Transcriptional inhibition of type I collagen gene expression in scleroderma fibroblasts by the antineoplastic drug ecteinascidin 743.

We previously showed that COL1A1 expression is up-regulated at the transcriptional level in systemic sclerosis (SSc) fibroblasts and that the CCAAT-binding factor (CBF) is involved in this increased expression. Ecteinascidin 743 (ET-743) is a chemotherapeutic agent that binds with sequence specificity to the minor groove of DNA and inhibits CBF-mediated transcriptional activation of numerous genes. Therefore, we examined the effects of ET-743 on the increased COL1A1 expression in SSc fibroblasts. The drug caused a potent and dose-dependent inhibition of type I collagen biosynthesis, which reached 70-90% at 700 pM without affecting cell viability. The same drug concentration caused 60-80% reduction in COL1A1 mRNA levels. The stability of the corresponding transcripts was not affected. In vitro nuclear transcription assays demonstrated a 54% down-regulation of COL1A1 transcription. Transient transfections with COL1A1 promoter constructs containing the specific CBF binding sequence into SSc cells previously treated with 700 pM ET-743 failed to show an effect on COL1A1 promoter activity. Furthermore, ET-743 did not affect the binding of CBF or Sp1 transcription factors to their cognate COL1A1 elements. However, treatment with 700 pM ET-743 of stably transfected NIH 3T3 cells expressing a human type II procollagen gene under the control of the human COL1A1 promoter caused a greater than 50% reduction in the production of type II procollagen and a similar decrease in the corresponding type II procollagen transcripts. These results indicate that ET-743 is a potent inhibitor of COL1A1 transcription. However, this effect cannot be explained by a direct effect on CBF binding to the COL1A1 promoter. Although the exact mechanisms responsible for the transcriptional inhibition of COL1A1 by ET-743 are not apparent, our observations suggest that the drug may be an effective agent to decrease collagen overproduction in SSc and other fibrotic diseases

Dysbindin-1 in dorsolateral prefrontal cortex of schizophrenia cases is reduced in an isoform-specific manner unrelated to altered dysbindin-1 gene expression

DTNBP1 (dystrobrevin binding protein 1) remains one of the top candidate genes in schizophrenia. Reduced expression of this gene and the protein it encodes, dysbindin-1, has been reported in the dorsolateral prefrontal cortex (DLPFC) of schizophrenia cases. It has not been established, however, if all dysbindin-1 isoforms are reduced in the DLPFC or if the reduction is associated with reduced DTNBP1 gene expression. Using Western blotting of whole-tissue lysates of the DLPFC with antibodies differentially sensitive to the three major isoforms of this protein (dysbindin-1A, -1B, and -1C), we found no significant differences between our schizophrenia cases and matched controls in dysbindin-1A or -1B, but did find a mean 46% reduction in dysbindin-1C in 71% of 28 case-control pairs (p = 0.022). This occurred in the absence of the one DTNBP1 risk haplotype for schizophrenia reported in the US and without alteration in levels of dysbindin-1C transcripts. Conversely, the absence of changes in the dysbindin-1A and -1B isoforms was accompanied by increased levels of their transcripts. We thus found no correspondence between alterations in dysbindin-1 gene and protein expression, the latter of which might be due to posttranslational modifications such as ubiquitination. Reduced DLPFC dysbindin-1C in schizophrenia probably occurs in PSDs, where we find dysbindin-1C to be heavily concentrated in the human brain. Given known postsynaptic effects of dysbindin-1 reductions in the rodent homolog of the prefrontal cortex, these findings suggest that reduced dysbindin-1C in the DLPFC may contribute to cognitive deficits of schizophrenia by promoting NMDA receptor hypofunction

PDE-4 inhibition rescues aberrant synaptic plasticity in Drosophila and mouse models of fragile X syndrome.

Fragile X syndrome (FXS) is the leading cause of both intellectual disability and autism resulting from a single gene mutation. Previously, we characterized cognitive impairments and brain structural defects in a Drosophila model of FXS and demonstrated that these impairments were rescued by treatment with metabotropic glutamate receptor (mGluR) antagonists or lithium. A well-documented biochemical defect observed in fly and mouse FXS models and FXS patients is low cAMP levels. cAMP levels can be regulated by mGluR signaling. Herein, we demonstrate PDE-4 inhibition as a therapeutic strategy to ameliorate memory impairments and brain structural defects in the Drosophila model of fragile X. Furthermore, we examine the effects of PDE-4 inhibition by pharmacologic treatment in the fragile X mouse model. We demonstrate that acute inhibition of PDE-4 by pharmacologic treatment in hippocampal slices rescues the enhanced mGluR-dependent LTD phenotype observed in FXS mice. Additionally, we find that chronic treatment of FXS model mice, in adulthood, also restores the level of mGluR-dependent LTD to that observed in wild-type animals. Translating the findings of successful pharmacologic intervention from the Drosophila model into the mouse model of FXS is an important advance, in that this identifies and validates PDE-4 inhibition as potential therapeutic intervention for the treatment of individuals afflicted with FXS

Synaptic Dysbindin-1 Reductions in Schizophrenia Occur in an Isoform-Specific Manner Indicating Their Subsynaptic Location

Background: An increasing number of studies report associations between variation in DTNBP1, a top candidate gene in schizophrenia, and both the clinical symptoms of the disorder and its cognitive deficits. DTNBP1 encodes dysbindin-1, reduced levels of which have been found in synaptic fields of schizophrenia cases. This study determined whether such synaptic reductions are isoform-specific. Methodology/Principal Findings: Using Western blotting of tissue fractions, we first determined the synaptic localization of the three major dysbindin-1 isoforms (A, B, and C). All three were concentrated in synaptosomes of multiple brain areas, including auditory association cortices in the posterior half of the superior temporal gyrus (pSTG) and the hippocampal formation (HF). Tests on the subsynaptic tissue fractions revealed that each isoform is predominantly, if not exclusively, associated with synaptic vesicles (dysbindin-1B) or with postsynaptic densities (dysbindin-1A and -1C). Using Western blotting on pSTG (n = 15) and HF (n = 15) synaptosomal fractions from schizophrenia cases and their matched controls, we discovered that synaptic dysbindin-1 is reduced in an isoform-specific manner in schizophrenia without changes in levels of synaptophysin or PSD-95. In pSTG, about 92% of the schizophrenia cases displayed synaptic dysbindin-1A reductions averaging 48% (p = 0.0007) without alterations in other dysbindin-1 isoforms. In the HF, by contrast, schizophrenia cases displayed normal levels of synaptic dysbindin-1A, but 67% showed synaptic reductions in dysbindin-1B averaging 33% (p = 0.0256), while 80% showed synaptic reductions in dysbindin-1C averaging 35% (p = 0.0171). Conclusions/Significance: Given the distinctive subsynaptic localization of dysbindin-1A, -1B, and -1C across brain regions, the observed pSTG reductions in dysbindin-1A are postsynaptic and may promote dendritic spine loss with consequent disruption of auditory information processing, while the noted HF reductions in dysbindin-1B and -1C are both presynaptic and postsynaptic and could promote deficits in spatial working memory

Inhibition of systemic sclerosis dermal fibroblast type I collagen production and gene expression by simvastatin

OBJECTIVE: To examine whether statins are capable of modulating collagen gene expression in cultured systemic sclerosis dermal fibroblasts. METHODS: Cultured dermal fibroblasts from 3 patients with diffuse systemic sclerosis of recent onset were treated with 5 microM and 10 microM of simvastatin for 3 or 4 days. Morphologic features, cytotoxicity, and type I collagen production and messenger RNA (mRNA) levels in the fibroblasts were examined. The effects of mevalonate, geranylgeranyl pyrophosphate (GGPP), and farnesyl pyrophosphate (FPP), which are lipids downstream from the hydroxymethylglutaryl-coenzyme A block, were also examined. Transient transfections with COL1A1 promoter-reporter constructs and electrophoretic gel mobility shift assays were utilized to examine COL1A1 transcription and Sp1 and CCAAT-box binding factor (CBF) binding. RESULTS: Simvastatin did not cause morphologic changes or cytotoxicity in the fibroblasts, even after 4 days of treatment. Type I collagen production and mRNA levels showed a potent and dose-related inhibition following 3 and 4 days of treatment. The inhibition of collagen gene expression by simvastatin was completely reversed by mevalonate and GGPP, but not by FPP. The statin effects occurred at the transcriptional level and involved the proximal COL1A1 promoter region encompassing -174 bp. A significant reduction in Sp1 and CBF binding activity was also found in simvastatin-treated cells. CONCLUSION: Simvastatin is a powerful inhibitor of type I collagen gene expression in normal and systemic sclerosis fibroblasts. The pleiotropic protective effects of statins on various endothelial and immune cell functions in conjunction with their potent inhibitory effects on type I collagen gene expression suggest that statins may be effective therapeutic agents in systemic sclerosis

Caspase-3 Is Enriched in Postsynaptic Densities and Increased in Alzheimer’s Disease

Progressive synaptic degeneration and neuron loss are major structural correlates of cognitive impairment in Alzheimer’s disease (AD). The mechanisms by which synaptic degeneration in AD occurs have not been established. The activation of proteins within the caspase family has been implicated in AD-associated neurodegeneration, and synaptically localized caspase activity could play a role in the synaptic degeneration and loss found in AD. We used synaptosomal fractionation with Western blotting and immunohistochemistry to examine the anatomical, subcellular, and subsynaptic expression patterns of caspase 3 in both the anterior cingulate cortex and hippocampus of control and AD patients. In both control and AD cases, there was a selective enrichment of caspase- 3 at synapses, particularly in the postsynaptic density (PSD) fractions. Compared with controls, AD patients exhibited significant increases in synaptic procaspase- 3 and active caspase-3 expression levels that were most evident in the PSD fractions. These data demonstrate for the first time the preferential localization and increase of caspase-3 in the PSD fractions in AD and suggest an important role for caspase 3 in synapse degeneration during disease progression