Genetic Ablation of Nrf2/Antioxidant Response Pathway in Alexander Disease Mice Reduces Hippocampal Gliosis but Does Not Impact Survival

In Alexander disease (AxD) the presence of mutant glial fibrillary acidic protein (GFAP), the major intermediate filament of astrocytes, triggers protein aggregation, with marked induction of a stress response mediated by the transcription factor, Nrf2. To clarify the role of Nrf2 in AxD, we have crossed Gfap mutant and transgenic mouse models into an Nrf2 null background. Deletion of Nrf2 eliminates the phase II stress response normally present in mouse models of AxD, but causes no change in body weight or lifespan, even in a severe lethal model. AxD astrocytes without Nrf2 retain features of reactivity, such as expression of the endothelin-B receptor, but have lower Gfap levels, a decrease in p62 protein and reduced iron accumulation, particularly in hippocampus. Microglial activation, indicated by Iba1 expression, is also diminished. Although the Nrf2 response is generally considered beneficial, these results show that in the context of AxD, loss of the antioxidant pathway has no obvious negative effects, while actually decreasing Gfap accumulation and pathology. Given the attention Nrf2 is receiving as a potential therapeutic target in AxD and other neurodegenerative diseases, it will be interesting to see whether induction of Nrf2, beyond the endogenous response, is beneficial or not in these same models

BRAF V600E Mutations Are Common in Pleomorphic Xanthoastrocytoma: Diagnostic and Therapeutic Implications

Pleomorphic xanthoastrocytoma (PXA) is low-grade glial neoplasm principally affecting children and young adults. Approximately 40% of PXA are reported to recur within 10 years of primary resection. Upon recurrence, patients receive radiation therapy and conventional chemotherapeutics designed for high-grade gliomas. Genetic changes that can be targeted by selective therapeutics have not been extensively evaluated in PXA and ancillary diagnostic tests to help discriminate PXA from other pleomorphic and often more aggressive astrocytic malignancies are limited. In this study, we apply the SNaPshot multiplexed targeted sequencing platform in the analysis of brain tumors to interrogate 60 genetic loci that are frequently mutated in 15 cancer genes. In our analysis we detect BRAF V600E mutations in 12 of 20 (60%) WHO grade II PXA, in 1 of 6 (17%) PXA with anaplasia and in 1 glioblastoma arising in a PXA. Phospho-ERK was detected in all tumors independent of the BRAF mutation status. BRAF duplication was not detected in any of the PXA cases. BRAF V600E mutations were identified in only 2 of 71 (2.8%) glioblastoma (GBM) analyzed, including 1 of 9 (11.1%) giant cell GBM (gcGBM). The finding that BRAF V600E mutations are common in the majority of PXA has important therapeutic implications and may help in differentiating less aggressive PXAs from lethal gcGBMs and GBMs

BRAF V600E Mutations Are Common in Pleomorphic Xanthoastrocytoma: Diagnostic and Therapeutic Implications

Pleomorphic xanthoastrocytoma (PXA) is low-grade glial neoplasm principally affecting children and young adults. Approximately 40% of PXA are reported to recur within 10 years of primary resection. Upon recurrence, patients receive radiation therapy and conventional chemotherapeutics designed for high-grade gliomas. Genetic changes that can be targeted by selective therapeutics have not been extensively evaluated in PXA and ancillary diagnostic tests to help discriminate PXA from other pleomorphic and often more aggressive astrocytic malignancies are limited. In this study, we apply the SNaPshot multiplexed targeted sequencing platform in the analysis of brain tumors to interrogate 60 genetic loci that are frequently mutated in 15 cancer genes. In our analysis we detect BRAF V600E mutations in 12 of 20 (60%) WHO grade II PXA, in 1 of 6 (17%) PXA with anaplasia and in 1 glioblastoma arising in a PXA. Phospho-ERK was detected in all tumors independent of the BRAF mutation status. BRAF duplication was not detected in any of the PXA cases. BRAF V600E mutations were identified in only 2 of 71 (2.8%) glioblastoma (GBM) analyzed, including 1 of 9 (11.1%) giant cell GBM (gcGBM). The finding that BRAF V600E mutations are common in the majority of PXA has important therapeutic implications and may help in differentiating less aggressive PXAs from lethal gcGBMs and GBMs

A multimodal approach to improving human papillomavirus vaccination in a community pharmacy setting

Background: Community pharmacy has become a major access point for several types of vaccinations. Despite the success of vaccination programs like influenza, pneumococcal, and herpes zoster, the rates of human papillomavirus vaccination continue to lag. Objectives: The primary objective is to describe and report on the impact of a multimodal series of pharmacist-led educational interventions on human papillomavirus vaccination rates in a community pharmacy setting. The primary outcome of this study was change in pharmacist-delivered human papillomavirus vaccination throughout a corresponding 8-week period in 2014 and 2015. Methods: A single-center, quasi-experimental interrupted time series mixed-methods pilot study was used to investigate a pharmacist-led, multimodal educational intervention approach to improve human papillomavirus vaccination rates in the community. Results: During the 2014 control period, there were no human papillomavirus vaccines dispensed or administered according to the internal prescription dispensing software. In 2015, a total of 10 patients indicated that they were vaccinated, with 9 patients receiving their first dose and 1 patient receiving his or her second dose at the pharmacy. Pharmacist recommendation was the most reported education method for increasing patient awareness of the human papillomavirus vaccine (n = 10). Conclusion: This study demonstrates pharmacist designed, educational interventions may impact human papillomavirus vaccination rates in the community. Further community-based research with larger sample sizes is warranted to verify these results. Due to the unique barriers to human papillomavirus vaccination, a multimodal and inter-professional approach such as the one presented here is warranted

Knockout of Nrf2 in Gfap mutant or transgenic mice eliminates ARE (antioxidant response element) activation.

<p>(<b>A</b>) Quantitation of brain transcripts from Nrf2 and its target gene Nqo1 for both GFAP^Tg and Gfap^+/R236H mice (3 mos of age) shows reduced expression in Nrf2^+/− background and further reduction in Nrf2^−/−. (<b>C, D</b>) Histochemical staining for alkaline phosphatase activity in GFAP^Tg mice crossed with an ARE-alkaline phosphatase reporter line shows wide-spread activation of Nrf2 (<b>C</b>), whereas GFAP^Tg/Nrf2^−/− mice show virtually no ARE-reporter activity (<b>D</b>; 6 wks of age). (<b>B</b>) Quantification of reporter activity in brains from either GFAP^Tg or GFAP^+/R236H mice shows a marked reduction in Nrf2^+/+ vs. Nrf2^+/− mice and virtually no detectable activity in Nrf2^−/− animals (3 mos of age). Error bars =  standard deviation. For Gfap^+/+ versus Gfap^+/R236H or GFAP^Tg comparisons (all Nrf2^+/+), significance is indicated with a (⁺) plus sign. For Nrf2^+/+ vs Nrf2^+/− or ^−/− comparisons, significance is indicated by an (*) asterisk (unpaired t-test: ^{+ or} *p<0.05; ^{++ or} **p<0.01; ^{+++ or} ***p<0.001, n≥3).</p

Primer sets for quantitative PCR.

<p>Primer sets for quantitative PCR.</p

Microglia are less reactive in Gfap^+/R236H mice without Nrf2.

<p>(<b>A</b>) Compared to wild-type mice, Gfap mutant mice show elevated Iba1 immunoreactivity in hippocampus either with or without Nrf2. (<b>B</b>) Western analysis of hippocampal protein shows reduced Iba1 expression in Gfap^+/R236H/Nrf2^−/− compared with Gfap^+/R236H/Nrf2^+/+ mice (8 wks), although levels remain elevated above wild-type (**p<0.01, unpaired t-test comparing Gfap^+/R236H/Nrf2^+/+ with Gfap^+/R236H/Nrf2^−/− mice, n = 4). Lanes 5 and 6 for Nrf2^−/− samples are from a different region of the same gel as lanes 1–4 (Nrf2^+/+ samples).</p

Iron accumulation in astrocytes from Gfap mutants is reduced with loss of Nrf2.

<p>(<b>A</b>) Modified Perl’s stain shows iron in small round cell bodies (brown) with oligodendrocyte morphology separate from Gfap immunostained astrocytes (blue/green) in Gfap^+/+ olfactory bulb, whereas iron staining is prominent in astrocytes with reactive morphology in Gfap^+/R236H mice and in some cases appears to localize with Gfap aggregates (3 mos of age). (<b>B</b>) Ferritin immunofluorescence shows occasional colocalization with Mac1 (hippocampus, 3 mos of age) suggesting some iron storage in microglia. (<b>C-H</b>) Iron histochemistry shows accumulation in astrocytes of both olfactory bulb (<b>C-E</b>) and hippocampus (<b>F-H</b>) in Gfap^+/R236H mice (<b>D, G</b>) compared with Gfap^+/+ brains (<b>C, F</b>). Loss of Nrf2 reduces iron staining in Gfap^+/R236H/Nrf2^−/− mice (<b>E, H</b>). (<b>I</b>-<b>N</b>) Immunostaining for ferritin (red) and Gfap (green) in olfactory bulb (<b>I-K</b>) and hippocampus (<b>L-N</b>) shows ferritin labeling in Gfap negative cells with oligodendrocyte morphology, especially in olfactory bulb (<b>I</b>) and sparse labeling in hippocampus (<b>L</b>) in wild-type mice at 8 wks of age. In Gfap^+/R236H/Nrf2^+/+ mice, ferritin colocalizes with Gfap in astrocytes in both olfactory bulb (<b>J</b>) and hippocampus (<b>M</b>). Gfap^+/R236H/Nrf2^−/− mice (<b>K, N</b>) show an intermediate pattern with less staining in Gfap positive cells, and in olfactory bulb (<b>K</b>), cells that appear to be oligodendrocytes. Scale bars = 25 µm (<b>A</b>); 50 µm (<b>B, </b><b>I</b>).</p

p62 in Gfap^+/R236H mice colocalizes with Gfap aggregates and is reduced with loss of Nrf2.

<p>(<b>A</b>) p62 immunohistochemistry in hippocampus from Gfap^+/R236H/Nrf2^+/+ mice shows astrocytic cells that are not apparent in Gfap^+/+ mice. Gfap^+/R236H/Nrf2^−/− mice show similarly stained cells (3 mos of age). (<b>B</b>) Confocal microscopy with immunofluorescence for p62 (green) shows colocalization with Gfap aggregates (red) in both Nrf2^+/+ and Nrf2^−/− Gfap^+/R236H mice (8 wks of age). (<b>C</b>) Western blot analysis of hippocampal protein shows increased p62 in Gfap mutant mice compared to wild-type, with Gfap^+/R236H/Nrf2^−/− mice having levels reduced to that of wild-type mice (8 wks of age, n≥4, t-test **p>0.01, comparing Gfap^+/R236H/Nrf2^+/+ with Gfap^+/R236H/Nrf2^−/−). Scale bar = 25 µm (shown in <b>B</b>, applies to both confocal images).</p