Fibrinogen regulates lesion border-forming reactive astrocyte properties after vascular damage.

Reactive astrocytes at the border of damaged neuronal tissue organize into a barrier surrounding the fibrotic lesion core, separating this central region of inflammation and fibrosis from healthy tissue. Astrocytes are essential to form the border and for wound repair but interfere with neuronal regeneration. However, the mechanisms driving these astrocytes during central nervous system (CNS) disease are unknown. Here we show that blood-derived fibrinogen is enriched at the interface of lesion border-forming elongated astrocytes after cortical brain injury. Anticoagulant treatment depleting fibrinogen reduces astrocyte reactivity, extracellular matrix deposition and inflammation with no change in the spread of inflammation, whereas inhibiting fibrinogen conversion into fibrin did not significantly alter astrocyte reactivity, but changed the deposition of astrocyte extracellular matrix. RNA sequencing of fluorescence-activated cell sorting-isolated astrocytes of fibrinogen-depleted mice after cortical injury revealed repressed gene expression signatures associated with astrocyte reactivity, extracellular matrix deposition and immune-response regulation, as well as increased gene expression signatures associated with astrocyte metabolism and astrocyte-neuron communication. Systemic pharmacologic depletion of fibrinogen resulted in the absence of elongated, border-forming astrocytes and increased the survival of neurons in the lesion core after cortical injury. These results identify fibrinogen as a critical trigger for lesion border-forming astrocyte properties in CNS disease.

P75 neurotrophin receptor controls subventricular zone neural stem cell migration after stroke.

Stroke is the leading cause of adult disability. Endogenous neural stem/progenitor cells (NSPCs) originating from the subventricular zone (SVZ) contribute to the brain repair process. However, molecular mechanisms underlying CNS disease-induced SVZ NSPC-redirected migration to the lesion area are poorly understood. Here, we show that genetic depletion of the p75 neurotrophin receptor (p75NTR-/-) in mice reduced SVZ NSPC migration towards the lesion area after cortical injury and that p75NTR-/- NSPCs failed to migrate upon BDNF stimulation in vitro. Cortical injury rapidly increased p75NTR abundance in SVZ NSPCs via bone morphogenetic protein (BMP) receptor signaling. SVZ-derived p75NTR-/- NSPCs revealed an altered cytoskeletal network- and small GTPase family-related gene and protein expression. In accordance, BMP-treated non-migrating p75NTR-/- NSPCs revealed an altered morphology and α-tubulin expression compared to BMP-treated migrating wild-type NSPCs. We propose that BMP-induced p75NTR abundance in NSPCs is a regulator of SVZ NSPC migration to the lesion area via regulation of the cytoskeleton following cortical injury.

Id proteins: emerging roles in CNS disease and targets for modifying neural stemcell behavior.

Neural stem/progenitor cells (NSPCs) are found in the adult brain and spinal cord, and endogenous or transplanted NSPCs contribute to repair processes and regulate immune responses in the CNS. However, the molecular mechanisms of NSPC survival and integration as well as their fate determination and functionality are still poorly understood. Inhibitor of DNA binding (Id) proteins are increasingly recognized as key determinants of NSPC fate specification. Id proteins act by antagonizing the DNA-binding activity of basic helix-loop-helix (bHLH) transcription factors, and the balance of Id and bHLH proteins determines cell fate decisions in numerous cell types and developmental stages. Id proteins are central in responses to environmental changes, as they occur in CNS injury and disease, and cellular responses in adult NSPCs implicate Id proteins as prime candidates for manipulating stemcell behavior. Here, we outline recent advances in understanding Id protein pleiotropic functions in CNS diseases and propose an integrated view of Id proteins and their promise as potential targets in modifying stemcell behavior to ameliorate CNS disease.

The E2A splice variant E47 regulates the differentiation of projection neurons via p57(KIP2) during cortical development.

During corticogenesis, distinct classes of neurons are born from progenitor cells located in the ventricular and subventricular zones, from where they migrate towards the pial surface to assemble into highly organized layer-specific circuits. However, the precise and coordinated transcriptional network activity defining neuronal identity is still not understood. Here, we show that genetic depletion of the basic helix-loop-helix (bHLH) transcription factor E2A splice variant E47 increased the number of Tbr1-positive deep layer and Satb2-positive upper layer neurons at E14.5, while depletion of the alternatively spliced E12 variant did not affect layer-specific neurogenesis. While ChIP-Seq identified a big overlap for E12- and E47-specific binding sites in embryonic NSCs, including sites at the cyclin-dependent kinase inhibitor (CDKI) Cdkn1c gene locus, RNA-Seq revealed a unique transcriptional regulation by each splice variant. E47 activated the expression of the CDKI Cdkn1c through binding to a distal enhancer. Finally, overexpression of E47 in embryonic NSCs in vitro impaired neurite outgrowth, and overexpression of E47 in vivo by in utero electroporation disturbed proper layer-specific neurogenesis and upregulated p57(KIP2) expression. Overall, this study identifies E2A target genes in embryonic NSCs and demonstrates that E47 regulates neuronal differentiation via p57(KIP2).

Systemic Delivery and Biodistribution of Cisplatin in Vivo.

Cisplatin is widely used to treat a variety of cancers. However, ototoxicity and nephrotoxicity remain serious side effects of cisplatin-based chemotherapy. In order to inform the study of cisplatin's off-target effects, a new drug-fluorophore conjugate was synthesized that exhibited utility as a tracer to determine the cellular uptake and in vivo distribution of cisplatin. This probe will serve as a useful tool to facilitate investigations into the kinetics and biodistribution of cisplatin and its associated side effects in preclinical models after systemic administration.

Rhodamine analogues for molecular ruler applications.

A series of geometrically well-defined cationic fluorophores were designed based on molecular mechanics. They contain biaryl linkers to impart rigidity preventing intramolecular folding between a conjugated biomolecule and fluorophore. All probes have absorption and emission maxima within 20 nm from Texas Red, as predicted by TDDFT calculations and validated experimentally.

Templated polymers enable selective capture and release of lysophosphatidic acid in human plasma via optimization of non-covalent binding to functional monomers.

The first solid phase extraction materials for selective lysophosphatidic acid (LPA) enrichment from human plasma are described. Molecularly imprinted polymers were designed, synthesized and evaluated as cartridge fillings. They enabled a relatively rapid and simple extraction protocol for LPA without any need for multiple liquid-liquid extraction steps. The five major subspecies of lysophosphatidic acid are readily separated from all other native plasma phospholipids, including those well-known to interfere with LPA quantitation, such as phosphatidylcholine and lysophosphatidylcholine. Outstanding LPA purity is obtained via these solid phase materials in a tandem extraction setup.

Simple enrichment and analysis of plasma lysophosphatidic acids.

A simple and highly efficient technique for the analysis of lysophosphatidic acid (LPA) subspecies in human plasma is described. The streamlined sample preparation protocol furnishes the five major LPA subspecies with excellent recoveries. Extensive analysis of the enriched sample reveals only trace levels of other phospholipids. This level of purity not only improves MS analyses, but enables HPLC post-column detection in the visible region with a commercially available fluorescent phospholipids probe. Human plasma samples from different donors were analyzed using the above method and validated by LC-ESI/MS/MS.

The Wilms' tumor suppressor Wt1 regulates Coronin 1B expression in the epicardium.

Coronin 1B has been shown to be critical for cell motility and various actin-dependent processes. To understand its role more extensively, the expression and transcriptional regulation of Coro1b gene during mouse development were explored. Coronin 1B is ubiquitously expressed in the whole embryo but nevertheless shows distinct expression pattern in developing heart. In addition to the localization in endocardium, Coronin 1B is specifically expressed in the endocardial cushion and epicardium where cardiac EMT processes take place as the heart develops. Promoter deletion analysis identified the positions between -1038 and -681 is important for Coro1b basal promoter activity. In addition to a correlation of Coronin 1B localization with Wt1 expression in the epicardium, we also identified putative Wt1 binding sequences within Coro1b promoter. Direct binding of Wt1 to GC-rich sequences within the Coro1b promoter is required for the regulation of Coro1b gene expression. In accordance with the motility defect found in Coronin 1B-knockdown cells, a modest decrease in expression of Coronin 1B in the remaining epicardium of Wt1(EGFPCre/EGFPCre) mutant embryos was observed. These findings seem to shed light on the role of Wt1 during cell migration and suggest that, at least in part, this involves transcriptional control of Coro1b gene expression.

Live cell imaging of a fluorescent gentamicin conjugate.

Understanding cellular mechanisms of ototoxic and nephrotoxic drug uptake, intracellular distribution, and molecular trafficking across cellular barrier systems aids the study of potential uptake blockers that preserve sensory and renal function during critical life-saving therapy. Herein we report the design, synthesis characterization and evaluation of a fluorescent conjugate of the aminoglycoside antibiotic gentamicin. Live cell imaging results show the potential utility of this new material. Related gentamicin conjugates studied to date quench in live kindney cells, and have been largely restricted to use in fixed (delipidated) cells.