Identification and characterization of two novel alternatively spliced E2F1 transcripts in the rat CNS.

E2F1 is a transcription factor classically known to regulate G0/G1 to S phase progression in the cell cycle. In addition, E2F1 also regulates a wide range of apoptotic genes and thus has been well studied in the context of neuronal death and neurodegenerative diseases. However, its function and regulation in the mature central nervous system are not well understood. Alternative splicing is a well-conserved post-transcriptional mechanism common in cells of the CNS and is necessary to generate diverse functional modifications to RNA or protein products from genes. Heretofore, physiologically significant alternatively spliced E2F1 transcripts have not been reported. In the present study, we report the identification of two novel alternatively spliced E2F1 transcripts: E2F1b, an E2F1 transcript retaining intron 5, and E2F1c, an E2F1 transcript excluding exon 6. These alternatively spliced transcripts are observed in the brain and neural cell types including neurons, astrocytes, and undifferentiated oligodendrocytes. The expression of these E2F1 transcripts is distinct during maturation of primary hippocampal neuroglial cells. Pharmacologically-induced global translation inhibition with cycloheximide, anisomycin or thapsigargin lead to significantly reduced expression of E2F1a, E2F1b and E2F1c. Conversely, increasing neuronal activity by elevating the concentration of potassium chloride selectively increased the expression of E2F1b. Furthermore, experiments expressing these variants in vitro show the transcripts can be translated to generate a protein product. Taken together, our data suggest that the alternatively spliced E2F1 transcript behave differently than the E2F1a transcript, and our results provide a foundation for future investigation of the function of E2F1 splice variants in the CNS.

TNF-α/TNFR2 Regulatory Axis Stimulates EphB2-Mediated Neuroregeneration Via Activation of NF-κB.

HIV-1 infected individuals are at high risk of developing HIV-associated neurocognitive disorders (HAND) as HIV infection leads to neuronal injury and synaptic loss in the central nervous system (CNS). The neurotoxic effects of HIV-1 are primarily a result of viral replication leading to the production of inflammatory chemokines and cytokines, including TNF-α. Given an important role of TNF-α in regulating synaptic plasticity, we investigated the effects of TNF-α on the development of neuronal processes after mechanical injury, and we showed that TNF-α treatment stimulates the regrowth of neuronal processes. To investigate transcriptional effects of TNF-α on synaptic plasticity, we analyzed both human neurosphere and isolated neuronal cultures for the regulation of genes central to synaptic alterations during learning and memory. TNF-α treatment upregulated Ephrin receptor B2 (EphB2), which is strongly involved in dendritic arborization and synaptic integrity. TNF-α strongly activates the NF-κB pathway, therefore, we propose that TNF-α-induced neurite regrowth occurs primarily through EphB2 signaling via stimulation of NF-κB. EphB2 promoter activity increased with TNF-α treatment and overexpression of NF-κB. Direct binding of NF-κB to the EphB2 promoter occurred in the ChIP assay, and site-directed mutagenesis identified binding sites involved in TNF-α-induced EphB2 activation. TNF-α induction of EphB2 was determined to occur specifically through TNF-α receptor 2 (TNFR2) activation in human primary fetal neurons. Our observations provide a new avenue for the investigation on the impact of TNF-α in the context of HIV-1 neuronal cell damage as well as providing a potential therapeutic target in TNFR2 activation of EphB2.

Isolation and Propagation of Primary Human and Rodent Embryonic Neural Progenitor Cells and Cortical Neurons.

The research on human neural progenitor cells holds great potential for the understanding of the molecular programs that control differentiation of cells of glial and neuronal lineages, as well as pathogenetic mechanisms of neurological diseases. Stem cell technologies also provide opportunities for the pharmaceutical industry to develop new approaches for regenerative medicine. Here, we describe the protocol for the isolation and maintenance of neural progenitor cells and cortical neurons using human fetal brain tissue. This protocol can be successfully adapted for the preparation of rodent neural and oligodendrocyte progenitor cells. While several methods for isolating neural and oligodendrocyte progenitors from rodent brain tissue have been described, including techniques utilizing gene transfer and magnetic resonance beads, few methods are specifically focused on deriving human oligodendrocyte progenitor cells. Development of the human cultures provides the most physiologically relevant system for investigating mechanisms which regulate the function of oligodendrocytes, specifically of human origin.

Compartmentalized Neuronal Cultures.

The culturing of neurons results in formation of the layer of neurons with random extensive overlapping outgrowth. To understand specific roles of somas, axons, and dendrites in complex function of neurons and to identify molecular mechanisms of biological processes in these cellular compartments, various methods were developed. We utilized AXon Investigation System (AXIS™) manufactured by Millipore. This device provides an opportunity to orient neuronal outgrowth and spatially isolate neuronal processes from neuronal bodies. AXIS device is a slide-mounted microfluidic system, which consists of four wells. Two of the wells are connected by a channel on each side of the device. Channels are connected by microgrooves (approximately 120). The size of microgrooves (10µm in width and 5µm in height) does not permit passage of cell through while allowing extension of neurites. The microfluidic design also allows for an establishment of a hydrostatic gradient when the volume in one chamber is greater than that in the other (Park et al., Nat Protoc 1:2128-2136, 2006). This feature allows for studying of the effect of specific compounds on selected compartments of neurons.

TNF-α/NF-κB signaling in the CNS: possible connection to EPHB2.

Tumor necrosis factor-alpha, TNF-α, is a cytokine that is a well-known factor in multiple disease conditions and is recognized for its major role in central nervous system signaling. TNF-α signaling is most commonly associated with neurotoxicity, but in some conditions it has been found to be neuroprotective. TNF-α has long been known to induce nuclear factor-kappa B, NF-κB, signaling by, in most cases, translocating the p65 (RelA) DNA binding factor to the nucleus. p65 is a key member of NF-κB, which is well established as a family of transcription factors that regulates many signaling events, including growth and process development, in neuronal cell populations. NF-κB has been shown to affect both the receiving aspect of neuronal signaling events in dendritic development as well as the sending of neuronal signals in axonal development. In both cases, NK-κB functions as a promoter and/or inhibitor of growth, depending on the environmental conditions and signaling cascade. In addition, NF-κB is involved in memory formation or neurogenesis, depending on the region of the brain in which the signaling occurs. The ephrin (Eph) receptor family represents a subfamily of receptor tyrosine kinases, RTKs, which received much attention due to its potential involvement in neuronal cell health and function. There are two subsets of ephrin receptors, Eph A and Eph B, each with distinct functions in cardiovascular and skeletal development and axon guidance and synaptic plasticity. The presence of multiple binding sites for NF-κB within the regulatory region of EphB2 gene and its potential regulation by NF-κB pathway suggests that TNF-α may modulate EphB2 via NF-κB and that this may contribute to the neuroprotective activity of TNF-α.