Rita Giuliano

Synuclein activates microglia in a model of Parkinson's disease

Parkinsons disease (PD) is the most common neurodegenerative movement disorder afflicting >500,000 patients in the United States alone. This age-related progressive disorder is typified by invariant loss of dopaminergic substantia nigra neurons (DAN), dystrophic neurites, the presence of alpha-synuclein (SYN) positive intracytoplasmic inclusions (Lewy bodies) in the remaining DAN, and activated microglia. As such, microglial activation and resultant increase in proinflammatory molecules have moved to the forefront of PD research as a potential pathobiologic mechanism of disease. Herein, we present data demonstrating early microglial activation in mice that over-express wild-type SYN, the release of SYN from a SYN overexpressing MN9D cell line, and dose-dependent SYN-mediated activation of primary microglial cultures with consequent increases in proinflammatory molecules. Furthermore, we provide evidence that the CD36 scavenger receptor and downstream kinases are involved in SYN-mediated microglial activation. Together, our data suggest an early role for SYN and inflammation in PD pathogenesis.

Development of mRNAs for glucocorticoid and mineralocorticoid receptors in rat hippocampus

The hippocampus plays an important role in mediating glucocorticoid effects on the brain. Glucocorticoids are also implicated in neurogenesis and age-related neuronal death in the hippocampus. The effects of glucocorticoids in the hippocampus are elicited through two receptors with high-affinity for corticosterone, the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR). In this study, we used a sensitive RNase protection assay to quantify the ontogeny of GR mRNA and MR mRNA in hippocampus from embryonic day 18 (E18) to postnatal day 60 (P60). GR mRNA and MR mRNA are expressed at approximately equal levels in the E18 hippocampus. However, by birth, the level of MR mRNA is three-fold that of GR mRNA and remains elevated up to P60. The levels of both mRNAs increase gradually during the period of postnatal neurogenesis after which they markedly increase to adult levels. In addition, the levels of hippocampal MR mRNA are the same in male and female rats, whereas the levels of GR mRNA are significantly higher in the P60 female rat hippocampus, but not in younger female rats. Our data on the development of mRNA levels do not parallel the levels of glucocorticoid and mineralocorticoid receptors as reported in a number of binding studies. Therefore, our studies, when considered together with previous reports, suggest that posttranscriptional mechanisms play a major role in regulating the levels of glucocorticoid-binding sites in the hippocampus.

In Vitro Studies of Glucocorticoid Effects on Neurons and Astrocytes

Studies using immunocytochemistry and RNase protection assay demonstrate that glucocorticoid and mineralocorticoid receptors (GR, MR) and their corresponding mRNAs are co-expressed in hippocampal neurons cultured in serum-free, defined medium and at lower levels in cultured astrocytes. Addition of serum or medium conditioned by astrocytes increases the levels of MR mRNA, but has little effect on the levels of GR mRNA. Cellular levels of both GR mRNA and MR mRNA are upregulated by growth of embryonic hippocampal neurons in corticosterone. This is in distinct contrast to regulation of receptor expression in vivo where mRNAs for these receptors are downregulated in the rat hippocampus by corticosterone treatment of the adult adrenalectomized rat. However, in cultured astrocytes, GR and MR mRNAs are also downregulated by corticosterone. To begin to define the role of glucocorticoids in gene expression in astrocytes, we have used giant two-dimensional (2D) gel electrophoresis to separate astrocyte cellular proteins and translation products synthesized in vitro from astrocyte poly A+ RNA. Analysis of approximately 1,500 in vitro translation products by giant 2D gel electrophoresis reveals 11 protein inductions and 1 repression that occur at the level of mRNA in the absence of protein synthesis following treatment of astrocytes with corticosterone. Interestingly, these changes appear to be mediated by GR, but not by MR. The in vitro studies described here are relevant to identifying the role of GR and MR in gene expression in specific cell types in the hippocampus.

Multipotent human hematopoietic cell line K562: Lineage-specific constitutive and inducible antigens

K562 cells have been reported to display a variety of non-erythroid properties. Using 28 lineage-specific monoclonal antibodies, we analysed which antigens are present spontaneously and which are inducible by a variety of agents. The data suggest that (1) antigens of a given lineage are preferentially responsive to certain inducers, e.g. megakaryocytic antigens to phorbol ester, and (2) a given inducer may influence antigens of different lineages in opposite directions, e.g. phorbol dibutyrate, not only induces megakaryocytic antigens, but also decreases granulocyte and erythroid antigens. We conclude that the K562 cell, despite its malignant origin, retains some capacity for expression of alternative programs of differentiation, a characteristic of the normal multipotent hematopoietic stem cell.

VIP is a transcriptional target of Nurr1 in dopaminergic cells.

The orphan nuclear receptor Nurr1 is required for the development of the ventral mesencephalic dopaminergic neurons. These are the same neurons that are invariantly lost in patients with Parkinsons disease. Nurr1 mRNA expression is not confined to the developing midbrain, and yet Nurr1 appears to be essential for either the maturation of progenitors into fully post-mitotic dopaminergic neurons and/or once formed, their survival. The function of Nurr1 in the transactivation of gene(s) important for neuronal development and/or maintenance is uncharacterized. To characterize potential downstream target genes of Nurr1, we sought to identify mRNAs that are differentially affected by Nurr1 expression. Using a dopaminergic cell line in which Nurr1 content was tightly regulated, differential display analysis identified transcripts altered by Nurr1 expression, including the mRNA encoding vasoactive intestinal peptide (VIP). Herein, we demonstrate that Nurr1 regulates VIP mRNA and protein levels, and transactivates the VIP promoter through Nurr1-responsive cis elements. In addition, dopaminergic cells release and utilize VIP to mediate survival when challenged with paraquat. Nurr1 regulation of VIP is also demonstrated in vivo as loss of Nurr1 function results in diminished VIP mRNA levels within the developing midbrain.

Temporal and spatial localization of nectin‐1 and l‐afadin during synaptogenesis in hippocampal neurons

Nectins are cell adhesion molecules that, together with the intracellular binding partner afadin, mediate adhesion and signaling at a variety of intercellular junctions. In this work we studied the distribution of nectin‐1 and afadin during hippocampal synapse formation using cultured primary hippocampal neurons. Nectin‐1 and afadin cluster at developing synapses between hippocampal neurons. These nectin‐afadin clusters uniformly colocalize with N‐cadherin–catenin pairs, suggesting that formation of developing synapses involves participation of both bimolecular systems. Nectin‐1 is initially expressed at excitatory and inhibitory synapses but is progressively lost at inhibitory synapses during their maturation. Treatment of neurons with actin depolymerizing agents disrupts the synaptically localized nectin‐1 and afadin cluster at an early stage and elicits nectin‐1 ectodomain shedding. These data indicate that the synaptic localization of nectin‐1 and l‐afadin are F‐actin‐dependent and that the shedding of nectin‐1 is a mechanism contributing to synaptic plasticity. J. Comp. Neurol. 507:1228–1244, 2008.

Ectodomain shedding of nectin‐1 regulates the maintenance of dendritic spine density

J. Neurochem. (2012) 120, 741–751.

In-tube transfection improves the efficiency of gene transfer in primary neuronal cultures

To facilitate genetic studies in primary neurons, we analyzed the efficiency of cationic lipid-mediated plasmid DNA transfection using adherent and acutely dissociated neuronal suspensions derived from embryonic mouse cortical tissue. Compared to transfections using adherent cultures, the in-tube procedure enhanced the delivery of a GFP reporter plasmid between four- to eightfold depending on the age of the harvested embryo. The procedure required relatively brief complex incubation times, and supported the transfection of cells expressing the neuronal markers NeuN and TuJ1 with improved uniformity in transfection events across the well surface. To demonstrate the utility of this approach in studying the genetic mechanisms controlling neuron development, we provide data regarding the role of the bZIP transcription factor c/EBP-beta in regulating neurite outgrowth. It is anticipated that this in vitro protocol will facilitate the identification of novel genes involved in both developmental and disease-relevant signaling pathways.

Induction of the FMS proto-oncogene product in HL-60 cells by vitamin D: A flow cytometric analysis☆

Agents which induce monocytic characteristics in HL-60 human acute promyelocytic leukemia cells induce mRNA for the fms proto-oncogene, which encodes the receptor for M-CSF. Previous studies of fms expression in HL-60 cells have characterized chiefly induction by phorbol esters of fms mRNA. Our studies of fms expression in HI-60 cells have characterized induction by vitamin D3 of the fms protein. We have used flow cytometry to correlate fms antigen with a monocyte-specific differentiation antigen recognized by antibody MO2 (CD14), with DNA content, and with the nuclear antigen Ki-67, a marker of cell cycling. HL-60 cells were cultured with or without 1 microM vitamin D for 7 days. fms antigen was found on 42 +/- 5.8% of the cells cultured without vitamin D, but on 63 +/- 4.3% of the cells cultured with vitamin D. MO2 binding was detected on only 2 +/- 0.5% of the cells without vitamin D, but on 59 +/- 9% with vitamin D. Cells cultured with vitamin D that were fms-positive were also predominantly (83%) MO2-positive. Analysis of DNA content, measured by propidium iodide staining, showed that 57 +/- 1.5% of cells cultured without vitamin D, but 93 +/- 0.5% of cells cultured with vitamin D, were in the G0/G1 cell cycle phase. Analysis of nuclear antigen Ki-67 revealed that, of the vitamin D-treated cells that were fms-positive, a significant proportion (37%) were still cycling. We conclude that (1) fms is demonstrable on some uninduced HL-60 cells, (2) when HL-60 cells are induced to develop monocytic characteristics by vitamin D, fms induction is part of the program for monocytic differentiation that includes MO2 expression, yet (3) some induced cells expressing fms are still cycling.

Improved HSV-1 amplicon packaging using virion host shutoff mutants lacking mRNAse activity.

Given their generous transgene capacity and inherent neurotropism, herpes simplex virus (HSV‐1)‐based viral vectors are promising tools for gene delivery to the central nervous system. Despite their widespread pre‐clinical use, vector toxicity remains a concern with regard to the use of herpes vectors in humans. One potential source of toxicity stems from the tegument‐associated virion host shutoff protein (vhs), which induces translational arrest in the host cell through non‐specific mRNAse activity. In the current study we utilized a series of HSV‐1 viruses containing a deletion in the UL41 open reading frame to investigate: (1) the requirement of intact vhs function in amplicon packaging and (2) whether vhs influences the post‐transduction survival of dissociated cortical neurons. Our results demonstrate that while amplicon yield was reduced an order of magnitude, UL41 deletion was associated with reduced vector toxicity. Furthermore, partial reconstitution of vhs function using mRNAse‐inactive point mutants improved amplicon titers without imparting the toxicity observed with wild‐type controls. These findings offer a novel approach to improving the titer and toxicity profiles of HSV‐based viral vectors. Copyright