Cocaine promotes primary human astrocyte proliferation via JNK-dependent up-regulation of cyclin A2.

PURPOSE: Astrocytes perform a plethora of important functions in the central nervous system (CNS) and are involved in cocaine-evoked synaptic plasticity. Previously, we showed that while cocaine decreased cyclin A2 expression in primary human neural progenitor cells, it increased cyclin A2 expression in human astrocytes. Since cyclin A2 is an essential regulator of the cell cycle, the aim of the present study is to clarify the effect of cocaine on proliferation of human astrocytes and elucidate the underlying molecular mechanisms. METHODS: Primary human astrocytes were treated with either 1, 10, or 100 µM cocaine for 48 hr, and cell proliferation was measured using the CyQUANT cell proliferation assay. To elucidate the molecular mechanisms through which cocaine affects the proliferation of astrocytes, we analyzed gene expression profiles in cocaine-treated primary human astrocytes using a human focused cDNA array. Gene ontology/pathway enrichment analysis, STRING protein-protein interaction analysis, RT-qPCR, and western blotting were used to identify signal transduction pathways that are involved in cocaine-induced astrocyte dysfunction. RESULTS: Cocaine at 10 and 100 µM significantly increased human astrocyte proliferation. Gene expression profiling revealed the JNK MAP kinase pathway as a driver of cell proliferation affected by cocaine in human astrocytes. Further experiments showed that cocaine-induced JNK activation induced up-regulation of cyclin A2, leading to enhanced proliferation of human astrocytes. CONCLUSION: Cocaine-induced abnormal increases in the number of astrocytes may cause disruption in neuron-glia signaling and contribute to synaptic impairment in the CNS. Understanding the mechanisms of cocaine's effects on human astrocytes may help to reveal the involvement of glial cells in addictive behaviors.

Novel changes in gene expression following axotomy of a sympathetic ganglion: a microarray analysis.

Neurons of the peripheral nervous system are capable of extensive regeneration following axonal injury. This regenerative response is accompanied by changes in gene expression in axotomized neurons and associated nonneuronal cells. In the sympathetic nervous system, a few of the genes affected by axonal injury have been identified; however, a broad sampling of genes that could reveal additional and unexpected changes in expression has been lacking. We have used DNA microarray technology to study changes in gene expression within 48 h of transecting the postganglionic trunks of the adult rat superior cervical ganglion (SCG). The expression of more than 200 known genes changed in the ganglion, most of these being genes not previously associated with the response to injury. In contrast, only 10 genes changed following transection of the preganglionic cervical sympathetic trunk. Real-time RT-PCR analysis verified the upregulation of a number of the axotomy-induced genes, including activating transcription factor-3 (ATF-3), arginase I (arg I), cardiac ankyrin repeat protein, galanin, osteopontin, pituitary adenylate cyclase-activating polypeptide (PACAP), parathyroid hormone-related peptide, and UDP-glucoronosyltransferase. Arg I mRNA and protein were shown to increase within neurons of the axotomized SCG. Furthermore, increases in the levels of putrescine and spermidine, a diamine and polyamine produced downstream of arg I activity, were also detected in the axotomized SCG. Our results identified many candidate genes to be studied in the context of peripheral nerve regeneration. In addition, the data suggest a potential role for putrescine and spermidine, acting downstream of arg I, in the regenerative process.

A comparison of the changes in the non-neuronal cell populations of the superior cervical ganglia following decentralization and axotomy.

Transecting the axons of neurons in the adult superior cervical ganglion (SCG; axotomy) results in the survival of most postganglionic neurons, the influx of circulating monocytes, proliferation of satellite cells, and changes in neuronal gene expression. In contrast, transecting the afferent input to the SCG (decentralization) results in nerve terminal degeneration and elicits a different pattern of gene expression. We examined the effects of decentralization on macrophages in the SCG and compared the results to those previously obtained after axotomy. Monoclonal antibodies were used to identify infiltrating (ED1+) and resident (ED2+) macrophages, as well as macrophages expressing MHC class II molecules (OX6+). Normal ganglia contained ED2+ cells and OX6+ cells, but few infiltrating macrophages. After decentralization, the number of infiltrating ED1+ cells increased in the SCG to a density about twofold greater than that previously seen after axotomy. Both the densities of ED2+ and OX6+ cells were essentially unchanged after decentralization, though a large increase in OX6+ cells occurred after axotomy. Proliferation among the ganglion's total non-neuronal cell population was examined and found to increase about twofold after decentralization and about fourfold after axotomy. Double-labeling experiments indicated that some of these proliferating cells were macrophages. After both surgical procedures, the percentage of proliferating ED2+ macrophages increased, while neither procedure altered the proliferation of ED1+ macrophages. Axotomy, though not decentralization, increased the proliferation of OX6+ cells. Future studies must address what role(s) infiltrating and/or resident macrophages play in regions of decentralized and axotomized neurons and, if both are involved, whether they play distinct roles.