Phosphatase and tensin homolog, deleted on chromosome 10 deficiency in brain causes defects in synaptic structure, transmission and plasticity, and myelination abnormalities

Abstract

The phosphatidylinositol 3-kinase (PI3K) signaling pathway modulates growth, proliferation and cell survival in diverse tissue types and plays specialized roles in the nervous system including influences on neuronal polarity, dendritic branching and synaptic plasticity. The tumor-suppressor phosphatase with tensin homology (PTEN) is the central negative regulator of the PI3K pathway. Germline PTEN mutations result in cancer predisposition, macrocephaly and benign hamartomas in many tissues, including Lhermitte-Duclos disease, a cerebellar growth disorder. Neurological abnormalities including autism, seizures and ataxia have been observed in association with inherited PTEN mutation with variable penetrance. It remains unclear how loss of PTEN activity contributes to neurological dysfunction. To explore the effects of Pten deficiency on neuronal structure and function, we analyzed several ultra-structural features of Pten-deficient neurons in Pten conditional knockout mice. Using Golgi stain to visualize full neuronal morphology, we observed that increased size of nuclei and somata in Pten-deficient neurons was accompanied by enlarged caliber of neuronal projections and increased dendritic spine density. Electron microscopic evaluation revealed enlarged abnormal synaptic structures in the cerebral cortex and cerebellum. Severe myelination defects included thickening and unraveling of the myelin sheath surrounding hypertrophic axons in the corpus callosum. Defects in myelination of axons of normal caliber were observed in the cerebellum, suggesting intrinsic abnormalities in Pten-deficient oligodendrocytes. We did not observe these abnormalities in wild-type or conditional Pten heterozygous mice. Moreover, conditional deletion of Pten drastically weakened synaptic transmission and synaptic plasticity at excitatory synapses between CA3 and CA1 pyramidal neurons in the hippocampus. These data suggest that Pten is involved in mechanisms that control development of neuronal and synaptic structures and subsequently synaptic function.

Fig. 1

Hypertrophy of Pten cKO neurons was associated with increased ribosome density and increased nucleolar size. A,B. Electron microscopy of neurons in layer 5 of the cerebral cortex of control (A) and Pten cKO (B) mice. The boundary of the cell membrane with extra cellular matrix is less defined in the Pten cKO tissue compared with the control. C,D. Electron microscopy of the boxed areas from Figure 1A and 1B of control (C) and Pten cKO mice (D). The nucleus and mitochondria are labeled with an N and arrow, respectively. There is a dramatic increase in ribosome density and mitochondrial size in the Pten cKO (D) mice compared with the control (C). The increased soma size is indicated with red color and an S in between two arrows. E. The area of nucleoli in cerebral cortex neurons in control (white bar) and Pten cKO mice (black bar) was measured. Nucleoli were enlarged in the Pten cKO mice compared with the controls. F. The number of nucleoli per cell in control (white bar) and Pten cKO (black bar) mice were counted. There was no significant difference in the number of nucleoli per cell between control and Pten cKO mice. G,H. H&E stained sections of cerebral cortex pyramidal neurons from control (G) and Pten cKO mice (H). The increased nucleolar size is apparent in the Pten cKO mice. Scale bar for A,B=5 μm, C,D=1μm and G,H=10μm. Mice were >10 weeks old. Paired t-test P values for E=0.0108 and F=0.4773.

Fig. 2

Neuronal hypertrophy in Pten cKO mice. A,B. Golgi stained neurons in the cerebral cortex of adult control (A) and Pten cKO (B) mice. Hypertrophy of Pten cKO neurons (B) involved the somata and all projections as compared to control neurons (A). C,D. At higher magnification, dendritic branches from control (C) and Pten cKO (D) mice are shown. The dendritic spines (arrow) in the control animal are spaced out along the dendrite and contain the rounded termini characteristic of mature spines. The spines (arrow) in the Pten cKO mice are densely packed and many lack the terminal rounded structure observed in the control spines. Mice were >10 weeks old. Scale bar for A,B=50μm and for C,D=10μm.

Fig. 3

Abnormal synaptic connections in Pten cKO mice. A,B,C,D. Electron micrographs of the cerebral cortex from control (A,C) and Pten cKO mice (B,D). In the control mice, the pre- (PS1) and post- (PS2) synaptic terminals are visible. They are labeled with brown and green color, respectively. The pre-synaptic terminal is identified by the presence of synaptic vesicles. The postsynaptic density (black arrow) is also clearly visible in the control cortex. In the Pten cKO mice (B), the majority of the structures observed were enlarged pre-synaptic terminals, which contained a large number of synaptic vesicles (PS1) with no visible postsynaptic density. A second type of synapse was observed which had a postsynaptic density (black arrow) that extended past the region of the pre-synaptic terminal (D). E,F. Immunofluorescent staining with synaptophysin in the cerebral cortex of control (E) and Pten cKO mice (F) shows increased synaptophysin expression in the Pten cKO mice. Mice were >10 weeks old. Scale bar for A,B,C,D=0.3μm and E,F=10μm.

Fig. 4

Structural abnormalities in Pten-deficient cerebellar granule neurons. A,B. Electron micrographs of the inner granule layer of control (A) and Pten cKO (B) mice. There is a significant enlargement of the granule neurons in the Pten cKO neurons compared with the control. The increased soma size is indicated with red color. These cells also have an increased ribosome density in the Pten cKO mice. C,D,E,F. Electron micrographs of the molecular layer from control (C,E) and Pten cKO (D,F) mice. Normal synapses containing a postsynaptic density (arrow) and pre-synaptic terminals containing synaptic vesicles (arrow head) are shown. The pre and post-synaptic terminals are indicated with brown and green color, respectively. The synapses in the Pten cKO (D,F) mice showed greatly enlarged pre-synaptic terminals with increased synaptic vesicles (arrow head) and postsynaptic densities (arrow) that engulfed the Purkinje cells. One pre-synaptic terminal is able to synapse with multiple post-synaptic terminals (F). Mice were >10 weeks old. Scale bar for A,B=2μm, C,D=0.3μm and E,F=0.5μm.

Fig. 5

Synaptic transmission and synaptic plasticity are defective in Pten cKO mice. A. Mean fEPSPs as a function of stimulation intensity in Pten cKO mice and wild-type littermates. B. Pair-pulse ratio of two mean fEPSPs as a function of interstimulus interval measured in slices from Pten cKO and WT mice. C. Average fEPSPs vs time before and after 200 Hz tetanizations measured in Pten cKO and WT mice (downward arrows). Mice were 3.5 to 4 weeks of age.

Fig. 6

Abnormal myelination in Pten cKO mouse brains. A,B,C,D. Electron micrographs at two magnifications of the corpus callosum from adult control (A,C) and Pten cKO mice (B,D). Myelin thickness is increased and the myelin sheath is unraveling in Pten cKO mice compared with control mice. Mice were >10 weeks old. Scale bar for A,B=1μm and C,D=0.5μm.

Fig. 7

Cre activity in oligodendrocytes leads to hypertrophy. A,B,C. Immunofluorescence in the corpus callosum of adult GFAP-cre;ROSA26R mice shows β-Gal expression (A), CC-1 (B) and the overlay of the two images (C). D,E,F. Immunofluorescence in the white matter tracks of the cerebellum of adult GFAP-cre;ROSA26R brains showing β-Gal (D) and CC-1 (E), and the overlapping images (F). G,H. CC-1 immunofluorescence in the corpus callosum control (G) and Pten cKO mice (H). A population of CC-1 positive cells in the Pten cKO mice are significantly enlarged compared with the control CC-1 positive cells in the same region. I,J. CC-1 immunofluorescence in the white matter track of the cerebellum of control (I) and Pten cKO mice (J). Again, a population of oligodendrocytes are significantly enlarged in size in the Pten cKO mice. Mice were 4 weeks of age for A,B,C,D,E,F and mice were >10 weeks old for G,H,I,J. Scale bar for all=30μm.

Fig. 8

Pten loss in oligodendrocytes leads to cell autonomous defects in myelination in the cerebellum. A,B. Pten immunohistochemistry shows loss of Pten in granule neurons of the IGL (arrow head), and persistent Pten expression in Purkinje cells (arrows) from Pten cKO cerebellum (B) compared to control (A). C,D. Electron micrographs showing myelination in the cerebellar white matter tracks of control (A) and Pten cKO (B). Mice were >10 weeks old. Scale bar for A,B=50μm and C,D=1μm.