Deregulation of the phosphatidylinositol-3 kinase signaling cascade is associated with neurodegeneration in Npc1-/- mouse brain

Abstract

Niemann-Pick type C (NPC) disease is caused by mutations to genes that encode proteins critical to intracellular lipid homeostasis. The events underlying NPC progressive neurodegeneration are poorly understood but include neurofibrillary tangles of the type found in Alzheimer's disease. Here we investigated possible contributions of a phosphatidylinositol-3 kinase cascade [PI3K, Akt, glycogen synthase kinase-3beta (GSK-3beta)] that is linked to apoptosis and various degenerative conditions. Brain concentrations of phosphorylated Akt, which phosphorylates and inactivates GSK-3beta, were significantly elevated in Npc1-/- mice relative to Npc1+/+ mice. Accordingly, levels of inactive GSK-3beta were 50 to 100% higher in mutant brains than in controls. Increases in inactive GSK-3beta occurred early in postnatal development, well before neuronal loss, and were most prominent in structures with intracellular cholesterol accumulation, suggesting a contribution to subsequent degeneration. Perturbations of nuclear factor (NF)-kappaB, which is regulated by GSK-3beta, occurred in Npc1-/- mouse brains. Nuclear concentrations and DNA binding activity of NF-kappaB's transactivation subunit, p65, were significantly reduced in Npc1-/- mice compared to Npc1+/+ mice. Cytoplasmic levels of the p50 subunit and its precursor, p105, were higher in Npc1-/- mice. These results suggest that excessive activity in the PI3K-Akt pathway depresses GSK-3beta, thereby disrupting the formation and/or nuclear import of p50/p65 NF-kappaB dimers and contributing to neuronal degeneration.

Figure 1

Increased Akt1 phosphorylation in developing brains of Npc1−/− mice. Samples from different brain regions (brainstem, cerebellum, neocortex, hippocampus) were collected at postnatal weeks 2 and 4 and subjected to immunoblotting analyses using anti-Akt1-Thr308, anti-Akt1-Ser473, and anti-Akt1 antibodies. The blots were then analyzed by using NIH image analysis software. A, B, and C are quantitative results of blots labeled with anti-Akt1-Thr308, anti-Akt1-Ser473, or anti-Akt1 antibodies, respectively. Data are presented as percent of values from Npc1+/+ mice; values shown in C are normalized to actin. *P < 0.05 and **P < 0.01. D: Representative images of blots labeled with anti-Akt1-Thr308 (top), anti-Akt1 (middle), or anti-actin (bottom) antibodies.

Figure 2

Changes in GSK-3 levels in brains of Npc1−/− mice during postnatal development. A, top: Representative immunoblots labeled with anti-GSK-3βSer9 antibodies (that recognize GSK-3αSer21, too) from brain tissues of 2- to 3-week-old wild-type (WT) or Npc1−/− mice. A, bottom: Representative immunoblots labeled with an anti-GSK-3β antibody that recognizes both active and inactive forms of GSK-3β (total GSK-3β). Levels of total GSK-3β were not different between wild-type and Npc1−/− mice. B–D: Quantitative results of phosphorylated GSK-3α (B) and GSK-3β (C) and total GSK-3β (D, values normalized to actin). Data are expressed as percentage of control and represent means ± SEM (n = 4 to 5 animals; **P < 0.01).

Figure 3

Distribution of GSK-3βSer9 in thalamus and neocortex of Npc1−/− mice at postnatal week 1. Coronal brain sections were prepared from 1-week-old Npc1−/− mice and immunostained with anti-GSK-3βSer9 antibodies. Staining was mainly present in layers IV and VI of auditory (AUD), visual (VIS), and somatosensory (SS) cortex, medial geniculate (MG), lateral geniculate (LG), and ventral posteromedial nucleus (VPM) of the thalamus. Inset: A higher magnification image of GSK-3βSer9-immunopositive cortical neurons. Scale bars, 40 μm.

Figure 4

Subcellular localization of GSK-3βSer9 in developing neocortex of Npc1−/− mice. GSK-3βSer9 in auditory (A–C) and motor cortex (D–F) of Npc1−/− mice at postnatal week 1 (A–E) and week 2 (F). Arrows indicate meganeurite-like structures; arrowheads indicate spines growing out of a meganeurite (B).

Figure 5

GSK-3βSer9 immunoreactivity in brains of 2- and 4-week-old Npc1−/− and wild-type mice. SS/2W: Deep layers of somatosensory cortex from a 2-week-old Npc1−/− mouse; arrow indicates a labeled axonal swelling resembling meganeurites found in human NPC neocortex. Mop/2W: Deep layers of primary motor cortex from a 2-week-old Npc1−/− mouse; arrows indicate labeled meganeurites. CeB/2W: Cerebellar cortex from a 2-week-old Npc1−/− mouse; note that numerous small cells were stained in the molecular layer (ML), but Purkinje cells (PC) were not stained. CeB/4W: GSK-3βSer9 staining in cerebellar cortex of a 4-week-old Npc1−/− mouse was located mainly in Purkinje cells. Insets show clusters of GSK-3βSer9-immunoreactive products. Hilus/4W: Npc1−/− hilar neurons at 4 weeks. VPM/2W WT, ventrolateral thalamic nuclei from a 2-week-old Npc1+/+ mouse (representative images from four 2- and 4-week-old Npc1+/+ and Npc1−/− mice). Scale bars, 40 μm.

Figure 6

Subcellular and cellular localization of GSK-3βSer9 in brains of 4-week-old Npc1−/− mice. A–C: GSK-3βSer9 (A) and cathepsin D (B) were co-localized (C) in meganeurite-like structures in cortical neurons of Npc1−/− mice. D: Confocal microscopic image showing the co-localization (yellow granules) of GSK-3βSer9 (red) and cathepsin D (green). E: GSK-3βSer9 immunoreactivity in cerebellar cortex where it was localized in small-sized cells (arrowheads and inset). F: GSK-3βSer9 (red)-immunopositive cells were not labeled by a microglial marker, F4/80 antigen (green). gl, granular layer; ml, molecular layer; pcl, Purkinje cell layer. Scale bar = 20 μm in E, 40 μm in F.

Figure 7

Co-localization of GSK-3βSer9 with cholesterol and FJB in degenerating brain areas. Brain tissue sections from 4-week-old Npc1−/− mice were first stained with filipin or FJB (both green), then processed for immunostaining with anti-GSK-3βSer9 antibody. GSK-3βSer9 immunoreactivity was revealed with Alexa Fluor 594-conjugated anti-rabbit IgG (red; for double staining with filipin) or with ABC method (for double staining with FJB). Top: Lower power images of medial geniculate nucleus. Middle and bottom: Higher power images of ventral lateral thalamic nuclei. GSK-3βSer9 is co-localized with filipin-labeled vesicles and in FJB-labeled neurons. GSK-3βSer9-immunopositive products often accumulated in meganeurite-like structures (arrows at bottom). Inset in right middle panel shows co-localization of GSK-3βSer9 with cholesterol in axonal spheroids in corpus callosum.

Figure 8

Changes in NF-κB in Npc1−/− mouse brain. A: Representative Western blots of nuclear fractions and whole homogenates prepared from brains of 2- to 3-week-old Npc1+/+ and Npc1−/− mice; blots were labeled with anti-p65 and anti-p50 antibodies. B: Quantification of Western blots labeled with anti-p65 and anti-p50 antibodies. There was a 30% decrease in levels of nuclear p65 in brains of Npc1−/− mice but not in whole homogenates (top). Levels of both p50 and its precursor (p105) increased in the cytosolic fractions and whole homogenates (bottom). **P < 0.01, n = 4 ∼ 5. No significant difference was observed with levels of nuclear p50. C: DNA binding activity assay showed a marked decrease in p65/p50 binding activity in nuclear fractions from brains of Npc1−/− mice. **P < 0.01, n = 5.

Figure 9

Negative correlation between p65 and GSK-3βSer9 immunoreactivity in brains of 4-week-old Npc1+/+ and Npc1−/− mice. Adjacent tissue sections were immunostained with anti-GSK-3βSer9 (A, B, H) or anti-p65 subunit of NF-κB (C–G). A, C, E: Tissue sections from ventral-posteromedial nuclei of thalamus (VPM) from Npc1+/+ mice. B, D, F: Tissue sections from VPM in Npc1−/− mice. G and H: Hippocampus from Npc1−/− mice. Note the loss of neuronal staining and the appearance of p65 immunoreactivity in glia in VPM from Npc1−/− mice. Note also that the loss of p65 immunostaining was obvious in VPM that exhibited high levels of GSK-3βSer9 immunoreactivity, but not in pyramidal neurons of hippocampus that showed very low levels of inactive GSK-3β.

Figure 10

Deregulation of the PI3K-Akt-GSK-3β pathway and neurodegeneration in NPC: a hypothesis. Activation of PI3K/Akt by the cytokine TNF-α or other factors, induces the phosphorylation and inactivation of GSK-3β in neurons, which in turn 1) impairs nuclear import of p65, 2) decreases the processing of p105 to p50, and 3) reduces proteasome (prot)-mediated degradation of p50. The net result would be a deficiency in p65/p50 nuclear dimers of NF-κB and decreased transcription of trophic factors, anti-apoptotic factors, and anti-oxidant protein/enzymes, which would lead to neurodegeneration. In addition, inactivation of GSK-3β may also be involved in autophagy, impairment in axonal trafficking, and formation of dystrophic neurites. On the other hand, cytokines and ATP released from damaged neurons bind to receptors that are specifically expressed in glia and activate NF-κB, which would induce glial proliferation and production of cytokines and free radicals that further facilitate neurodegeneration. Arrows indicate stimulating and ⊥ signs indicate inhibitory effects; thicker solid lines indicate enhanced, whereas broken lines indicate decreased levels of activation in NPC.