Hugh J. L. Fryer

Brain‐Derived Neurotrophic Factor Induces Excitotoxic Sensitivity in Cultured Embryonic Rat Spinal Motor Neurons Through Activation of the Phosphatidylinositol 3‐Kinase Pathway

Abstract: Neurotrophic factors (NTFs) can protect against or sensitize neurons to excitotoxicity. We studied the role played by various NTFs in the excitotoxic death of purified embryonic rat motor neurons. Motor neurons cultured in brain‐derived neurotrophic factor, but not neurotrophin 3, glial‐derived neurotrophic factor, or cardiotrophin 1, were sensitive to excitotoxic insult. BDNF also induces excitotoxic sensitivity (ES) in motor neurons when BDNF is combined with these other NTFs. The effect of BDNF depends on de novo protein and mRNA synthesis. Reagents that either activate or inhibit the 75‐kDa NTF receptor p75NTR do not affect BDNF‐induced ES. The low EC50 for BDNF‐induced survival and ES suggests that TrkB mediates both of these biological activities. BDNF does not alter glutamate‐evoked rises of intracellular Ca2+, suggesting BDNF acts downstream. Both wortmannin and LY294002, which specifically block the phosphatidylinositol 3‐kinase (PI3K) intracellular signaling pathway in motor neurons, inhibit BDNF‐induced ES. We confirm this finding using a herpes simplex virus (HSV) that expresses the dominant negative p85 subunit of PI3K. Infecting motor neurons with this HSV, but not a control HSV, blocks activation of the PI3K pathway and BDNF‐induced ES. Through the activation of TrkB and the PI3K signaling pathway, BDNF renders developing motor neurons susceptible to glutamate receptor‐mediated cell death.

Excitotoxic Death of a Subset of Embryonic Rat Motor Neurons In Vitro

Abstract : We have used cultures of purified embryonic rat spinal cord motor neurons to study the neurotoxic effects of prolonged ionotropic glutamate receptor activation. NMDA and non‐NMDA glutamate receptor agonists kill a maximum of 40% of the motor neurons in a concentration‐ and time‐dependent manner, which can be blocked by receptor subtype‐specific antagonists. subunit‐specific antibodies stain all of the motor neurons with approximately the same intensity and for the same repertoire of subunits, suggesting that the survival of the nonvulnerable population is unlikely to be due to the lack of glutamate receptor expression. Extracellular Ca2+ is required for excitotoxicity, and the route of entry initiated by activation of non‐NMDA, but not NMDA, receptors is L‐type Ca2+ channels. Ca2+ imaging of motor neurons after application of specific glutamate receptor agonists reveals a sustained rise in intracellular Ca2+ that is present to a similar degree in most motor neurons, and can be blocked by appropriate receptor/channel antagonists. Although the lethal effects of glutamate receptor agonists are seen in only a subset of cultured motor neurons, the basis of this selectivity is unlikely to be simply the glutamate receptor phenotype or the level/pattern of rise in agonist‐evoked intracellular Ca2+.

The role of polysialic acid and other carbohydrate polymers in neural structural plasticity.

Polysialic acid (PSA) fulfills several criteria for a molecule involved in structural plasticity, including expression in regions capable of plasticity, re-expression in structures undergoing synaptic rearrangement in the adult, downregulation following innervation, and regulation by activity. In addition, removal of PSA reduces the capacity for structural plasticity. PSA may be paradigmatic for other large polymeric carbohydrates, such as glycosaminoglycans and proteoglycans, which also are highly charged and can be extensively hydrated. These carbohydrates may affect structural plasticity by altering cell-cell and/or cell-matrix interactions by increasing intermolecular spacing through hydration.

Identification and characterization of novel developmentally regulated proteins in rat spinal cord.

We previously used 2-dimensional (2-D) gel electrophoresis to identify novel proteins that may be involved in the genesis of the mammalian nervous system [1]. Several novel proteins that were up- or down-regulated coincident with neurogenesis and neuronal migration in rat neocortex were identified. To further investigate the expression of some of these developmentally regulated proteins during a comparable period in spinal cord development, 2-D electrophoresis is used to study their regulation in the crude membrane and soluble fractions of spinal cord at embryonic day 12 (E12) and embryonic day 21 (E21). This analysis indicates that 7 of the proteins that exhibited large changes in their synthesis in cerebral cortex between embryonic day 14 (E14) and embryonic day 21 (E21) demonstrate similar up- or down-regulation during spinal cord neurogenesis. However, two proteins are restricted in their expression or developmental regulation. One of these, 667-800, appears cortex-specific, while the up-regulation of protein SC.1 appears to be spinal cord specific. Several of these proteins also appear to be enriched in both the cortex and spinal cord relative to non-neural tissues (117, 162, 182, 310 [TOAD-64], 667-800) and may be neural specific. To further characterize its expression, one of these neural-specific, up-regulated proteins, TOAD-64 (protein 310) [2-4], is studied throughout embryonic and postnatal spinal cord development using peptide-specific polyclonal antibodies. As suggested by the 2-D gel analysis and the previously reported expression pattern in cerebral cortex [3], TOAD-64 is transiently expressed in postmitotic spinal cord neurons early in their development and sharply down-regulated after the second postnatal week. In the adult spinal cord, TOAD-64 expression is remarkably restricted to a subset of primary afferents to the spinal cord. This expression pattern, coupled with its recently discovered homology to two proteins implicated in axon pathfinding in the chick and nematode [5,3], suggests that TOAD-64 may have a fundamental role in axon pathfinding.

Reduction of functional N-methyl-D-aspartate receptors in neurons by RNase P-mediated cleavage of the NR1 mRNA.

One approach to studying the functional role of individual NMDA receptor subunits involves the reduction in the abundance of the protein subunit in neurons. We have pursued a strategy to achieve this goal that involves the use of a small guide RNA which can lead to the destruction of the mRNA for a specific receptor subunit. We designed a small RNA molecule, termed ‘external guide sequence’ (EGS), which binds to the NR1 mRNA and directs the endonuclease RNase P to cleave the target message. This EGS has exquisite specificity and directed the RNase P‐dependent cleavage at the targeted location within the NR1 mRNA. To improve the efficiency of this EGS, an in vitro evolution strategy was employed which led to a second generation EGS that was 10 times more potent than the parent molecule. We constructed an expression cassette by flanking the EGS with self‐cleaving ribozymes and this permitted generation of the specified EGS RNA sequence from any promoter. Using a recombinant Herpes simplex virus (HSV), we expressed the EGS in neurons and showed the potency of the EGS to reduce NR1 protein within neurons. In an excitotoxicity assay, we showed that expression of the EGS in cortical neurons is neuroprotective. Our results demonstrate the utility of EGSs to reduce the expression of any gene (and potentially any splice variant) in neurons.