Studying synapses in human brain with array tomography and electron microscopy

Postmortem studies of synapses in human brain are problematic due to the axial resolution limit of light microscopy and the difficulty preserving and analyzing ultrastructure with electron microscopy. Array tomography overcomes these problems by embedding autopsy tissue in resin and cutting ribbons of ultrathin serial sections. Ribbons are imaged with immunofluorescence, allowing high-throughput imaging of tens of thousands of synapses to assess synapse density and protein composition. The protocol takes approximately 3 days per case, excluding image analysis, which is done at the end of the study. Parallel processing for transmission electron microscopy (TEM) using a protocol modified to preserve structure in human samples allows complimentary ultrastructural studies. Incorporation of array tomography and TEM into brain banking is a potent way of phenotyping synapses in well-characterized clinical cohorts to develop clinico-pathological correlations at the synapse level. This will be important for research in neurodegenerative disease, developmental diseases, and psychiatric illness

The androgen receptor can signal through Wnt/β-Catenin in prostate cancer cells as an adaptation mechanism to castration levels of androgens

<p>Abstract</p> <p>Background</p> <p>A crucial event in Prostate Cancer progression is the conversion from a hormone-sensitive to a hormone-refractory disease state. Correlating with this transition, androgen receptor (AR) amplification and mutations are often observed in patients failing hormonal ablation therapies. β-Catenin, an essential component of the canonical Wnt signaling pathway, was shown to be a coactivator of the AR signaling in the presence of androgens. However, it is not yet clear what effect the increased levels of the AR could have on the Wnt signaling pathway in these hormone-refractory prostate cells.</p> <p>Results</p> <p>Transient transfections of several human prostate cancer cell lines with the AR and multiple components of the Wnt signaling pathway demonstrate that the AR overexpression can potentiate the transcriptional activities of Wnt/β-Catenin signaling. In addition, the simultaneous activation of the Wnt signaling pathway and overexpression of the AR promote prostate cancer cell growth and transformation at castration levels of androgens. Interestingly, the presence of physiological levels of androgen or other AR agonists inhibits these effects. These observations are consistent with the nuclear co-localization of the AR and β-Catenin shown by immunohistochemistry in human prostate cancer samples. Furthermore, chromatin immunoprecipitation assays showed that Wnt3A can recruit the AR to the promoter regions of Myc and Cyclin D1, which are well-characterized downstream targets of the Wnt signalling pathway. The same assays demonstrated that the AR and β-Catenin can be recruited to the promoter and enhancer regions of a known AR target gene PSA upon Wnt signaling. These results suggest that the AR is promoting Wnt signaling at the chromatin level.</p> <p>Conclusion</p> <p>Our findings suggest that the AR signaling through the Wnt/β-Catenin pathway should be added to the well established functional interactions between both pathways. Moreover, our data show that via this interaction the AR could promote prostate cell malignancy in a ligand-independent manner.</p

The ongoing pursuit of neuroprotective therapies in Parkinson disease

Many agents developed for neuroprotective treatment of Parkinson disease (PD) have shown great promise in the laboratory, but none have translated to positive results in patients with PD. Potential neuroprotective drugs, such as ubiquinone, creatine and PYM50028, have failed to show any clinical benefits in recent high-profile clinical trials. This 'failure to translate' is likely to be related primarily to our incomplete understanding of the pathogenic mechanisms underlying PD, and excessive reliance on data from toxin-based animal models to judge which agents should be selected for clinical trials. Restricted resources inevitably mean that difficult compromises must be made in terms of trial design, and reliable estimation of efficacy is further hampered by the absence of validated biomarkers of disease progression. Drug development in PD dementia has been mostly unsuccessful; however, emerging biochemical, genetic and pathological evidence suggests a link between tau and amyloid-β deposition and cognitive decline in PD, potentially opening up new possibilities for therapeutic intervention. This Review discusses the most important 'druggable' disease mechanisms in PD, as well as the most-promising drugs that are being evaluated for their potential efficiency in treatment of motor and cognitive impairments in PD

The androgen receptor can signal through Wnt/β-Catenin in prostate cancer cells as an adaptation mechanism to castration levels of androgens-3

<p><b>Copyright information:</b></p><p>Taken from "The androgen receptor can signal through Wnt/β-Catenin in prostate cancer cells as an adaptation mechanism to castration levels of androgens"</p><p>http://www.biomedcentral.com/1471-2121/9/4</p><p>BMC Cell Biology 2008;9():4-4.</p><p>Published online 24 Jan 2008</p><p>PMCID:PMC2246119.</p><p></p>thout Wnt3A 100 ng/ml for four days in 96 well plates. The cells were then labeled with [H]-thymidine and harvested to measure the thymidine incorporation. Cells that received no DHT treatment were used as control. The percentage of [H]-thymidine incorporation was calculated compared with control. (B). LNCaP and LNCaP-Flag-AR cells were plated in soft agar with no treatment as control or with 100 ng/ml Wnt3A, or 10 nM DHT. After approximately 4 weeks, colonies were fixed with 10% formaldehyde in PBS. A representative field of cells was photographed for each cell type, with or without treatment using bright-field microscopy. Upper panel LNCaP or LNCaP-Flag-AR cells received no treatment, while lower panel, LNCaP-Flag-AR cells were either treated with Wnt3A or DHT

The androgen receptor can signal through Wnt/β-Catenin in prostate cancer cells as an adaptation mechanism to castration levels of androgens-4

<p><b>Copyright information:</b></p><p>Taken from "The androgen receptor can signal through Wnt/β-Catenin in prostate cancer cells as an adaptation mechanism to castration levels of androgens"</p><p>http://www.biomedcentral.com/1471-2121/9/4</p><p>BMC Cell Biology 2008;9():4-4.</p><p>Published online 24 Jan 2008</p><p>PMCID:PMC2246119.</p><p></p>n normal prostate tissues, the low levels of AR expression, compared with the high levels in prostate cancer cells, were indicated by lack of AR staining in cells (A); β-Catenin was predominantly located at the normal cytoplasmic membrane (B). In late stage prostate cancer samples, AR was substantially overexpressed in the nuclei of the prostate cancer cells (C) where nuclear β-Catenin staining was also observed in some of these cells (D)

The androgen receptor can signal through Wnt/β-Catenin in prostate cancer cells as an adaptation mechanism to castration levels of androgens-1

<p><b>Copyright information:</b></p><p>Taken from "The androgen receptor can signal through Wnt/β-Catenin in prostate cancer cells as an adaptation mechanism to castration levels of androgens"</p><p>http://www.biomedcentral.com/1471-2121/9/4</p><p>BMC Cell Biology 2008;9():4-4.</p><p>Published online 24 Jan 2008</p><p>PMCID:PMC2246119.</p><p></p>lates. 7 days after transfection, the number of cells was determined with a Guava proliferation assay. Cells transfected with GFP were used as control. The percentage of cell numbers was calculated for cells transfected with Wnt or AR or both compared with control samples. (B) AR expression and Flag-tagged AR in LNCaP-Flag-AR cells and LNCaP cells were detected by Western blot. Actin was used as loading control. (C). LNCaP cells and LNCaP-Flag-AR cells in phenol-red free RPMI with 5% charcoal-stripped serum were treated with 100 ng/ml Wnt3A for four days in 96 well plates. The cells were then labelled with [H]-thymidine and harvested to measure the amount of thymidine incorporation. LNCaP cells which received no Wnt3A treatment were used as control. The percentage of [H]-thymidine incorporation was calculated compared with control

The androgen receptor can signal through Wnt/β-Catenin in prostate cancer cells as an adaptation mechanism to castration levels of androgens-5

<p><b>Copyright information:</b></p><p>Taken from "The androgen receptor can signal through Wnt/β-Catenin in prostate cancer cells as an adaptation mechanism to castration levels of androgens"</p><p>http://www.biomedcentral.com/1471-2121/9/4</p><p>BMC Cell Biology 2008;9():4-4.</p><p>Published online 24 Jan 2008</p><p>PMCID:PMC2246119.</p><p></p>6 hours before cross-linking. Anti-β-Catenin (A, B, G and H) and anti-AR (C, D, E and F) antibodies, together with negative control IgG were then used for immunoprecipitation. After reverse crosslinking and DNA purification, PCR products were analyzed using the Agilent 2100 bioanalyzer. Percent of input is shown here to compare the levels of β-Catenin or AR at the promoter or enhancer region of PSA, or the promoter region of Cyclin D1 or Myc