12 research outputs found

    Inhibition of prostate cancer proliferation by interference with SONIC HEDGEHOG-GLI1 signaling

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    Prostate cancer is the most common solid tumor in men, and it shares with all cancers the hallmark of elevated, nonhomeostatic cell proliferation. Here we have tested the hypothesis that the SONIC HEDGEHOG (SHH)ā€“GLI signaling pathway is implicated in prostate cancer. We report expression of SHHā€“GLI pathway components in adult human prostate cancer, often with enhanced levels in tumors versus normal prostatic epithelia. Blocking the pathway with cyclopamine or anti-SHH antibodies inhibits the proliferation of GLI1(+)/PSA(+) primary prostate tumor cultures. Inversely, SHH can potentiate tumor cell proliferation, suggesting that autocrine signaling may often sustain tumor growth. In addition, pathway blockade in three metastatic prostate cancer cell lines with cyclopamine or through GLI1 RNA interference leads to inhibition of cell proliferation, suggesting cell-autonomous pathway activation at different levels and showing an essential role for GLI1 in human cells. Our data demonstrate the dependence of prostate cancer on SHHā€“GLI function and suggest a novel therapeutic approach

    Hyperactivity and Hypermotivation Associated With Increased Striatal mGluR1 Signaling in a Shank2 Rat Model of Autism

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    Mutations in the SHANK family of genes have been consistently identified in genetic and genomic screens of autism spectrum disorder (ASD). The functional overlap of SHANK with several other ASD-associated genes suggests synaptic dysfunction as a convergent mechanism of pathophysiology in ASD. Although many ASD-related mutations result in alterations to synaptic function, the nature of those dysfunctions and the consequential behavioral manifestations are highly variable when expressed in genetic mouse models. To investigate the phylogenetic conservation of phenotypes resultant of Shank2 loss-of-function in a translationally relevant animal model, we generated and characterized a novel transgenic rat with a targeted mutation of the Shank2 gene, enabling an evaluation of gene-associated phenotypes, the elucidation of complex behavioral phenotypes, and the characterization of potential translational biomarkers. The Shank2 loss-of-function mutation resulted in a notable phenotype of hyperactivity encompassing hypermotivation, increased locomotion, and repetitive behaviors. Mutant rats also expressed deficits in social behavior throughout development and in the acquisition of operant tasks. The hyperactive phenotype was associated with an upregulation of mGluR1 expression, increased dendritic branching, and enhanced long-term depression (LTD) in the striatum but opposing morphological and cellular alterations in the hippocampus (HP). Administration of the mGluR1 antagonist JNJ16259685 selectively normalized the expression of striatally mediated repetitive behaviors and physiology but had no effect on social deficits. Finally, Shank2 mutant animals also exhibited alterations in electroencephalography (EEG) spectral power and event-related potentials, which may serve as translatable EEG biomarkers of synaptopathic alterations. Our results show a novel hypermotivation phenotype that is unique to the rat model of Shank2 dysfunction, in addition to the traditional hyperactive and repetitive behaviors observed in mouse models. The hypermotivated and hyperactive phenotype is associated with striatal dysfunction, which should be explored further as a targetable mechanism for impairment in ASD

    A platform to investigate the neuronal determinants of <i>APOE </i>genotype on neuron function

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    Alzheimerā€™s disease (AD), the most prevalent form of dementia worldwide, affects a growing proportion of the elderly and poses a significant societal burden. Pathological hallmarks include the appearance of misfolded proteins in the brain. This is evidenced by the deposition of amyloid-Ī² (AĪ²) plaques and tau-containing neurofibrillary tangles leading to neuron loss.Advanced age is the single most important factor that leads to the dementia associated with AD. Additionally, genetic factors underlie the severity and/or age of onset. APOE &#x1d700;4 is the strongest genetic risk factor for sporadic AD. Subtle changes in the gene lead to three common variants: APOE e4, APOE e3, and APOE e2. In terms of AD, e4 is associated with increased while e2 is associated with decreased incidence compared with &#x1d700;3. The protein from this gene, apolipoprotein E (ApoE), is involved in numerous functions in the brain including cholesterol homeostasis and clearance of AĪ². Functional complexity is increased as ApoE is primarily synthesized and secreted from astrocytes while neuronal expression has also been identified in the context of injury, damage, or stress. ApoE is a secreted molecule, which raises questions regarding which cell source and what fundamental functions of ApoE modulate AD susceptibility. Is there a neuron-intrinsic disease trigger and/or disproportionately high neuron-autonomous contribution to AD?I compared isogenic cell lines in which the genome is engineered to carry homozygosity for each of the three APOE alleles listed above. I interrogated the impact of genotype on neuronal differentiation, morphology, and potential variation in vulnerability to stress. This latter aspect was modeled by exposing mature cultures to kainate-induced excitotoxic insult. These approaches revealed that APOE had little impact on neuron differentiation, except for subtly affecting connectivity-related gene expression in the e4/e4 versus neurons of the other two genotypes. Upon kainic acid exposure, e4/e4 neurons again showed a small difference; compared to e3/e3 and e2/e2 neurons, there was a delay in mounting an ATF4 response, a stress-protectivemechanism. No other differential response to excitotoxicity emerged. Although these neurons did show a detectable low level of APOE transcript, there was no upregulation of APOE upon kainate exposure. My established neuronal platform of APOE variants is now available for further studies to more deeply probe genotype-specific mechanistic differences based on my initial observations.These subtle but clear changes in e4-related altered neurodevelopment and delayed stress response suggest that the e4 allele may have limited effect early in life. However, over multiple stress events that may occur over a lifetime, these changes could provoke neurodegeneration. Another implication is that because e2/e2 neurons did not simply show the opposite phenotype of &#x1d700;4 neurons, the neuroprotective mechanism of e2 does not overlap with the pathways responsible for &#x1d700;4ā€™s detrimental effects

    Dataset supporting the University of Southampton Doctoral Thesis &quot;A platform to investigate the neuronal determinants of APOE genotype on neuron function&quot;.

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    Dataset supporting the University of Southampton Doctoral Thesis &quot;A platform to investigate the neuronal determinants of APOE genotype on neuron function&quot;. The dataset includes raw data for RT-PCR, NanoString, ATP levels for iPSC derived neurons 4-week timecourse and 18-hour KA treatment. The data is presented in Excel and CSV files. </span

    Inhibition of prostate cancer proliferation by interference with SONIC HEDGEHOG-GLI1 signaling

    No full text
    Prostate cancer is the most common solid tumor in men, and it shares with all cancers the hallmark of elevated, nonhomeostatic cell proliferation. Here we have tested the hypothesis that the SONIC HEDGEHOG (SHH)-GLI signaling pathway is implicated in prostate cancer. We report expression of SHH-GLI pathway components in adult human prostate cancer, often with enhanced levels in tumors versus normal prostatic epithelia. Blocking the pathway with cyclopamine or anti-SHH antibodies inhibits the proliferation of GLI1+/PSA+ primary prostate tumor cultures. Inversely, SHH can potentiate tumor cell proliferation, suggesting that autocrine signaling may often sustain tumor growth. In addition, pathway blockade in three metastatic prostate cancer cell lines with cyclopamine or through GLI1 RNA interference leads to inhibition of cell proliferation, suggesting cell-autonomous pathway activation at different levels and showing an essential role for GLI1 in human cells. Our data demonstrate the dependence of prostate cancer on SHH-GLI function and suggest a novel therapeutic approach

    Class I HDAC inhibition is a novel pathway for regulating astrocytic apoE secretion

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    <div><p>Despite the important role of apolipoprotein E (apoE) secretion from astrocytes in brain lipid metabolism and the strong association of apoE4, one of the human apoE isoforms, with sporadic and late onset forms of Alzheimerā€™s disease (AD) little is known about the regulation of astrocytic apoE. Utilizing annotated chemical libraries and a phenotypic screening strategy that measured apoE secretion from a human astrocytoma cell line, inhibition of pan class I histone deacetylases (HDACs) was identified as a mechanism to increase apoE secretion. Knocking down select HDAC family members alone or in combination revealed that inhibition of the class I HDAC family was responsible for enhancing apoE secretion. Knocking down LXRĪ± and LXRĪ² genes revealed that the increase in astrocytic apoE in response to HDAC inhibition occurred via an LXR-independent pathway. Collectively, these data suggest that pan class I HDAC inhibition is a novel pathway for regulating astrocytic apoE secretion.</p></div

    A pan class I HDAC inhibitor increases astrocytic apoE expression and secretion differently from a prototypic pan LXR agonist.

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    <p>Concentration-dependent effect of MS275 (o) and TO901317 (ā—), on secreted apoE protein (<i>A</i>) and relative levels of apoE mRNA (<i>B</i>), ABCA1 mRNA (<i>C</i>), LXRĪ± mRNA (<i>D</i>) and LXRĪ² mRNA (<i>E</i>) in CCF-STTG1 cells. The amount of apoE protein secreted into the media and the relative mRNA levels of apoE, ABCA1, LXRĪ± and LXRĪ² after compound treatment were normalized to those obtained from vehicle treated controls. The average of GAPDH and 18S mRNA were used as endogenous controls for mRNA normalization. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194661#pone.0194661.s010" target="_blank">S3 Table</a> for gene expression assays. TO901317 and MS275 were synthesized at Pfizer and their binding to LXRĪ± (<i>F</i>) and LXRĪ² (<i>G</i>) was determined using the Lanthascreen assay (Life Technologies). All data are represented as values Ā±SEM.</p

    Table_1_Hyperactivity and Hypermotivation Associated With Increased Striatal mGluR1 Signaling in a Shank2 Rat Model of Autism.xlsx

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    <p>Mutations in the SHANK family of genes have been consistently identified in genetic and genomic screens of autism spectrum disorder (ASD). The functional overlap of SHANK with several other ASD-associated genes suggests synaptic dysfunction as a convergent mechanism of pathophysiology in ASD. Although many ASD-related mutations result in alterations to synaptic function, the nature of those dysfunctions and the consequential behavioral manifestations are highly variable when expressed in genetic mouse models. To investigate the phylogenetic conservation of phenotypes resultant of Shank2 loss-of-function in a translationally relevant animal model, we generated and characterized a novel transgenic rat with a targeted mutation of the Shank2 gene, enabling an evaluation of gene-associated phenotypes, the elucidation of complex behavioral phenotypes, and the characterization of potential translational biomarkers. The Shank2 loss-of-function mutation resulted in a notable phenotype of hyperactivity encompassing hypermotivation, increased locomotion, and repetitive behaviors. Mutant rats also expressed deficits in social behavior throughout development and in the acquisition of operant tasks. The hyperactive phenotype was associated with an upregulation of mGluR1 expression, increased dendritic branching, and enhanced long-term depression (LTD) in the striatum but opposing morphological and cellular alterations in the hippocampus (HP). Administration of the mGluR1 antagonist JNJ16259685 selectively normalized the expression of striatally mediated repetitive behaviors and physiology but had no effect on social deficits. Finally, Shank2 mutant animals also exhibited alterations in electroencephalography (EEG) spectral power and event-related potentials, which may serve as translatable EEG biomarkers of synaptopathic alterations. Our results show a novel hypermotivation phenotype that is unique to the rat model of Shank2 dysfunction, in addition to the traditional hyperactive and repetitive behaviors observed in mouse models. The hypermotivated and hyperactive phenotype is associated with striatal dysfunction, which should be explored further as a targetable mechanism for impairment in ASD.</p

    Image_2_Hyperactivity and Hypermotivation Associated With Increased Striatal mGluR1 Signaling in a Shank2 Rat Model of Autism.TIF

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    <p>Mutations in the SHANK family of genes have been consistently identified in genetic and genomic screens of autism spectrum disorder (ASD). The functional overlap of SHANK with several other ASD-associated genes suggests synaptic dysfunction as a convergent mechanism of pathophysiology in ASD. Although many ASD-related mutations result in alterations to synaptic function, the nature of those dysfunctions and the consequential behavioral manifestations are highly variable when expressed in genetic mouse models. To investigate the phylogenetic conservation of phenotypes resultant of Shank2 loss-of-function in a translationally relevant animal model, we generated and characterized a novel transgenic rat with a targeted mutation of the Shank2 gene, enabling an evaluation of gene-associated phenotypes, the elucidation of complex behavioral phenotypes, and the characterization of potential translational biomarkers. The Shank2 loss-of-function mutation resulted in a notable phenotype of hyperactivity encompassing hypermotivation, increased locomotion, and repetitive behaviors. Mutant rats also expressed deficits in social behavior throughout development and in the acquisition of operant tasks. The hyperactive phenotype was associated with an upregulation of mGluR1 expression, increased dendritic branching, and enhanced long-term depression (LTD) in the striatum but opposing morphological and cellular alterations in the hippocampus (HP). Administration of the mGluR1 antagonist JNJ16259685 selectively normalized the expression of striatally mediated repetitive behaviors and physiology but had no effect on social deficits. Finally, Shank2 mutant animals also exhibited alterations in electroencephalography (EEG) spectral power and event-related potentials, which may serve as translatable EEG biomarkers of synaptopathic alterations. Our results show a novel hypermotivation phenotype that is unique to the rat model of Shank2 dysfunction, in addition to the traditional hyperactive and repetitive behaviors observed in mouse models. The hypermotivated and hyperactive phenotype is associated with striatal dysfunction, which should be explored further as a targetable mechanism for impairment in ASD.</p

    Pan class I HDAC inhibition increases astrocytic apoE secretion independent of LXR activation.

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    <p>Specific siRNA oligonucleotides were used to knock down LXRĪ± (Ī±KD) and LXRĪ² (Ī²KD) either separately or in combination (Ī±Ī²KD) in CCF-STTG1 cells (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194661#pone.0194661.s009" target="_blank">S2 Table</a> for siRNA oligonucleotides). Concentration-dependent effect of exposure to TO901317 on apoE protein secreted into the media (<i>A</i>); and on apoE mRNA levels (<i>B</i>) upon knocking down LXRĪ± and LXRĪ² separately or in combination. Concentration-dependent effect of exposure to MS275 on relative apoE protein secreted into the media (<i>C</i>); and on apoE mRNA levels (<i>D</i>) upon knocking down LXRĪ± and LXRĪ² separately or in combination. Concentration-dependent effect of exposure to TO901317 (<i>E</i>) and MS275 (<i>F</i>) on relative ABCA1 mRNA level upon knocking down LXRĪ± and LXRĪ² separately and in combination. (<i>G</i>) Concentration-dependent response of apoE protein secreted from primary human astrocytes (lot number 06589) after exposure to MS275 upon knocking down LXRĪ± and LXRĪ² separately or in combination. For all, levels were normalized to the respective vehicle treated control. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194661#pone.0194661.s006" target="_blank">S6 Fig</a> for knock-down efficiencies of LXRĪ± and LXRĪ² genes and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194661#pone.0194661.s010" target="_blank">S3 Table</a> for gene expression assays. All data are represented as values Ā±SEM.</p
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