Cyclooxygenase-2 (COX-2), a key enzyme in the biosynthesis of prostaglandins, is induced in brain blood vessels by pyrogens, and its essential role in fever has been hypothesized. In this study, we determined (1) the type of cells that express cyclooxygenase-2 in brain blood vessels of lipopolysaccharide-treated rats, and (2) the precise relationship between the time course of fever and that of cyclooxygenase-2 protein expression in these cells. Five hours after the lipopolysaccharide injection (100 microg/kg, i.p.), cyclooxygenase-2-like immunoreactive cells were found in the parenchymal and subarachnoidal blood vessels. In these blood vessels, the cyclooxygenase-2-like immunoreactivity was restricted to the perinuclear region of the endothelial cells as revealed by a laser confocal microscopy, double-immunofluorescence staining with an endothelial marker, and immunoelectron microscopy. On the other hand, the cyclooxygenase-2-like immunoreactive cells were distinct from microglia or perivascular/meningeal macrophages as revealed by double immunostaining with macrophage/microglia-specific antibodies. Cyclooxygenase-2-like immunoreactive cells were first found at 1.5 hr after the lipopolysaccharide injection, at which time the fever had not been developed. After that, the number of cyclooxygenase-2-like immunoreactive cells and fever followed a similar time course, both being highest at 5 hr after the lipopolysaccharide injection and both returning to the baseline by 24 hr. These results demonstrate that brain endothelial cells are the primary sites where the activation of arachidonic acid cascade takes place during fever after intraperitoneal injection of lipopolysaccharide.

Neuronal growth-associated proteins (nGAPs) are in general neuron-specific gene products whose expression correlates tightly with neuronal process outgrowth and/or regeneration, and are mostly good downstream targets of neurotrophin stimulation. Expression of genes encoding nGAPs such as GAP-43, SCG10, and stathmin is upregulated following lesioning of cortical and hippocampal regions of the adult rat brain. In the brains of aged animals, however, the magnitude of the response is reduced, whereas the time course of the response is mostly unchanged when compared with that for brains of young ones. Expression of GAP-43 and stathmin is reduced by aging, and is also changed in age-related neurodegenerative conditions such as Alzheimer's disease in humans. Certain nGAPs are induced during long-term potentiation (LTP) and also during critical periods of song-learning and ocular dominance column formation in birds and cats, respectively. Recent evidence further supports the idea that functional synaptic modulation is often associated with remodeling of synaptic structures. These results suggest that neurotrophin-responsive nGAPs serve as molecular markers of neuronal plasticity during development and aging, and that the neuronal plasticity decreases, at least in certain neuronal circuits, in the aged brain and neurodegenerative diseases. Recent findings on the roles of stathmin and SCG10-related proteins in microtubule destabilization and its functional block by phosphorylation further support the importance of the SCG10 family proteins in neuronal cytoskeletal regulation, particularly as to microtubule dynamics. We summarize here a decade of research on SCG10 and its related molecules with special interests to brain aging and disease.

Microtubule dynamics, one of the key elements in neurite outgrowth, is regulated by various regulatory factors to determine the behavior of the neuronal growth cone and to form the specialized neuronal shape. SCG10 is a neuron-specific stathmin protein with a potent microtubule destabilizing factor and is enriched in the growth cones of the developing neurons. We investigated the functional role of SCG10 in neurite outgrowth using rat hippocampal primary cultured neurons. Genetic manipulation of SCG10 using a short-interfering RNA duplex markedly decreased the SCG10 expression level and significantly suppressed neurite outgrowth. This result was confirmed by immunodepletion experiments. On the other hand, the protein transduction of SCG10 using a polyarginine tag stimulated neurite outgrowth. Such manipulation of the SCG10 expression level affected microtubule morphology within the growth cones. A decrease in the SCG10 level converted the morphology to a more stable state, while an increase converted the morphology to a more dynamic state. However, an excess of SCG10 induced neurite retraction due to an excess of microtubule disassembly. These results suggest that SCG10 serves as an important regulatory factor of growth cone motility by enhancing microtubule dynamics, possibly through increasing the catastrophe frequency.

Neurotrophins are key regulators of the fate and shape of neuronal cells and act as guidance cues for growth cones by remodeling the actin cytoskeleton. Actin dynamics is controlled by Rho GTPases. We identified a novel Rho GTPase-activating protein (Grit) for Rho/Rac/Cdc42 small GTPases. Grit was abundant in neuronal cells and directly interacted with TrkA, a high-affinity receptor for nerve growth factor (NGF). Another pool of Grit was recruited to the activated receptor tyrosine kinase through its binding to N-Shc and CrkL/Crk, adapter molecules downstream of activated receptor tyrosine kinases. Overexpression of the TrkA-binding region of Grit inhibited NGF-induced neurite elongation. Further, we found some tendency for neurite promotion in full-length Grit-overexpressing PC12 cells upon NGF stimulation. These results suggest that Grit, a novel TrkA-interacting protein, regulates neurite outgrowth by modulating the Rho family of small GTPases.