Pten loss in Olig2 expressing neural progenitor cells and oligodendrocytes leads to interneuron dysplasia and leukodystrophy

Abstract

Therapeutic modulation of phosphatidylinositol 3-kinase (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:Pten(fl/fl) mice that showed a unique pattern of Pten loss and PI3K activation in Olig2-lineage cells. Olig2-cre:Pten(fl/fl) animals progressively developed central nervous system white matter hypermyelination by 3 weeks of age leading to later onset leukodystrophy, chronic neurodegeneration, and death by 9 months. In contrast, during immediate postnatal 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:Pten(fl/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; Interneurons; Leukodystrophy; Olig2; Pten.

Figure 1

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 Pten^fl/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 Pten^fl and Pten^wt primers detect appropriate bands at 335 and 225 bp, respectively. (D) Olig2-cre:Pten^fl/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:Pten^fl/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:Pten^fl/fl mice. Scale bar = 50μm.

Figure 2

Pten deletion in Olig2+ cells leads to hypermyelination and oligodendrocyte loss. (A) Histological analysis of 3 week old Olig2-cre:Pten^fl/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:Pten^fl/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:Pten^fl/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:Pten^fl/fl animals. Scale bar = 100 μm (D) mRNA extracted from 9 month old Olig2:Pten^fl/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:Pten^fl/fl mice reveals excessive myelin wrapping and increased axonal diameters (bar graph), which are associated with decreased g-ratios (scatter plot).

Figure 3

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:Pten^fl/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:Pten^fl/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.

Figure 4

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:Pten^fl/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:Pten^fl/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:Pten^fl/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:Pten^fl/fl animals compared to controls. Scale bars = 50 μm.

Figure 5

In vitro E14.5 Olig2-cre:Pten^fl/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:Pten^fl/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.

Figure 6

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.

Figure 7

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.