Cocultures of neurons and astrocytes as a model for examining hypoxia-induced neuronal death.

Astrocytes perform critical functions necessary for neuronal survival. Thus, examining the influence of astrocyte function on neuronal cell death during disease, including hypoxia/ischemia, has become an important avenue of investigation. In this chapter we detail the methodology and potential pitfalls for establishing cocultures of astrocytes and cortical neurons for studying hypoxia-induced neuronal death.

Ectodomain shedding of nectin-1 regulates the maintenance of dendritic spine density.

Synaptic remodeling has been postulated as a mechanism underlying synaptic plasticity and cell adhesion molecules are thought to contribute to this process. We examined the role of nectin-1 ectodomain shedding on synaptogenesis in cultured rat hippocampal neurons. Nectins are Ca(2+) -independent immunoglobulin-like adhesion molecules, involved in cell-cell adherens junctions.

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.

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.

Proteolytic processing of proNGF is necessary for mature NGF regulated secretion from neurons.

Nerve growth factor mediates neuronal survival, synaptogenesis, and synaptic remodeling. We utilized primary hippocampal cultures to investigate the intrinsic motifs of proNGF that might contribute to its processing and subsequent allocation to a regulated versus constitutive secretory pathway. The addition of a carboxypeptidase E motif to proNGF did not alter the secretion of NGF.

Synuclein activates microglia in a model of Parkinson's disease.

Parkinson's 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.

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 Parkinson's 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.

Herpes simplex virus (HSV) amplicon-mediated codelivery of secondary lymphoid tissue chemokine and CD40L results in augmented antitumor activity.

Development of effective antitumor immune responses depends on timely interactions of effector cells. A bimodal approach that involves coexpression of chemokines and costimulatory molecules within the tumor bed may elaborate a more optimal antitumor response. One candidate includes secondary lymphoid tissue chemokine (SLC), which promotes the colocalization of naïve, nonpolarized memory T cells and dendritic cells (DCs) within lymph nodes and Peyer's patches.

Reporter gene transfer induces apoptosis in primary cortical neurons.

Modern cell biologists typically use reporter genes either alone or co-expressed with a protein of interest to facilitate the localization or quantification of protein expression. Our work demonstrates that reporter genes should be used cautiously, as several common reporter gene products are toxic to primary cortical neuronal cultures. We used the herpes simplex virus-based viral amplicon vector to transduce cortical neurons with three different reporter genes and assessed whether any reporter gene products were toxic over time, by monitoring neurite disintegration and apoptosis.