MAB326 Sigma-AldrichAnti-CNPase Antibody, clone 11-5B

Prior work showed that whole blood, plasma, and serum injections are damaging to the neonatal rodent brain in a model of intracerebral/periventricular hemorrhage. Thrombin alone is also damaging. In adult animal models of hemorrhagic stroke, the protease-activated (thrombin) receptor PAR1 mediates some of the brain damage. We hypothesized that PAR1 interference will reduce the adverse effects of blood products on immature rodent brain and cells.Cultured oligodendrocyte precursor cells from rats and mice were exposed to blood plasma with and without the PAR1 antagonists SCH-79797 or BMS-200261. In concentrations previously shown to have activity on brain cells, neither drug showed evidence of protection against the toxicity of blood plasma. Newborn mice (wild type, heterozygous, and PAR1 knockout) were subjected to intracerebral injection of autologous whole blood into the periventricular region of the frontal lobe. Cell proliferation, measured by Ki67 immunoreactivity in the subventricular zone, was suppressed at 1 and 2 days, and was not normalized in the knockout mice. Cell apoptosis, measured by activated caspase 3 immunoreactivity, was not apparent in the subventricular zone. Increased apoptosis in periventricular striatal cells was not normalized in the knockout mice.Interference with the thrombin-PAR1 system does not reduce the adverse effects of blood on germinal cells of the immature rodent brain. PAR1 interference is unlikely to be a useful treatment for reducing the brain damage that accompanies periventricular (germinal matrix) hemorrhage, a common complication of premature birth.

Astrocytes can mediate neurovascular coupling, modulate neuronal excitability, and promote synaptic maturation and remodeling. All these functions are likely to be modulated by the sleep/wake cycle, because brain metabolism, neuronal activity and synaptic turnover change as a function of behavioral state. Yet, little is known about the effects of sleep and wake on astrocytes.Here we show that sleep and wake strongly affect both astrocytic gene expression and ultrastructure in the mouse brain. Using translating ribosome affinity purification technology and microarrays, we find that 1.4 % of all astrocytic transcripts in the forebrain are dependent on state (three groups, sleep, wake, short sleep deprivation; six mice per group). Sleep upregulates a few select genes, like Cirp and Uba1, whereas wake upregulates many genes related to metabolism, the extracellular matrix and cytoskeleton, including Trio, Synj2 and Gem, which are involved in the elongation of peripheral astrocytic processes. Using serial block face scanning electron microscopy (three groups, sleep, short sleep deprivation, chronic sleep restriction; three mice per group, greater than 100 spines per mouse, 3D), we find that a few hours of wake are sufficient to bring astrocytic processes closer to the synaptic cleft, while chronic sleep restriction also extends the overall astrocytic coverage of the synapse, including at the axon-spine interface, and increases the available astrocytic surface in the neuropil.Wake-related changes likely reflect an increased need for glutamate clearance, and are consistent with an overall increase in synaptic strength when sleep is prevented. The reduced astrocytic coverage during sleep, instead, may favor glutamate spillover, thus promoting neuronal synchronization during non-rapid eye movement sleep.

The skin is a rich source of readily accessible stem cells. The level of plasticity afforded by these cells is becoming increasingly important as the potential of stem cells in Cell Therapy and Regenerative Medicine continues to be explored. Several protocols described single type stem cell isolation from skin; however, none of them afforded simultaneous isolation of more than one population. Herein, we describe the simultaneous isolation and characterization of three stem cell populations from the dermis and epidermis of murine skin, namely Epidermal Stem Cells (EpiSCs), Skin-derived Precursors (SKPs) and Mesenchymal Stem Cells (MSCs). The simultaneous isolation was possible through a simple protocol based on culture selection techniques. These cell populations are shown to be capable of generating chondrocytes, adipocytes, osteocytes, terminally differentiated keratinocytes, neurons and glia, rendering this protocol suitable for the isolation of cells for tissue replenishment and cell based therapies. The advantages of this procedure are far-reaching since the skin is not only the largest organ in the body, but also provides an easily accessible source of stem cells for autologous graft.

The stratum lacunosum moleculare (SLM) is the connection hub between entorhinal cortex and hippocampus, two brain regions that are most vulnerable in Alzheimer's disease. We recently identified a specific synaptic deficit of Nectin-3 in transgenic models for tauopathy. Here we defined cognitive impairment and electrophysiological problems in the SLM of Tau.P301L mice, which corroborated the structural defects in synapses and dendritic spines. Reduced diffusion of DiI from the ERC to the hippocampus indicated defective myelinated axonal pathways. Ultrastructurally, myelinated axons in the temporoammonic pathway (TA) that connects ERC to CA1 were damaged in Tau.P301L mice at young age. Unexpectedly, the myelin defects were even more severe in bigenic biGT mice that co-express GSK3β with Tau.P301L in neurons. Combined, our data demonstrate that neuronal expression of protein Tau profoundly affected the functional and structural organization of the entorhinal-hippocampal complex, in particular synapses and myelinated axons in the SLM. White matter pathology deserves further attention in patients suffering from tauopathy and Alzheimer's disease.

We reported previously that amoeboid microglial cells in the postnatal rat brain expressed 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) both in vivo and in vitro; however, the functional role of CNPase in microglia has remained uncertain. This study extended the investigation to determine CNPase expression in activated microglia derived from cell culture and animal models of brain injury with the objective to clarify its putative functions.Three-day-old Wistar rats were given an intraperitoneal injection of lipopolysaccharide to induce microglial activation, and the rats were killed at different time points. Along with this, primary cultured microglial cells were subjected to lipopolysaccharide treatment, and expression of CNPase was analyzed by real-time reverse transcription PCR and immunofluorescence. Additionally, siRNA transfection was employed to downregulate CNPase in BV-2 cells. Following this, inducible nitric oxide synthase, IL-1β and TNF-α were determined at mRNA and protein levels. Reactive oxygen species and nitric oxide were also assessed by flow cytometry and colorimetric assay, respectively. In parallel to this, CNPase expression in activated microglia was also investigated in adult rats subjected to fluid percussion injury as well as middle cerebral artery occlusion.In vivo, CNPase immunofluorescence in activated microglia was markedly enhanced after lipopolysaccharide treatment. A similar feature was observed in the rat brain after fluid percussion injury and middle cerebral artery occlusion. In vitro, CNPase protein and mRNA expression was increased in primary microglia with lipopolysaccharide stimulation. Remarkably, inducible nitric oxide synthase, IL-1β, TNF-α, reactive oxygen species and nitric oxide were significantly upregulated in activated BV-2 cells with CNPase knockdown. siRNA knockdown of CNPase increased microglia migration; on the other hand, microglial cells appeared to be arrested at G1 phase.The present results have provided the first morphological and molecular evidence that CNPase expression is increased in activated microglia. CNPase knockdown resulted in increased expression of various inflammatory mediators. It is concluded that CNPase may play an important role as a putative anti-inflammatory gene both in normal and injured brain.

Purifying and culturing cells from the central nervous system (CNS) has proved to be an incredibly powerful tool for dissecting fundamental neuron and glial properties, and especially powerful in understanding neuronal-glial interactions. In a series of detailed protocols, we have provided step-by-step instructions for purifying and culturing specific types of neurons, glia, and vascular cells from the CNS by immunopanning. This article discusses common pitfalls and errors as well as important design considerations for the immunopanning procedure.

Postmortem studies have revealed a downregulation of the transcription factor Pax5 in GABAergic neurons in bipolar disorder, a neurodevelopmental disorder, raising the question whether Pax5 in GABAergic neurons has a role in normal brain development. In a genetic approach to study functions of Pax5 in GABAergic neurons, Pax5 was specifically deleted in GABAergic neurons from Pax5 floxed mice using a novel Gad1-Cre transgenic mouse line expressing Cre recombinase in Gad1-positive, that is, GABAergic neurons. Surprisingly, these mice developed a marked enlargement of the lateral ventricles at approximately 7 weeks of age, which was lethal within 1-2 weeks of its appearance. This hydrocephalus phenotype was observed in mice homozygous or heterozygous for the Pax5 conditional knockout, with a gene dosage-dependent penetrance. By QTL (quantitative trait loci) mapping, a 3.5 Mb segment on mouse chromosome 4 flanked by markers D4Mit237 and D4Mit214 containing approximately 92 genes including Pax5 has previously been linked to differences in lateral ventricular size. Our findings are consistent with Pax5 being a relevant gene underlying this QTL phenotype and demonstrate that Pax5 in GABAergic neurons is essential for normal ventricular development.

The subventricular zone (SVZ) is one of the two major neurogenic regions in the adult mammalian brain. Its close proximity to the striatum suggests that a cell-based therapeutic strategy for the treatment of Huntington's disease (HD) is possible. To achieve this, it is important to understand how adult cell production, migration and differentiation may be altered in the HD brain. In this study, we quantified the number of adult-born striatal cells and characterized their fate in the R6/2 transgenic mouse model of HD. We found that the number of new striatal cells was approximately two-fold greater in R6/2 vs. wild type mice, while SVZ cell proliferation was not affected. Using cell-type specific markers, we demonstrated that the majority of new striatal cells were mature oligodendrocytes or oligodendroglial precursors that were intrinsic to the striatum. We also detected a significant increase in the number of migrating neuroblasts that appeared to be recruited from the SVZ to the striatum. However, these neuroblasts did not mature into neurons and most were lost between 1 and 2 weeks of cell age. Crossing the R6/2 mice with mice the over-expressing brain-derived neurotrophic factor in the striatum increased the numbers of neuroblasts that survived to 2 weeks, but did not promote their differentiation. Together, our data indicate that the potential treatment of HD based on manipulating endogenous progenitor cells should take into consideration the apparent enhancement in striatal oligodendrogliogenesis and the limited ability of recruited SVZ neuroblasts to survive long-term and differentiate in the diseased striatum.

Cell adhesion molecules are important structural substrates, required for synaptic plasticity and synaptogenesis. CAMs differ widely in their expression throughout different brain regions and their specific structural and functional roles in the brain remain to be elucidated. Here, we investigated selected cell adhesion molecules for alterations in expression levels and neuronal localization in validated mouse models for Alzheimer's disease that mimic the age-related progression of amyloid accumulation and tauopathy. Among the cell adhesion molecules analyzed, Nectin-3 expression was affected most and specifically in all mouse models with tauopathy. In particular was Nectin-3 depleted from the specific region of the hippocampus, known as the stratum lacunosum and moleculare, in mice that express wild-type or mutant human protein Tau, either chronically or sub-acutely. Tauopathy progresses from the entorhinal cortex to the hippocampus by unknown mechanisms that could involve transport by the myelinated axons of the temporoammonic and perforant pathways. The decreased expression of Nectin-3 in the stratum lacunosum moleculare is an early marker of impaired transport, and eventual synaptic problems, caused by beginning tauopathy.

Previous studies of differential gene expression in sleep and wake pooled transcripts from all brain cells and showed that several genes expressed at higher levels during sleep are involved in the synthesis/maintenance of membranes in general and of myelin in particular, a surprising finding given the reported slow turnover of many myelin components. Other studies showed that oligodendrocyte precursor cells (OPCs) are responsible for the formation of new myelin in both the injured and the normal adult brain, and that glutamate released from neurons, via neuron-OPC synapses, can inhibit OPC proliferation and affect their differentiation into myelin-forming oligodendrocytes. Because glutamatergic transmission is higher in wake than in sleep, we asked whether sleep and wake can affect oligodendrocytes and OPCs. Using the translating ribosome affinity purification technology combined with microarray analysis in mice, we obtained a genome-wide profiling of oligodendrocytes after sleep, spontaneous wake, and forced wake (acute sleep deprivation). We found that hundreds of transcripts being translated in oligodendrocytes are differentially expressed in sleep and wake: genes involved in phospholipid synthesis and myelination or promoting OPC proliferation are transcribed preferentially during sleep, while genes implicated in apoptosis, cellular stress response, and OPC differentiation are enriched in wake. We then confirmed through BrdU and other experiments that OPC proliferation doubles during sleep and positively correlates with time spent in REM sleep, whereas OPC differentiation is higher during wake. Thus, OPC proliferation and differentiation are not perfectly matched at any given circadian time but preferentially occur during sleep and wake, respectively.