Brain abnormalities and glioma-like lesions in mice overexpressing the long isoform of PDGF-A in astrocytic cells

Abstract

Background: Deregulation of platelet-derived growth factor (PDGF) signaling is a hallmark of malignant glioma. Two alternatively spliced PDGF-A mRNAs have been described, corresponding to a long (L) and a short (S) isoform of PDGF-A. In contrast to PDGF-A(S), the PDGF-A(L) isoform has a lysine and arginine rich carboxy-terminal extension that acts as an extracellular matrix retention motif. However, the exact role of PDGF-A(L) and how it functionally differs from the shorter isoform is not well understood.

Methodology/principal findings: We overexpressed PDGF-A(L) as a transgene under control of the glial fibrillary acidic protein (GFAP) promoter in the mouse brain. This directs expression of the transgene to astrocytic cells and GFAP expressing neural stem cells throughout the developing and adult central nervous system. Transgenic mice exhibited a phenotype with enlarged skull at approximately 6-16 weeks of age and they died between 1.5 months and 2 years of age. We detected an increased number of undifferentiated cells in all areas of transgene expression, such as in the subependymal zone around the lateral ventricle and in the cerebellar medulla. The cells stained positive for Pdgfr-α, Olig2 and NG2 but this population did only partially overlap with cells positive for Gfap and the transgene reporter. Interestingly, a few mice presented with overt neoplastic glioma-like lesions composed of both Olig2 and Gfap positive cell populations and with microvascular proliferation, in a wild-type p53 background.

Conclusions: Our findings show that PDGF-A(L) can induce accumulation of immature cells in the mouse brain. The strong expression of NG2, Pdgfr-α and Olig2 in PDGF-A(L) brains suggests that a fraction of these cells are oligodendrocyte progenitors. In addition, accumulation of fluid in the subarachnoid space and skull enlargement indicate that an increased intracranial pressure contributed to the observed lethality.

Figure 1. Detection and validation of transgene expression.

(A) The hGFAPp-PDGFA_L-IRES-βGeo construct as depicted in a schematic drawing. (Abbreviations: hGFAPp, human glial fibrillary acidic protein promoter; hPDGFAl, human platelet-derived growth factor A long; IRES, internal ribosome entry site; βGEO, beta-galactosidase/neomycine fusion; pA, polyadenylation signal.) (B) Results from PCR using genomic DNA from mouse tail biopsies (transgenic line, A_L8) and primers for human PDGF-A_L, resulting in a 561 base pair fragment. PCR primers unique for PDGF-A_L are situated in a way that the reverse primer corresponds to a sequence in exon 6, which is only present in PDGF-A_L, not -A_S. Controls are the hGFAPp-PDGFA_L-IRES-βGEO construct (pA_L) and DNA from wild type (wt) tails. (C) Whole mount X-gal staining of P0 transgenic and wt brains reveals activity of the transgene on the brain surface (blue color). (D–F) Presence of the β-galactosidase (β-gal) reporter in the transgenic mouse brain compared to a wt brain as visualized by IHC (brown color). A higher magnification image (F) reveals the individual β-gal+ cells, here exemplified by cells in a section from the temporal lobe of a transgenic mouse brain (40x obj.).

Figure 2. PDGF-A_L transgenic mice die from abnormalities in the brain.

(A) Skull enlargement as exemplified in PDGF-A_L transgenic mice of line 8 (A_L8). Compared to wt littermate controls there was an abnormal increase in the amount of fluid in the subarachnoid space situated between the brain surface and the skull in transgenic mice. (B) Kaplan-Meier survival curve showing the life span of transgenic mice that included data from 25 mice of line #8 and the founder mouse #12.

Figure 3. Increased cellularity in PDGF-A_L brains.

An overview of coronal sections of a PDGF-A_L transgenic mouse brain (A_L8 line, #4012) and wt littermate control (H&E staining). Higher magnification images in lower panels of selected areas from the overviews serve to illustrate areas of increased cellularity (20x and 40x objectives, respectively). A rather loose texture of the accumulating “clear” cells is indicated with arrows in pia and cerebellum. Strikingly, the borders of the inner granular layer in cerebellum appeared to be diffuse compared to wt control brain. Abbreviations: Hp-hippocampus, CC-Corpus callosum, SVZ- subventricular zone, Cblm – cerebellum.

Figure 4. Morphology of accumulating cells in the PDGF-A_L brain.

Light microscopy images of an H&E stained section, in low (A) and high (B) magnification, illustrating the presence of immature cells in the roof of the lateral ventricle in a PDGF-A_L transgenice mouse brain (A_L8 line, #4012).

Figure 5. Quantification of cell numbers and of cells expressing different lineage markers in brain areas with increased cellularity.

(A) Quantification of total cell numbers in hippocampus (Hp), subventricular zone (SVZ), corpus callosum (CC), temporal lobe area (TL), and cerebellum (Cblm). The counting is based on the comparison of one high power field (40x obj.) from each anatomical region in transgenic (A_L8) and wt mice. Mean and standard error of the mean is presented for 5 transgenic mice (A_L8) and 5 wt mice. Differences at p<0.05 were considered significant and are marked with (*). Given that the protruding lesions in Pia did not have a corresponding wt area to count it was excluded from the analysis (a.u =  area unit). (B–F) Frequencies of Ki67, Olig2, Pdgfr-α, Sox2, and Gfap immunoreactive cells in wt and transgenic mice. Quantification was based on the counting of both the total number of cells and positively stained cells for each individual marker in one high power field (40x obj.). Mean and standard error of the mean is presented for 3 transgenic mice (A_L8) and 3 wt mice. Differences at p<0.05 were considered significant and are marked with (*). P-values<0.01 are marked with (**).

Figure 6. Presence of different lineage marker proteins in brain areas with increased cellularity.

(A) IHC for β-gal and Gfap in the SVZ from an A_L8 mouse (#4012). The stainings revealed strong expression of the transgene (β-gal marker) and the location of Gfap⁺ astrocyte-like cells matched the expression of β-gal in parallel sections. Corresponding SVZ areas are shown for wt control brain (40x obj.). (B) Presence of Olig2 positive and Pdgfr-α positive cells in the SVZ (40x obj.) (C) IHC for β-gal, Gfap, Olig2 and Pdgfr-α in a pial lesion of a transgenic mouse of line A_L8 (#4016). Abbreviation: SVZ-subventricular zone.

Figure 7. Sox2 positive cells in the areas with increased cellularity.

Presence of Sox2+ cells in different regions of a transgenic mouse brain (A_L8, # 4012), as shown in the upper two panels in low (20x obj.) and high magnification (40x obj.), respectively. Shown are also corresponding regions in a wt brain in the lower two panels (20x and 40x obj.). Abbreviations: Hp-hippocampus, CC-Corpus callosum, SVZ-subventricular zone.

Figure 8. Changes in the presence of different cell lineage marker proteins and in the structure of cerebellum in PDGF-A_L transgenic mice.

(A) IHC for Calbindin (marker for Purkinje cells) and NeuN (marker for terminally differentiated neuronal cells, here granular cells) in the cerebellum of the transgenic mouse A_L8, #4016 (upper panel) and in cerebellum of a wt mouse (lower panel). (B) H&E staining and expression of Sox2 in cerebellum of a transgenic brain (A_L8, #4012) compared to corresponding areas in a wt cerebellum (40x obj) (C) H&E staining and IHC for β-gal, Gfap, Olig2 and Pdgfr-α in cerebellum from a transgenic mouse (D) IHC for Pdgfr-β cerebellum of a transgenic (A_L8 #4011) and a wt mouse (20x obj.).

Figure 9. Accumulated cells expressing oligodendrocyte progenitor markers do not generally overlap with but are mixed with β-gal and Gfap positive cells.

(A–C) Co-immunostainings for β-gal (brown color) with Olig2, Pdgfr-α and NG2 (blue color) respectively. Only a fraction of the accumulated cells showed double positivity as indicated with arrows in β-gal/Olig2 double IHC (see text for details). (D) Co-immunostaining (IHC) of Gfap (brown) and Olig2 (blue). In D the section was also counterstained with Fast Red dye (pink) (40x obj.). The tissue sections represent the SVZ from A_L8 mice (#4007 and #4008).

Figure 10. Lineage marker protein expression in brain areas with glioma-like lesions.

(A) Different areas of a transgenic mouse brain with neoplastic glioma-like lesions (A_L8, #4000) as indicated (20x obj. upper panel and 40x obj. lower panel, H&E staining). (B) IHC for β-gal, Gfap and Olig2 in the temporal lobe area of the same transgenic mouse brain as in (A). (C) IHC for β-gal, Gfap and Olig2 in the pial area of transgenic mouse brain A_L8, #4000 (20x obj. left panels and 40x obj. right panels). Abbreviations: Hp-hippocampus, CC-Corpus callosum, SVZ-subventricular zone, TL-temporal lobe.

Figure 11. Ki67 in different areas of a transgenic mouse brain with glioma-like lesions.

IHC for Ki67 in different areas of a transgenic mouse brain (A_L8, #4000) compared to similar areas in a wt mouse brain. Abbreviations: Hp-hippocampus, SVZ-subventricular zone, TL-temporal lobe, LV-left ventricular wall, and Cblm-cerebellum.