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The neurofibromatosis 2 tumor suppressor protein, merlin or schwannomin, functions as a negative growth regulator; however, its mechanism of action is not known. In an effort to determine how merlin regulates cell growth, we analyzed a recently identified novel merlin interactor, hepatocyte growth factor-regulated tyrosine kinase substrate (HRS). We demonstrate that regulated overexpression of HRS in rat schwannoma cells results in similar effects as overexpression of merlin, including growth inhibition, decreased motility and abnormalities in cell spreading. Previously, we showed that merlin forms an intramolecular association between the N- and C-termini and exists in "open" and "closed" conformations. Merlin interacts with HRS in the unfolded, or open, conformation. This HRS binding domain maps to merlin residues 453-557. Overexpression of C-terminal merlin has no effect on HRS function, arguing that merlin binding to HRS does not negatively regulate HRS growth suppressor activity. These results suggest the possibility that merlin and HRS may regulate cell growth in schwannoma cells through interacting pathways.

The neurofibromatosis 2 tumor suppressor protein schwannomin/merlin is commonly mutated in schwannomas and meningiomas. Schwannomin, a member of the 4.1 family of proteins, which are known to link the cytoskeleton to the plasma membrane, has little known function other than its ability to suppress tumor growth. Using yeast two-hybrid interaction cloning, we identified the HGF-regulated tyrosine kinase substrate (HRS) as a schwannomin interactor. We verified the interaction by both immunoprecipitation of endogenous HRS with endogenous schwannomin in vivo as well as by using bacterially purified HRS and schwannomin in vitro. We narrowed the regions of interaction to include schwannomin residues 256-579 and HRS residues from 480 to the end of either of two HRS isoforms. Schwannomin molecules with a L46R, L360P, L535P or Q538P missense mutation demonstrated reduced affinity for HRS binding. As HRS is associated with early endosomes and may mediate receptor translocation to the lysosome, we demonstrated that schwannomin and HRS co-localize at endosomes using the early endosome antigen 1 in STS26T Schwann cells by indirect immunofluorescence. The identification of schwannomin as a HRS interactor implicates schwannomin in HRS-mediated cell signaling.

NF2 is the most commonly mutated gene in benign tumours of the human nervous system. The NF2 protein, called schwannomin or merlin, is absent in virtually all schwannomas, and many meningiomas and ependymomas. Using the yeast two-hybrid system, we identified betaII-spectrin (also known as fodrin) as a schwannomin-binding protein. Interaction occurred between the carboxy-terminal domain of schwannomin isoform 2 and the ankyrin-binding region of betaII-spectrin. Isoform 1 of schwannomin, in contrast, interacted weakly with betaII-spectrin, presumably because of its strong self-interaction. Thus, alternative splicing of NF2 may regulate betaII-spectrin binding. Schwannomin co-immunoprecipitated with betaII-spectrin at physiological concentrations. The two proteins interacted in vitro and co-localized in several target tissues and in STS26T cells. Three naturally occurring NF2 missense mutations showed reduced, but not absent, betaII-spectrin binding, suggesting an explanation for the milder phenotypes seen in patients with missense mutations. STS26T cells treated with NF2 antisense oligonucleotides showed alterations of the actin cytoskeleton. Schwannomin itself lacks the actin binding sites found in ezrin, radixin and moesin, suggesting that signalling to the actin cytoskeleton occurs via actin-binding sites on betaII-spectrin. Thus, schwannomin is a tumour suppressor directly involved in actin-cytoskeleton organization, which suggests that alterations in the cytoskeleton are an early event in the pathogenesis of some tumour types.

The expression patterns of presenilin 1 (PS1) and presenilin 2 (PS2) in human normal and Alzheimer disease (AD) brains were investigated using antibodies to specific N-terminal peptides of PS1 (Alzh14A and Alzh14B) and PS2 (Alzh1A-AB). The antibodies to peptides Alzh14A (Alzh14A-AB) and Alzh14B (Alzh14B-AB) detected the full-length protein (approximately 63 kDa) and the N-terminal-processed fragment (36 kDa) of PS1, while the Alzh1A-AB detected mainly the N-terminal-processed fragment (36 kDa) of PS2. Immunofluorescent staining detected by confocal microscopy suggested that both native PS1 and PS2 are localized mainly in the Golgi/ER apparatus. Immunohistochemical studies of human temporal lobes from 2 normal and 5 sporadic Alzheimer brains revealed high levels of PS1 and PS2 expression in the granule cell layer and pyramidal neurons of the hippocampus. Strong immunoreactivity was found in reactive astrocytes and neurofibrillary tangles of all 5 Alzheimer brains. In contrast, only 2 sporadic Alzheimer brains showed presenilin-positive neuritic plaques. These observations suggest that presenilins may be involved in the pathology of some cases of sporadic AD.

In addition to schwannomas, patients with neurofibromatosis type 2 (NF2) frequently develop meningiomas and occasionally, ependymomas. Using DNA and protein analyses, we have shown NF2 gene mutations and lack of the gene product schwannomin in 29 schwannomas, 10 meningiomas, and in 7 ependymomas. We have raised antibodies (ABs) to peptides from the C-terminal (5990-AB) and N-terminal (5991-AB) domains of schwannomin. The ABs specifically detected a 65 kDa protein in a Schwann cell line and recognized schwannomin in the cytoplasm of Schwann cells (SCH), perineurial cells, and vestibular ganglion neurons. None of the 29 schwannomas were stained by the 5990-AB. Only 4 schwannomas were stained by the 5991-AB, indicating that most truncated schwannomins were unstable or not expressed in schwannomas. Seven of 10 meningiomas, including 3 tumors from NF2 patients, were not stained by either 5990-AB or 5991-AB. Only 2 of 7 ependymomas lacked schwannomin. Complete lack of schwannomin in these tumors supports a tumor suppressor function for schwannomin in some meningiomas and ependymomas. All tumors showed staining with an antibody to a C-terminal peptide of neurofibromin, confirming that full-length neurofibromin is present in these vestibular schwannomas, meningiomas, and ependymomas. The presence of schwannomin in some meningiomas and in the majority of ependymomas indicates that additional genes are likely to play a role in tumorigenesis of these tumors.

The expression pattern of presenilin 1 (PS-1) in adult mouse brain was investigated using antibodies to specific peptides of PS-1. One antibody, Alz14A, specifically detected a 53 kDa protein in retinoic acid-treated P19 cells and mouse brain protein extracts consistent with the predicted PS-1 molecular weight. Immunohistochemical staining revealed that PS-1 was localized predominantly in large neurons in areas that were known to be affected by Alzheimer disease (AD) such as the hippocampal formation, entorhinal cortex and the subiculum. Selected neurons in other regions not known to be directly affected by AD, such as thalamic nuclei, Purkinje cells, large neurons in the brainstem and the gray matter of the spinal cord, and the dorsal root ganglion, also expressed PS-1. These observations suggest that other as yet identified factors might interact with mutated presenilins to cause neurodegeneration in AD-affected areas.

Neurofibromatosis 2 (NF2) is an autosomal dominant inherited disorder that predisposes to benign tumors of the nervous system as well as a variety of ocular abnormalities. In contrast to NF1, NF2 is associated with only minor developmental abnormalities. The human NF2 gene encodes a tumor suppressor protein, termed schwannomin or merlin, which is a member of a superfamily of proteins thought to link cytoskeletal elements to cell membrane components. To determine the pattern of NF2 gene expression in mouse embryos, we sequenced the mouse NF2 gene and used in situ hybridization and antischwannomin antibodies to determine the developmental expression of the NF2 gene. Schwannomin was detected in most differentiated tissues but was undetectable in undifferentiated tissues. In particular, schwannomin was not detectable in mitotic neuroepithelial cells, the perichondrium, the liver, the neocortex, and the ventricular zone of the developing cerebral cortex. In the heart, expression was observed in all developmental stages beginning on embryonic day 8. In the eye, which shows developmental abnormalities in NF2 patients, expression was detected in the cells of the lens and in the pigment epithelium but weakly detected in retinal neurons. The most striking example of tightly regulated NF2 expression was observed in cells migrating from the ventricular zone to the cortical plate on embryonic days 15 and 16. Only cells in the intermediate zone expressed schwannomin, indicating that schwannomin may play an important role in cellular migration.

Mutations in the neurofibromatosis 2 (NF2) gene are the predominant cause in the development of sporadic schwannomas and are also involved in the pathogenesis of meningiomas and ependymomas. The product of the NF2 gene, termed merlin or schwannomin, is thought to act as a tumor suppressor protein. Although its protein sequence shows homology to proteins that are known to link the cytoskeleton to the cell membrane, no direct evidence for this function has been obtained. We used antisense phosphorothioate oligodeoxynucleotides (pODNs) complementary to the human NF2 cDNA sequence and transfected them into Schwann-like STS26T cells permeabilized by streptolysin 0. Changes in cell morphology and attachment were observed at 12 to 24 h and continued up to 48 h post transfection. Cells were rounded and easily dislodged from the substratum at 12-24 h. These changes were reversible and cells became bipolar with thin protrusions and began to reattach to the substratum after 48 h. Normal morphology and adhesion were observed at 72 h post transfection. Morphological changes were due to suppression of schwannomin synthesis. Immunoprecipitations with antischwannomin antibodies showed schwannomin to be almost absent 3 h after treatment with antisense pODNs and to be significantly suppressed up to 12 h post transfection whereas beta-actin levels remained unchanged. The morphological changes were not the result of cell death, but resulted in increased cell proliferation. These data demonstrate that antisense oligonucleotides can be successfully employed to suppress schwannomin synthesis and indicate that schwannomin may belong to a class of tumor suppressor genes that provide a link between cell adhesion and tumorigenesis.

Mutations in the neurofibromatosis type 2 (NF2) gene predispose individuals to the development of nervous system tumors and ocular abnormalities. The NF2 gene product, schwannomin, is a member of a superfamily of proteins thought to link cytoskeletal elements to cell membrane components. These proteins share significant homologies in the N-terminal and alpha-helical domains, but diverge in the C-terminus. During our efforts to characterize mouse NF2 transcripts, we identified four different transcripts by cDNA analysis and reverse-transcribed PCR that contained different sequences in the 3' end of the coding sequences. In human cell lines three isoforms encoding two distinct schwannomins were detected. The mouse and human transcripts containing 61 and 60 bp inserts, respectively, have not been previously described. The isoforms encode schwannomins with significantly altered C-termini and were expressed at different relative levels in adult mouse tissues and during mouse embryogenesis. These results suggest that schwannomin isoforms have distinct functional roles and predict the existence of human mutations involving the C-terminus of schwannomin.