in electron microscopy. We also would like to thank the DF/HCC Rodent Histopathology Core as well as Edward Fox at the DF/HCC Microarray Core. Financial support for this study was provided by NIH R01 (K.L.L., C.L.M.), T32 (S.R.), and the American Brain Tumor Society (L.R.). COI: K.L.L. and S.R. are co-investigators on a clinical trial of BKM120 in glioblastoma sponsored by Novartis but do not receive financial support from this study.

Abbreviation

Olig2-cre:Ptenfl/fl

deletion of Pten in the Olig2+ cells

NPCs

neural progenitor cells

NSCs

neural stem cells

SVZ

subventricular zone

BKM120

PI3K inhibitor NVP-BKM120 from Novartis Inc

Footnotes

Author contributions: C.L.M.: Conception and design, Collection and/or assembly of data, Provision of study material or patients, Data analysis and interpretation, Manuscript writing, Final approval of manuscript; S.R.: Conception and design, Collection and/or assembly of data, Provision of study material or patients, Data analysis and interpretation, Manuscript writing, Final approval of manuscript; M.H.: Provision of study material or patients, Collection and/or assembly of data.; S.H.: Collection and/or assembly of data, Data analysis and interpretation; L.R.: Data analysis and interpretation, Manuscript writing, Final approval of manuscript; E.D.: Provision of study material or patients, Final approval of manuscript; K.L.L.: Financial support, Conception and design, Data analysis and interpretation, Manuscript writing, Final approval of manuscript.

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Associated Data

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Supplementary Materials

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