Calsenilin regulates presenilin 1/γ-secretase-mediated N-cadherin ε-cleavage and ß-catenin signaling.

Presenilin 1 (PS1) is a component of the γ-secretase complex that cleaves a variety of type I membrane proteins, including the ß-amyloid precursor protein (ß-APP), Notch, and neuronal (N)- and epithelial (E)-cadherins. N-cadherin is an essential adhesion molecule that forms a complex with, and is cleaved by, PS1/γ-secretase and ß-catenin in the plasma membrane. The purpose of this study was to determine whether calsenilin, a presenilin-interacting protein, has a functional role in PS1/γ-secretase-mediated N-cadherin ε-cleavage using Western blot analysis, RT-PCR, immunoprecipitation, subcellular fractionation, biotinylation, and a luciferase reporter assay in SH-SY5Y neuroblastoma cells. Here, we demonstrate that the expression of calsenilin leads to a disruption of PS1/γ-secretase-mediated ε-cleavage of N-cadherin, which results in the significant accumulation of N-cadherin C-terminal fragment 1 (Ncad/CTF1), the reduction of cytoplasmic Ncad/CTF2 release, and a deceleration of PS1-CTF delivery to the cell surface. Interestingly, we also found that the expression of calsenilin is associated with the redistribution of ß-catenin from the cell surface to a cytoplasmic pool, as well as with the negative regulation of genes that are targets of T-cell factor/ß-catenin nuclear signaling. Taken together, our findings suggest that calsenilin is a novel negative regulator of N-cadherin processing that plays an important role in ß-catenin signaling.

Increased neurogenesis in brains of scrapie-infected mice.

Persistent neurogenesis occurs in the adult brain throughout the life of all mammals. Recent studies have shown that neurogenesis was increased in adult gerbil and rat brains after ischemia. Neurogenesis has not been examined during neurodegenerative diseases such as scrapie. To investigate the regeneration of neurons after scrapie-infection, we infused 5-bromo-2'-deoxyuridine (BrdU), a DNA replication indicator, into both control and scrapie-infected mice. Mice were sacrificed at 150 days post-infection, i.e., at the start of clinical disease and a time when PrP(Sc) was readily detected in brain by both immunostaining and Western blot. We investigated expression of BrdU in each region of brain and observed cellular localization of BrdU using various cell markers such as neuronal nuclear (NeuN), microtubule-associated protein 2 (MAP2) and glial fibrillary acidic protein (GFAP). Immunohistochemically, BrdU-labeled cells were observed in the striatum, hippocampus, and brain stem of scrapie-infected brains. BrdU-labeled cells were much more prevalent in the hippocampus of scrapie-infected mice compared to hippocampus of control brains. In scrapie mice, there was more staining in hippocampus than in other brain regions. We also found that BrdU-positive cells colocalized with the neuronal markers NeuN and MAP2, whereas BrdU staining was not merged with GFAP, an astrocytic marker. Taken together, our results suggest that scrapie-infection induces region-specific increases in neuron regeneration.

Galectin-3 expression is correlated with abnormal prion protein accumulation in murine scrapie.

To investigate the involvement of galectin-3 in the process of neurodegeneration in prion diseases, the expression and cellular localization of galectin-3 in the brain were studied in scrapie, a mouse model of prion disease. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses showed that the expression of galectin-3 protein and mRNA was induced in scrapie-affected brains, particularly at the time when the abnormal prion protein PrP(Sc) began to accumulate in the brains. Immunohistochemically, immunostaining for galectin-3 was found mainly in B4-isolectin-positive cells (presumably activated microglia/macrophages), but not in astrocytes. Galectin-3 immunoreactivity was localized mainly in areas of PrP(Sc) accumulation and neuronal death in scrapie-infected brains. These findings suggest that the expression of galectin-3 by activated microglia/macrophages in prion disease correlates with abnormal prion protein accumulation.

Increased expression of osteopontin in the brain with scrapie infection.

The expression of osteopontin (OPN) was studied in the brains of mice with scrapie. Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis showed that the expression of OPN protein and mRNA was increased significantly in the scrapie-infected brains compared to the controls. The increased expression of OPN protein was largely matched with the PrP(Sc) accumulation. Immunohistochemically, OPN was intensely immunostained in neurons of the midbrain at the time of scrapie infection initiation. Particularly, OPN immunostaining was noted in the reactive astrocytes and some microglia in the scrapie brains, while those cells were devoid of OPN immunoreactivity in control brains. Overall, these findings suggest that some neurons affected by PrP(Sc) at an early stage of scrapie transiently express OPN but subsequently succumb to cell death at a later stage of scrapie; astroglial cells after scrapie infection are activated to express OPN; and increased OPN expression in these cells may play an important role in the pathology of scrapie. The precise role of OPN in scrapie needs further study.

Increased expression of the embryonic intermediate filament, nestin, in the brains of scrapie-infected mice.

This study investigated the immunohistochemical localization of nestin and its expression level in the brains of mice infected with scrapie, a disease which is characterized by spongiform neurodegeneration, neuronal vacuolation, and astrogliosis. Western blot analysis showed that the protein level of nestin was significantly increased in scrapie-infected brains compared with those of control brains. Nestin immunoreactivity in vascular endothelial cells was more intense in the brains of scrapie-infected mice compared with those of controls. There was marked nestin immunostaining in reactive astrocytes, but not in the neurons of the scrapie-infected brains. Considering all the findings, this study suggests that the nestin-positive cells, including glial cells and vascular endothelial cells, may have the potential to differentiate into mature glial cells and other neuronal elements, which would replenish lost neurons.

JAK-STAT signaling pathway mediates astrogliosis in brains of scrapie-infected mice.

Scrapie is characterized histologically, in part, by astrogliosis in brain and spinal cord. However, the mechanisms of astrogliosis in brain injury occurring during prion infection are not well understood. In this study, we investigated the expression levels and cellular localization of Janus kinase (JAK) -signal transducers and activators of transcription (STAT) signaling molecules and growth factors such as leukemia inhibitory factor (LIF) and ciliary neurotropic factor (CNTF) by western blot analysis and immunohistochemistry. We found that expression levels of LIF and CNTF were increased in scrapie-infected brains and phosphorylated (p)-JAK2, p-STAT1 (Ser727 and Tyr701), p-STAT3 (Tyr705), and glial fibrillary acidic protein were expressed strongly in scrapie-infected brains. Moreover, we found that p-STAT1 and p-STAT3 were found mainly in the nucleus in scrapie-infected brains. Immunohistochemically, p-STAT1 was colocalized with LIF and CNTF and p-JAK2 in many reactive astrocytes in scrapie-infected brains. In contrast, immunostaining for p-STAT3 was found in comparatively few astrocytes in limited regions; p-STAT3 staining merged with p-JAK2 in hippocampus sections of scrapie-infected brains. Taken together, our results suggest that activation of JAK2-STAT1 signaling pathway occurred in reactive astrocytes in hippocampus of scrapie-infected brains.

Phospholipase D1 is associated with amyloid precursor protein in Alzheimer's disease.

Amyloid precursor protein (APP) is a widely expressed transmembrane protein of unknown function that is involved in the pathogenesis of Alzheimer's disease (AD). We investigated the involvement of phospholipase D (PLD) in the pathophysiology of AD. We showed dramatic upregulation of PLD1 immunoreactivity in reactive astroglial cells in brain tissue sections from authentic AD patients. Expression and activity of PLD1 were up-regulated in brain tissues from AD patients, especially caveolae membrane fraction, compared with those of control brains. Interestingly, PLD1 physically interacts and colocalizes with APP and caveolin-3. We found that APP was associated with the pleckstrin homology domain of PLD1, and the amyloid region of APP interacted with PLD. Elevated expression of APP stimulated PLD activity in human astroglioma cells. These results suggest that up-regulation of PLD might have a role in the neuronal pathology associated with AD.