Expression of P2 nucleotide receptors varies with age and sex in murine brain microglia

Microglia are implicated in multiple neurodegenerative disorders, many of which display sexual dimorphisms and have symptom onsets at different ages. P2 purinergic receptors are critical for regulating various microglial functions, but little is known about how their expression varies with age or sex. Therefore, comprehensive information about purinergic receptor expression in normal microglia, in both sexes, over age is necessary if we are to better understand their roles in the healthy and diseased CNS. We analyzed the expression of all fourteen rodent P2X and P2Y receptors in CD11b+ cells freshly-isolated from the brains of C57Bl/6 mice at five different ages ranging from postnatal day 3 to 12 months, in males and females, using quantitative RT-PCR. We also compared purinergic receptor expression in microglia freshly-isolated from 3 day-old pups to that in primary neonatal microglial cultures created from mice of the same age. We observed patterns in P2 receptor expression with age, most notably increased expression with age and age-restricted expression. There were also several receptors that showed sexually dimorphic expression. Lastly, we noted that in vitro culturing of neonatal microglia greatly changed their P2 receptor expression profiles. These data represent the first complete and systematic report of changes in purinergic receptor expression of microglia with age and sex, and provide important information necessary for accurate in vitro modeling of healthy animals

The effect of low-abundance OTU filtering methods on the reliability and variability of microbial composition assessed by 16S rRNA amplicon sequencing

PCR amplicon sequencing may lead to detection of spurious operational taxonomic units (OTUs), inflating estimates of gut microbial diversity. There is no consensus in the analytical approach as to what filtering methods should be applied to remove low-abundance OTUs; moreover, few studies have investigated the reliability of OTU detection within replicates. Here, we investigated the reliability of OTU detection (% agreement in detecting OTU in triplicates) and accuracy of their quantification (assessed by coefficient of variation (CV)) in human stool specimens. Stool samples were collected from 12 participants 22–55 years old. We applied several methods for filtering low-abundance OTUs and determined their impact on alpha-diversity and beta-diversity metrics. The reliability of OTU detection without any filtering was only 44.1% (SE=0.9) but increased after filtering low-abundance OTUs. After filtering OTUs with <0.1% abundance in the dataset, the reliability increased to 87.7% (SE=0.6) but at the expense of removing 6.97% reads from the dataset. When filtering was based on individual sample, the reliability increased to 73.1% after filtering OTUs with <10 copies while removing only 1.12% of reads. High abundance OTUs (>10 copies in sample) had lower CV, indicating better accuracy of quantification than low-abundance OTUs. Excluding very low-abundance OTUs had a significant impact on alpha-diversity metrics sensitive to the presence of rare species (observed OTUs, Chao1) but had little impact on relative abundance of major phyla and families and alpha-diversity metrics accounting for both richness and evenness (Shannon, Inverse Simpson). To increase the reliability of microbial composition, we advise removing OTUs with <10 copies in individual samples, particularly in studies where only one subsample per specimen is available for analysis

Myelin-mediated inhibition of oligodendrocyte precursor differentiation can be overcome by pharmacological modulation of Fyn-RhoA and protein kinase C signalling

Failure of oligodendrocyte precursor cell (OPC) differentiation contributes significantly to failed myelin sheath regeneration (remyelination) in chronic demyelinating diseases. Although the reasons for this failure are not completely understood, several lines of evidence point to factors present following demyelination that specifically inhibit differentiation of cells capable of generating remyelinating oligodendrocytes. We have previously demonstrated that myelin debris generated by demyelination inhibits remyelination by inhibiting OPC differentiation and that the inhibitory effects are associated with myelin proteins. In the present study, we narrow down the spectrum of potential protein candidates by proteomic analysis of inhibitory protein fractions prepared by CM and HighQ column chromatography followed by BN/SDS/SDS–PAGE gel separation using Nano-HPLC-ESI-Q-TOF mass spectrometry. We show that the inhibitory effects on OPC differentiation mediated by myelin are regulated by Fyn-RhoA-ROCK signalling as well as by modulation of protein kinase C (PKC) signalling. We demonstrate that pharmacological or siRNA-mediated inhibition of RhoA-ROCK-II and/or PKC signalling can induce OPC differentiation in the presence of myelin. Our results, which provide a mechanistic link between myelin, a mediator of OPC differentiation inhibition associated with demyelinating pathologies and specific signalling pathways amenable to pharmacological manipulation, are therefore of significant potential value for future strategies aimed at enhancing CNS remyelination

Minocycline Inhibition of Monocyte Activation Correlates with Neuronal Protection in SIV NeuroAIDS

Background: Minocycline is a tetracycline antibiotic that has been proposed as a potential conjunctive therapy for HIV-1 associated cognitive disorders. Precise mechanism(s) of minocycline’s functions are not well defined. Methods: Fourteen rhesus macaques were SIV infected and neuronal metabolites measured by proton magnetic resonance spectroscopy (1H MRS). Seven received minocycline (4 mg/kg) daily starting at day 28 post-infection (pi). Monocyte expansion and activation were assessed by flow cytometry, cell traffic to lymph nodes, CD16 regulation, viral replication, and cytokine production were studied. Results: Minocycline treatment decreased plasma virus and pro-inflammatory CD14+CD16+ and CD14loCD16+ monocytes, and reduced their expression of CD11b, CD163, CD64, CCR2 and HLA-DR. There was reduced recruitment of monocyte/ macrophages and productively infected cells in axillary lymph nodes. There was an inverse correlation between brain NAA/ Cr (neuronal injury) and circulating CD14+CD16+ and CD14loCD16+ monocytes. Minocycline treatment in vitro reduced SIV replication CD16 expression on activated CD14+CD16+ monocytes, and IL-6 production by monocytes following LPS stimulation. Conclusion: Neuroprotective effects of minocycline are due in part to reduction of activated monocytes, monocyte traffic. Mechanisms for these effects include CD16 regulation, reduced viral replication, and inhibited immune activation. Citation: Campbell JH, Burdo TH, Autissier P, Bombardier JP, Westmoreland SV, et al. (2011) Minocycline Inhibition of Monocyte Activation Correlate