The contribution of inflammatory astrocytes to BBB impairments in a brain-chip model of Parkinson's disease.

Astrocyte dysfunction has previously been linked to multiple neurodegenerative disorders including Parkinson's disease (PD). Among their many roles, astrocytes are mediators of the brain immune response, and astrocyte reactivity is a pathological feature of PD. They are also involved in the formation and maintenance of the blood-brain barrier (BBB), but barrier integrity is compromised in people with PD. This study focuses on an unexplored area of PD pathogenesis by characterizing the interplay between astrocytes, inflammation and BBB integrity, and by combining patient-derived induced pluripotent stem cells with microfluidic technologies to generate a 3D human BBB chip. Here we report that astrocytes derived from female donors harboring the PD-related LRRK2 G2019S mutation are pro-inflammatory and fail to support the formation of a functional capillary in vitro. We show that inhibition of MEK1/2 signaling attenuates the inflammatory profile of mutant astrocytes and rescues BBB formation, providing insights into mechanisms regulating barrier integrity in PD. Lastly, we confirm that vascular changes are also observed in the human postmortem substantia nigra of both males and females with PD.

Folic acid supplementation reduces multigenerational sperm miRNA perturbation induced by in utero environmental contaminant exposure.

Persistent organic pollutants (POPs) can induce epigenetic changes in the paternal germline. Here, we report that folic acid (FA) supplementation mitigates sperm miRNA profiles transgenerationally following in utero paternal exposure to POPs in a rat model. Pregnant founder dams were exposed to an environmentally relevant POPs mixture (or corn oil) ± FA supplementation and subsequent F1-F4 male descendants were not exposed to POPs and were fed the FA control diet. Sperm miRNA profiles of intergenerational (F1, F2) and transgenerational (F3, F4) lineages were investigated using miRNA deep sequencing. Across the F1-F4 generations, sperm miRNA profiles were less perturbed with POPs+FA compared to sperm from descendants of dams treated with POPs alone. POPs exposure consistently led to alteration of three sperm miRNAs across two generations, and similarly one sperm miRNA due to POPs+FA; which was in common with one POPs intergenerationally altered sperm miRNA. The sperm miRNAs that were affected by POPs alone are known to target genes involved in mammary gland and embryonic organ development in F1, sex differentiation and reproductive system development in F2 and cognition and brain development in F3. When the POPs treatment was combined with FA supplementation, however, these same miRNA-targeted gene pathways were perturbed to a lesser extend and only in F1 sperm. These findings suggest that FA partially mitigates the effect of POPs on paternally derived miRNA in a intergenerational manner.

Quantitative profiling of the UGT transcriptome in human drug-metabolizing tissues.

Alternative splicing as a mean to control gene expression and diversify function is suspected to considerably influence drug response and clearance. We report the quantitative expression profiles of the human UGT genes including alternatively spliced variants not previously annotated established by deep RNA-sequencing in tissues of pharmacological importance. We reveal a comprehensive quantification of the alternative UGT transcriptome that differ across tissues and among individuals. Alternative transcripts that comprise novel in-frame sequences associated or not with truncations of the 5'- and/or 3'- termini, significantly contribute to the total expression levels of each UGT1 and UGT2 gene averaging 21% in normal tissues, with expression of UGT2 variants surpassing those of UGT1. Quantitative data expose preferential tissue expression patterns and remodeling in favor of alternative variants upon tumorigenesis. These complex alternative splicing programs have the strong potential to contribute to interindividual variability in drug metabolism in addition to diversify the UGT proteome.

Unravelling the transcriptomic landscape of the major phase II UDP-glucuronosyltransferase drug metabolizing pathway using targeted RNA sequencing.

A comprehensive view of the human UDP-glucuronosyltransferase (UGT) transcriptome is a prerequisite to the establishment of an individual's UGT metabolic glucuronidation signature. Here, we uncover the transcriptome landscape of the 10 human UGT gene loci in normal and tumoral metabolic tissues by targeted RNA next-generation sequencing. Alignment on the human hg19 reference genome identifies 234 novel exon-exon junctions. We recover all previously known UGT1 and UGT2 enzyme-coding transcripts and identify over 130 structurally and functionally diverse novel UGT variants. We further expose a revised genomic structure of UGT loci and provide a comprehensive repertoire of transcripts for each UGT gene. Data also uncover a remodelling of the UGT transcriptome occurring in a tissue- and tumor-specific manner. The complex alternative splicing program regulating UGT expression and protein functions is likely critical in determining detoxification capacity of an organ and stress-related responses, with significant impact on drug responses and diseases.

Establishment of a comprehensive reference transcriptome for vertebral bone tissue to study the impacts of nutritional phosphorus deficiency in rainbow trout (Oncorhynchus mykiss, Walbaum).

Reducing dietary phosphorus (P) is a common approach to reduce effluent P outputs. The potential resulting P-deficiency is known to negatively impact fish bone condition and might result in vertebral deformities. To date, no large-scale study involving deep sequencing of the bone transcriptome has been conducted in salmonids and vertebral molecular changes remain poorly described. This study aims to provide the first comprehensive vertebral transcriptome for rainbow trout (Oncorhynchus mykiss) to allow functional and quantitative expression studies. Fish weighing 60.8±1.6g, were fed for 27weeks using two practical diets having 0.29% (deficient) and 0.45% (sufficient) available phosphorus (P), respectively. Deep sequencing was conducted using HiSeq2000 Illumina 100 paired-end technology from pooled P-deficient and P-sufficient fish and individuals displaying vertebral deformities. Over 140 million trimmed paired-end reads were assembled de novo with Trinity and resulted in 679,869 transcripts with a mean length of 542.5bp. From these sequences, 340,747 matched with referenced ESTs from rainbow trout. Furthermore, 141,909 and 117,564 sequences were functionally annotated against Nr and Uniprot databases, respectively. Interestingly, we observed putative homologue sequences for most of the key components involved in bone formation and turnover in mammals.

Effect of sitagliptin therapy on triglyceride-rich lipoprotein kinetics in patients with type 2 diabetes.

AIM: To investigate the effects of sitagliptin therapy on the kinetics of triglyceride-rich lipoprotein (TRL) apolipoprotein (apo)B-48, VLDL apoB-100, apoE and apoC-III in patients with type 2 diabetes. METHODS: Twenty-two subjects with type 2 diabetes were recruited in this double-blind crossover study, during which the subjects received sitagliptin (100 mg/day) or placebo for a 6-week period each. At the end of each phase of treatment, the in vivo kinetics of the different apolipoproteins were assessed using a primed-constant infusion of l-[5,5,5-D3]leucine for 12 h, with the participants in a constantly fed state. RESULTS: Sitagliptin therapy significantly reduced fasting plasma triglyceride (-15.4%, p = 0.03), apoB-48 (-16.3%, p = 0.03) and free fatty acid concentrations (-9.5%, p = 0.04), as well as plasma HbA1c (placebo: 7.0% ± 0.8 vs. sitagliptin: 6.6% ± 0.7, p < 0.0001) and plasma glucose levels (-13.5%, p = 0.001), without any significant effect on insulin levels. Kinetic results showed that treatment with sitagliptin significantly reduced the pool size of TRL apoB-48 by -20.8% (p = 0.03), paralleled by a reduction in the production rate of these particles (-16.0%, p = 0.03). The VLDL apoB-100 pool size was also significantly decreased by sitagliptin therapy (-9.3%, p = 0.03), mainly because of a reduction in the hepatic secretion of these lipoproteins, although this difference did not reach statistical significance (-9.2%, p = 0.06). CONCLUSIONS: Treatment with sitagliptin for 6 weeks reduced triglyceride-rich apoB-containing lipoprotein levels by reducing the synthesis of these particles.

PARP-3 associates with polycomb group bodies and with components of the DNA damage repair machinery.

Poly(ADP-ribose) polymerase 3 (PARP-3) is a novel member of the PARP family of enzymes that synthesize poly(ADP-ribose) on themselves and other acceptor proteins. Very little is known about this PARP, which is closely related to PARP-1 and PARP-2. By sequence analysis, we find that PARP-3 may be expressed in two isoforms which we studied in more detail to gain insight into their possible functions. We find that both PARP-3 isoforms, transiently expressed as GFP or FLAG fusions, are nuclear. Detection of endogenous PARP-3 with a specific antibody also shows a widespread nuclear distribution, appearing in numerous small foci and a small number of larger foci. Through co-localization experiments and immunoprecipitations, the larger nuclear foci were identified as Polycomb group bodies (PcG bodies) and we found that PARP-3 is part of Polycomb group protein complexes. Furthermore, using a proteomics approach, we determined that both PARP-3 isoforms are part of complexes comprising DNA-PKcs, PARP-1, DNA ligase III, DNA ligase IV, Ku70, and Ku80. Our findings suggest that PARP-3 is a nuclear protein involved in transcriptional silencing and in the cellular response to DNA damage.

Regulation of poly(ADP-ribose) metabolism by poly(ADP-ribose) glycohydrolase: where and when?

Poly(ADP-ribose) glycohydrolase (PARG) is a catabolic enzyme that cleaves ADP-ribose polymers formed by members of the PARP family of enzymes. Despite its discovery and subsequent partial purification in the 1970s and the cloning of its single gene in the late 1990s, little is known about the role of PARG in cell function. Because of its low abundance within cells and its extreme sensitivity to proteases, PARG has been difficult to study. The existence of several PARG isoforms with different subcellular localizations is still debated today after more than 30 years of intensive research. In this article, we want to summarize and discuss the current knowledge related to PARG, its different forms and subcellular distribution. We also examine the possible biological roles of PARG in modulating chromatin structure, transcription, DNA repair and apoptosis.