Neural mechanisms underlying stress resilience in Ahi1 knockout mice: relevance to neuropsychiatric disorders.

The Abelson helper integration site 1 (AHI1) gene has a pivotal role in brain development. Studies by our group and others have demonstrated association of AHI1 with schizophrenia and autism. To elucidate the mechanism whereby alteration in AHI1 expression may be implicated in the pathogenesis of neuropsychiatric disorders, we studied Ahi1 heterozygous knockout (Ahi1(+/-)) mice. Although their performance was not different from wild-type mice on tests that model classical schizophrenia-related endophenotypes, Ahi1(+/-) mice displayed an anxiolytic-like phenotype across different converging modalities. Using behavioral paradigms that involve exposure to environmental and social stress, significantly decreased anxiety was evident in the open field, elevated plus maze and dark-light box, as well as during social interaction in pairs. Assessment of core temperature and corticosterone secretion revealed a significantly blunted response of the autonomic nervous system and the hypothalamic-pituitary-adrenal axis in Ahi1(+/-) mice exposed to environmental and visceral stress. However, response to centrally acting anxiogenic compounds was intact. On resting-state functional MRI, connectivity of the amygdala with other brain regions involved in processing of anxiogenic stimuli and inhibitory avoidance learning, such as the lateral entorhinal cortex, ventral hippocampus and ventral tegmental area, was significantly reduced in the mutant mice. Taken together, our data link Ahi1 under-expression with a defect in the process of threat detection. Alternatively, the results could be interpreted as representing an anxiety-related endophenotype, possibly granting the Ahi1(+/-) mouse relative resilience to various types of stress. The current knockout model highlights the contribution of translational approaches to understanding the genetic basis of emotional regulation and its associated neurocircuitry, with possible relevance to neuropsychiatric disorders.

Cell death in cultured human Saos2 osteoblasts exposed to low-density lipoprotein.

Osteoporosis (OP) and atherosclerotic-cardiovascular diseases (and possibly dementia) constitute emerging age-related co-morbidity states that might share risk factors. Blood-born lipids, like LDL involved in atherosclerosis and apolipoprotein-E4 (ApoE4) involved in dementia, may also be implicated in development of OP. We examined osteoblast cell lines as a culture model for OP by exposure to lipoproteins. ApoE expression in Saos2 and U2OS osteoblasts was confirmed by PCR. ApoE4 did decrease cell counts relatively to ApoE3, especially in Saos2 cells in which it was less selective for cells with higher alkaline phosphatase (ALP, an osteoblast marker) activity than ApoE3. This associates with ApoE4, being a risk factor for both dementia and OP. Saos2, but not U2OS, showed a decrease in cell counts after 48 h exposure to native LDL (NLDL). Both cell lines had decreased cell counts already after 24 h when exposed to oxidized-LDL (OxLDL) for which Saos2 also showed a higher sensitivity than U2OS. Exposure of Saos2 to both, OxLDL at low concentration (5 microg/ml) and NLDL revealed a shrunken size cell fraction of 17-23% on the fluorescence-activated cell sorter (FACS) analysis. Such shrunken cell fraction was not seen when Saos2 cells were exposed to 50 microg/ml of OxLDL or to OxLDL combined with 10 nM dexamethasone (DEX, a stimulator of osteoprogenitor differentiation). DEX treatment has lysed the cells earlier than 24 h post exposure and has selected more resistant cells that did not show apoptotic shrinkage in the FACS analysis done after 24 h. We interpret this as a failure to detect the apoptotic cell fraction due to their lysis prior to the FACS analysis. Western blots performed at different time points (10 min, 30 min, 4 h, 24 h, and 48 h) under OxLDL + DEX revealed a fall in the positive regulator of pp60Src-kinase phosphotyrosine (pY)418 relative to the DEX controls during the first 4 h. This is consistent with DEX osteogenic induction, known to be negatively regulated by c-Src, although the pY418/pY529 ratios (negative/positive kinase regulation) fell only at the 10 min time point. Contrarily the pY418/pY529 ratio increased, relative to untreated controls, under 5 microg/ml and 50 microg/ml of NLDL at the 4 h time point and under 50 microg/ml NLDL only at the 10 min time point, being consistent with the ability of a higher dose of LDL to antagonize osteoblast differentiation. This could be even more acceptable if the NLDL would have become minimally oxidized during its long purification procedure. Under NLDL, the Bcl-2/Bax ratio was pro-apoptotic at 10 min, 30 min, and 4 h only under 50 microg/ml, whereas under OxLDL + DEX it was pro-apoptotic only after 4 h suggesting that additional pathways contribute to cell death. These results indicate that lipid effects on human osteoblast lines in culture may be used as a model to identify molecular targets shared between OP and atherosclerosis for intervention in this co-morbidity.

Expression of the familial Mediterranean fever gene and activity of the C5a inhibitor in human primary fibroblast cultures.

Familial Mediterranean fever (FMF) is an inherited disease whose manifestations are acute but reversible attacks of sterile inflammation affecting synovial and serosal spaces. The FMF gene (MEFV) was recently cloned, and it codes for a protein (pyrin/marenostrin) homologous to known nuclear factors. We previously reported the deficient activity of a C5a/interleukin (IL)-8 inhibitor, a physiologic regulator of inflammatory processes, in FMF serosal and synovial fluids. We now describe the concomitant expression of MEFV and C5a/IL-8-inhibitor activity in primary cultures of human fibroblasts. Fibroblasts grown from synovial and peritoneal tissues displayed C5a/IL-8-inhibitor activity that could be further induced with phorbol myristate acetate (PMA) and IL-1 beta. Very low levels of chemotactic inhibitor were evident in skin fibroblast cultures or in peritoneal and skin fibroblasts obtained from FMF patients. MEFV was expressed in peritoneal and skin fibroblasts at a lower level than in neutrophils and could be further induced by PMA and IL-1 beta. In the FMF cultures, the MEFV transcript carried the M694V mutation, consistent with the genetic defect found in patients with this disease. MEFV was also expressed in other cell lines that do not produce C5a/IL-8 inhibitor. These findings suggest that human primary fibroblast cultures express MEFV and produce C5a/IL-8-inhibitor activity. The interrelationship between pyrin, the MEFV product, and the C5a/IL-8 inhibitor requires further investigation. (Blood. 2000;96:727-731)