Tumors derived from rat C6 cell implants into rat brain exhibit similar morphological characteristics and degree of vascularization to human glioblastomas. To establish a molecular basis for C6 gliosarcoma malignancy, we have constructed a molecular profile of the most abundantly expressed genes, using serial analysis of gene expression (SAGE). Sequence tags (1168) representing 738 individual transcripts were collected and tag-to-gene mapping was carried out using the UniGene data set for rat. Differentially expressed C6 transcripts were identified by comparison of tags collected for C6 cells with a similar number (1002) of tags from a rat primary astrocyte library. Genes found to be expressed at increased levels in C6 cells are associated with cell surface interactions, migration, or metastasis formation and proliferation. These include the receptor for hyaluronan-mediated motility (RHAMM), S-100 related protein 42A, galectin I, preproenkephalin, osteopontin, autocrine motility factor, alpha-tubulin, ad1 antigen, and cofilin. In addition, a tag with no database match probably representing a previously uncharacterized transcript was differentially expressed in C6 cells. Transcripts showing reduced expression in C6 cells relative to astrocytes included the extracellular matrix glycoprotein osteonectin/SPARC (secreted protein, acidic, rich in cysteine), actin-binding proteins thymosins beta-4 and beta-10, the cysteine protease inhibitor cystatin C, the actin-gelling protein SM22/transgelin, and ferritin-H. SAGE results were confirmed by Northern blot for all transcripts tested, reaffirming the value of the SAGE technique for expression profiling in cancer biology.

The Sez6 family consists of Sez6, Sez6L, and Sez6L2. Its members are expressed throughout the brain and have been shown to influence synapse numbers and dendritic morphology. They are also linked to various neurological and psychiatric disorders. All Sez6 family members contain 2-3 CUB domains and 5 complement control protein (CCP) domains, suggesting that they may be involved in complement regulation. We show that Sez6 family members inhibit C3b/iC3b opsonization by the classical and alternative pathways with varying degrees of efficacy. For the classical pathway, Sez6 is a strong inhibitor, Sez6L2 is a moderate inhibitor, and Sez6L is a weak inhibitor. For the alternative pathway, the complement inhibitory activity of Sez6, Sez6L, and Sez6L2 all equaled or exceeded the activity of the known complement regulator MCP. Using Sez6L2 as the representative family member, we show that it specifically accelerates the dissociation of C3 convertases. Sez6L2 also functions as a cofactor for Factor I to facilitate the cleavage of C3b; however, Sez6L2 has no cofactor activity toward C4b. In summary, the Sez6 family are novel complement regulators that inhibit C3 convertases and promote C3b degradation.

BACE inhibitors, which decrease BACE1 (β-secretase 1) cleavage of the amyloid precursor protein, are a potential treatment for Alzheimer’s disease. Clinical trials using BACE inhibitors have reported a lack of positive effect on patient symptoms and, in some cases, have led to increased adverse events, cognitive worsening and hippocampal atrophy. A potential drawback of this strategy is the effect of BACE inhibition on other BACE1 substrates such as Seizure-related gene 6 (Sez6) family proteins which are known to have a role in neuronal function. Mice were treated with an in-diet BACE inhibitor for 4–8 weeks to achieve a clinically-relevant level of amyloid-β40 reduction in the brain. Mice underwent behavioural testing and postmortem analysis of dendritic spine number and morphology with Golgi-Cox staining. Sez6 family triple knockout mice were tested alongside wild-type mice to identify whether any effects of the treatment were due to altered cleavage of Sez6 family proteins. Wild-type mice treated with BACE inhibitor displayed hyperactivity on the elevated open field, as indicated by greater distance travelled, but this effect was not observed in treated Sez6 triple knockout mice. BACE inhibitor treatment did not lead to significant changes in spatial or fear learning, reference memory, cognitive flexibility or anxiety in mice as assessed by the Morris water maze, context fear conditioning, or light–dark box tests. Chronic BACE inhibitor treatment reduced the density of mushroom-type spines in the somatosensory cortex, regardless of genotype, but did not affect steady-state dendritic spine density or morphology in the CA1 region of the hippocampus. Chronic BACE inhibition for 1–2 months in mice led to increased locomotor output but did not alter memory or cognitive flexibility. While the mechanism underlying the treatment-induced hyperactivity is unknown, the absence of this response in Sez6 triple knockout mice indicates that blocking ectodomain shedding of Sez6 family proteins is a contributing factor. In contrast, the decrease in mature spine density in cortical neurons was not attributable to lack of shed Sez6 family protein ectodomains. Therefore, other BACE1 substrates are implicated in this effect and, potentially, in the cognitive decline in longer-term chronically treated patients.