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Platelet-derived growth factor-BB (PDGF-BB):PDGF receptor-β (PDGFRβ) signalling in brain pericytes is critical to the development, maintenance and function of a healthy blood-brain barrier (BBB). Furthermore, BBB impairment and pericyte loss in Alzheimer’s disease (AD) is well documented. We found that PDGF-BB:PDGFRβ signalling components were altered in human AD brains, with a marked reduction in vascular PDGFB. We hypothesised that reduced PDGF-BB:PDGFRβ signalling in pericytes may impact on the BBB. We therefore tested the effects of PDGF-BB on primary human brain pericytes in vitro to define pathways related to BBB function. Using pharmacological inhibitors, we dissected distinct aspects of the PDGF-BB response that are controlled by extracellular signal-regulated kinase (ERK) and Akt pathways. PDGF-BB promotes the proliferation of pericytes and protection from apoptosis through ERK signalling. In contrast, PDGF-BB:PDGFRβ signalling through Akt augments pericyte-derived inflammatory secretions. It may therefore be possible to supplement PDGF-BB signalling to stabilise the cerebrovasculature in AD.

In situ hybridization (ISH) is a powerful tool that can be used to localize mRNA expression in tissue samples. Combining ISH with immunohistochemistry (IHC) to determine cell type provides cellular context of mRNA expression, which cannot be achieved with gene microarray or polymerase chain reaction. To study mRNA and protein expression on the same section we investigated the use of RNAscope® ISH in combination with fluorescent IHC on paraffin-embedded human brain tissue. We first developed a high-throughput, automated image analysis workflow for quantifying RNA puncta across the total cell population and within neurons identified by NeuN+ immunoreactivity. We then applied this automated analysis to tissue microarray (TMA) sections of middle temporal gyrus tissue (MTG) from neurologically normal and Alzheimer's Disease (AD) cases to determine the suitability of three commonly used housekeeping genes: ubiquitin C (UBC), peptidyl-prolyl cis-trans isomerase B (PPIB) and DNA-directed RNA polymerase II subunit RPB1 (POLR2A). Overall, we saw a significant decrease in total and neuronal UBC expression in AD cases compared to normal cases. Total expression results were validated with RT-qPCR using fresh frozen tissue from 5 normal and 5 AD cases. We conclude that this technique combined with our novel automated analysis pipeline provides a suitable platform to study changes in gene expression in diseased human brain tissue with cellular and anatomical context. Furthermore, our results suggest that UBC is not a suitable housekeeping gene in the study of post-mortem AD brain tissue.

Both in adult and children, high-grade gliomas (WHO grades III and IV) account for a high proportion of death due to cancer. This poor prognosis is a direct consequence of tumor recurrences occurring within few months despite a multimodal therapy consisting of a surgical resection followed by chemotherapy and radiotherapy. There is increasing evidence that glioma stem cells (GSCs) contribute to tumor recurrences. In fact, GSCs can migrate out of the tumor mass and reach the subventricular zone (SVZ), a neurogenic niche persisting after birth. Once nested in the SVZ, GSCs can escape a surgical intervention and resist to treatments. The present review will define GSCs and describe their similarities with neural stem cells, residents of the SVZ. The architectural organization of the SVZ will be described both for humans and rodents. The migratory routes taken by GSCs to reach the SVZ and the signaling pathways involved in their migration will also be described hereafter. In addition, we will debate the advantages of the microenvironment provided by the SVZ for GSCs and how this could contribute to tumor recurrences. Finally, we will discuss the clinical relevance of the SVZ in adult GBM and pediatric HGG and the therapeutic advantages of targeting that neurogenic region in both clinical situations.

Background Microglia play critical roles in the brain during homeostasis and pathological conditions. Understanding the molecular events underpinning microglial functions and activation states will further enable us to target these cells for the treatment of neurological disorders. The transcription factor PU.1 is critical in the development of myeloid cells and a major regulator of microglial gene expression. In the brain, PU.1 is specifically expressed in microglia and recent evidence from genome-wide association studies suggests that reductions in PU.1 contribute to a delayed onset of Alzheimer’s disease (AD), possibly through limiting neuroinflammatory responses. Methods To investigate how PU.1 contributes to immune activation in human microglia, microarray analysis was performed on primary human mixed glial cultures subjected to siRNA-mediated knockdown of PU.1. Microarray hits were confirmed by qRT-PCR and immunocytochemistry in both mixed glial cultures and isolated microglia following PU.1 knockdown. To identify attenuators of PU.1 expression in microglia, high throughput drug screening was undertaken using a compound library containing FDA-approved drugs. NanoString and immunohistochemistry was utilised to investigate the expression of PU.1 itself and PU.1-regulated mediators in primary human brain tissue derived from neurologically normal and clinically and pathologically confirmed cases of AD. Results Bioinformatic analysis of gene expression upon PU.1 silencing in mixed glial cultures revealed a network of modified AD-associated microglial genes involved in the innate and adaptive immune systems, particularly those involved in antigen presentation and phagocytosis. These gene changes were confirmed using isolated microglial cultures. Utilising high throughput screening of FDA-approved compounds in mixed glial cultures we identified the histone deacetylase inhibitor vorinostat as an effective attenuator of PU.1 expression in human microglia. Further characterisation of vorinostat in isolated microglial cultures revealed gene and protein changes partially recapitulating those seen following siRNA-mediated PU.1 knockdown. Lastly, we demonstrate that several of these PU.1-regulated genes are expressed by microglia in the human AD brain in situ. Conclusions Collectively, these results suggest that attenuating PU.1 may be a valid therapeutic approach to limit microglial-mediated inflammatory responses in AD and demonstrate utility of vorinostat for this purpose. Electronic supplementary material The online version of this article (10.1186/s13024-018-0277-1) contains supplementary material, which is available to authorized users.

Cancer cells are continually exposed to environmental stressors forcing them to adapt their protein production to survive. The translational machinery can be recruited by malignant cells to synthesize proteins required to promote their survival, even in times of high physiological and pathological stress. This phenomenon has been described in several cancers including in gliomas. Abnormal regulation of translation has encouraged the development of new therapeutics targeting the protein synthesis pathway. This approach could be meaningful for glioma given the fact that the median survival following diagnosis of the highest grade of glioma remains short despite current therapy. The identification of new targets for the development of novel therapeutics is therefore needed in order to improve this devastating overall survival rate. This review discusses current literature on translation in gliomas with a focus on the initiation step covering both the cap-dependent and cap-independent modes of initiation. The different translation initiation protagonists will be described in normal conditions and then in gliomas. In addition, their gene expression in gliomas will systematically be examined using two freely available datasets. Finally, we will discuss different pathways regulating translation initiation and current drugs targeting the translational machinery and their potential for the treatment of gliomas.

The neural cell adhesion molecule (NCAM) is a transmembrane protein involved in major cellular processes. The addition of polysialic acid (PSA), a post-translational modification (PTM) almost exclusively carried by NCAM, alters NCAM properties and functions and is therefore tightly regulated. Changes in NCAM and PSA-NCAM take place during development and ageing and occur in various diseases. The presence of PTMs can reduce the accessibility of antibodies to their epitopes and lead to false negative results. Thus, it is vital to identify antibodies that can specifically detect their target regardless of the presence of PTMs. In the present study, four commercially available NCAM antibodies were characterized by western blot and immunocytochemistry. Antibody specificity was determined by decreasing NCAM expression with small interfering RNA and subsequently determining whether the antibodies still produced a signal. In addition, PSA was digested with endoneuraminidase N to assess whether removing PSA improves NCAM detection with these antibodies. Our study revealed that the presence of PSA on NCAM reduced antibody accessibility to the epitope and consequently masked NCAM antigenicity for both techniques investigated. Moreover, three of the four antibodies tested were specific for the detection of NCAM by western blot and by immunocytochemistry. Altogether, this study demonstrates the importance of choosing the correct antibody to study NCAM depending on the technique of interest and underlines the importance of taking PTMs into account when using antibody-based techniques for the study of NCAM.

Background Neuroinflammation and blood-brain barrier (BBB) disruption are common features of many brain disorders, including Alzheimer’s disease, epilepsy, and motor neuron disease. Inflammation is thought to be a driver of BBB breakdown, but the underlying mechanisms for this are unclear. Brain pericytes are critical cells for maintaining the BBB and are immunologically active. We sought to test the hypothesis that inflammation regulates the BBB by altering pericyte biology. Methods We exposed primary adult human brain pericytes to chronic interferon-gamma (IFNγ) for 4 days and measured associated functional aspects of pericyte biology. Specifically, we examined the influence of inflammation on platelet-derived growth factor receptor-beta (PDGFRβ) expression and signalling, as well as pericyte proliferation and migration by qRT-PCR, immunocytochemistry, flow cytometry, and western blotting. Results Chronic IFNγ treatment had marked effects on pericyte biology most notably through the PDGFRβ, by enhancing agonist (PDGF-BB)-induced receptor phosphorylation, internalization, and subsequent degradation. Functionally, chronic IFNγ prevented PDGF-BB-mediated pericyte proliferation and migration. Conclusions Because PDGFRβ is critical for pericyte function and its removal leads to BBB leakage, our results pinpoint a mechanism linking chronic brain inflammation to BBB dysfunction. Electronic supplementary material The online version of this article (doi:10.1186/s12974-016-0722-4) contains supplementary material, which is available to authorized users.

Epigenetic alterations have been associated with several human diseases including Alzheimer's disease (AD). AD is a complex neurodegenerative disease characterized by a progressive decline in cognitive functions, neuronal cell loss and by the presence of β amyloid (Aβ) plaques and neurofibrillary tangles (NFTs) in the cortex. Mutations in specific genes have been identified but can only explain a small percentage of the AD cases. The origins of the sporadic cases of AD are still not known but there is evidence for a role of epigenetics in the etiology of the disease. In this review we focus on discussing the roles of DNA methylation and hydroxymethylation in the development and potential treatment of AD. We discuss papers showing that there are alterations in methylated cytosine (5mC) levels in AD and also highlight the potential role of hydroxylated methylcytosine (5hmC) in the epigenetic regulation of brain gene expression and perhaps in AD. We discuss the potential influence of environmental factors, working through epigenetic mechanisms, on increasing the risk of developing AD and their potential in treating this major neurodegenerative disorder.

DNA methylation (5-methylcytosine [5mC]) is one of several epigenetic markers altered in Alzheimer's disease (AD) brain. More recently, attention has been given to DNA hydroxymethylation (5-hydroxymethylcytosine [5hmC]), the oxidized form of 5mC. Whereas 5mC is generally associated with the inhibition of gene expression, 5hmC has been associated with increased gene expression and is involved in cellular processes such as differentiation, development, and aging. Recent findings point toward a role for 5hmC in the development of diseases including AD, potentially opening new pathways for treating AD through correcting methylation and hydroxymethylation alterations. In the present study, levels of 5mC and 5hmC were investigated in the human middle frontal gyrus (MFG) and middle temporal gyrus (MTG) by immunohistochemistry. Immunoreactivity for 5mC and 5hmC were significantly increased in AD MFG (N = 13) and MTG (N = 29) compared with age-matched controls (MFG, N = 13 and MTG, N = 29). Global levels of 5mC and 5hmC positively correlated with each other and with markers of AD including amyloid beta, tau, and ubiquitin loads. Our results showed a global hypermethylation in the AD brain and revealed that levels of 5hmC were also significantly increased in AD MFG and MTG with no apparent influence of gender, age, postmortem delay, or tissue storage time. Using double-fluorescent immunolabeling, we found that in control and AD brains, levels of 5mC and 5hmC were low in astrocytes and microglia but were elevated in neurons. In addition, our colocalization study showed that within the same nuclei, 5mC and 5hmC mostly do not coexist. The present study clearly demonstrates the involvement of 5mC and 5hmC in AD emphasizing the need for future studies determining the exact time frame of these epigenetic changes during the progression of AD pathology.

Background The use of RNAi to analyse gene function in vitro is now widely applied in biological research. However, several difficulties are associated with its use in vivo, mainly relating to inefficient delivery and non-specific effects of short RNA duplexes in animal models. The latter can lead to false positive results when real-time RT-qPCR alone is used to measure target mRNA knockdown. Findings We observed that detection of an apparent siRNA-mediated knockdown in vivo was dependent on the primers used for real-time RT-qPCR measurement of the target mRNA. Two siRNAs specific for RRM1 with equivalent activity in vitro were administered to A549 xenografts via intratumoural injection. In each case, apparent knockdown of RRM1 mRNA was observed only when the primer pair used in RT-qPCR flanked the siRNA cleavage site. This false-positive result was found to result from co-purified siRNA interfering with both reverse transcription and qPCR. Conclusions Our data suggest that using primers flanking the siRNA-mediated cleavage site in RT-qPCR-based measurements of mRNA knockdown in vivo can lead to false positive results. This is particularly relevant where high concentrations of siRNA are introduced, particularly via intratumoural injection, as the siRNA may be co-purified with the RNA and interfere with downstream enzymatic steps. Based on these results, using primers flanking the siRNA target site should be avoided when measuring knockdown of target mRNA by real-time RT-qPCR.

Silencing genes essential for replication and division using siRNA has potential as a therapeutic strategy for cancer treatment. In order to identify the most potent siRNA, target sequence and siRNA design must be considered together, as tolerance for structural changes can be sequence-dependent. Here we have used growth inhibition assays to investigate the effects of silencing of RRM1, RRM2, and PLK1 with standard siRNAs, Stealth() duplexes, and Dicer substrate siRNAs. The growth inhibitory effect of RRM1, RRM2, or PLK1 knockdown in A549 cells varied with mRNA target site and the format of the siRNA, with longer modified siRNAs generally more effective than standard siRNAs specific for the same target site. Standard siRNAs of varying activity became more potent inhibitors of growth when converted to Stealth() duplexes, and the increase in activity was due to a combination of chemical modification and length. In each case, the effect on activity of changing the siRNA format depended on the siRNA sequence. Taken together these results suggest that, in vitro, longer siRNAs with chemical modifications are in general more active than standard siRNAs targeting the same site, and that structure, chemical modification, and target site must be considered together to identify the most active siRNAs.

Specific detection of mRNA cleavage by 5′RACE is the only method to confirm the knockdown of mRNA by RNA interference, but is rarely reported for in vivo studies. We have combined 5′-RNA-linker-mediated RACE (5′-RLM-RACE) with real-time PCR using a molecular beacon to develop a rapid and specific method termed MBRACE, which we have used to detect small-interfering RNA (siRNA)-induced cleavage of ApoB, RRM1 and YBX1 transcripts in vitro, and ApoB in vivo. When RNA from siRNA-transfected cells was used for 5′-RLM-RACE and a cleavage site-specific molecular beacon probe was included in subsequent real-time PCR analysis, the specific mRNA cleavage product was detected. Detection of siRNA-mediated cleavage was also observed when RNA from mouse liver following administration of ApoB-specific siRNA was analysed, even in cases where ApoB knockdown measured by real-time PCR was

Ribonucleotide reductase (RR) has an essential role in DNA synthesis and repair and is a therapeutic target in a number of different cancers. Previous studies have shown that RNAi-mediated knockdown of either the RRM1 or RRM2 subunit sensitizes cells to the cytotoxic effects of the nucleoside analogs and more recently it has been shown that RRM2 knockdown itself has a growth inhibitory effect. Here we compare the effects of siRNA-mediated knockdown of both RRM1 and RRM2 subunits of RR in A549 and HCT-116 cells using an optimized transfection protocol. Growth of A549 cells was strongly inhibited by efficient siRNA-mediated silencing of either RRM1 or RRM2, and knockdown of each subunit led to long-term growth inhibition and cell-cycle arrest. Knockdown with sub growth inhibitory siRNA concentrations sensitized A549 and HCT-116 cells to gemcitabine when RRM1 was targeted, whereas RRM2 knockdown led to hydroxyurea sensitization. These results suggest that the inhibition of cell growth, rather than drug sensitization, is the major effect of RRM1 and RRM2 knockdown. In an A549 xenograft model, cells transfected with RRM1-specific siRNA failed to form tumors in 6 out of 8 CD1 nude mice, whereas those transfected with RRM2-specific siRNA grew but at a reduced rate. Taken together, these data demonstrate that siRNA-mediated knockdown of the RRM1 subunit is more effective than knockdown of RRM2 in inhibiting the growth of cancer cell lines and suggest that RRM1 is a potential target for nucleic acid-based cancer therapies, either alone or in combination with gemcitabine.