Glutamate Regulates the Activity of Topoisomerase I in Mouse Cerebellum

Abstract

Topoisomerase I (topo I) is a nuclear enzyme which participates in most DNA transactions. It was shown to be inhibited in depolarized neurons by poly adenosine diphosphate (ADP)-ribosylation of the enzyme protein. We demonstrated previously an age and sex dependent topo I activity and enzyme protein level in the various regions of mouse brain. A specific distribution pattern of topo I was observed and the inhibitory neurons exhibited the highest enzyme activity and protein level in both the nucleus and the cytoplasm. Here, we show that neurotransmitters (glutamate and gamma-aminobutyric acid (GABA)) regulate the activity of topo I in mouse cerebellum sections. Glutamate exhibited a significant time-dependent inhibition of topo I activity but no effect of the enzyme protein level. GABA in contrary only slightly and transiently inhibited topo I activity. The inhibitory effect of glutamate was mediated by Ca(+2) and by ADP-ribosylation of topo I protein and the glutamate ionotropic receptors were involved. Glutamate also diminished the inhibitory effect of topotecan on topo I. These results point to distinct and highly specific effects of the major neurotransmitters on topo I activity in the cerebellum suggesting that topo I possesses a specific role in the brain which differs from its known biological functions.

Topoisomerase 1 inhibition reversibly impairs synaptic function

“…Given these graded transcriptional effects, synaptic function and brain development are likely to be impacted in proportion to how strongly TOP1 is inhibited by chemicals or mutations. Moreover, TOP1 activity increases from birth to maturity (16) and can be inhibited by the activation of glutamate or GABA receptors in mouse cerebellum (45). Thus, several mechanisms may regulate the transcriptional activities of TOP1 in neurons in a graded manner.…”

Excitotoxic and Radiation Stress Increase TERT Levels in the Mitochondria and Cytosol of Cerebellar Purkinje Neurons

“…Brain slices were prepared as previously described [40]. Briefly, brains were quickly removed from the skull, washed with Ringer solution (150 mM NaCl, 4.6 mM KCl, 26 mM HCO 3 Cl, 1.3 mM Na 2 HPO 4 , 2.5 mM CaCl 2 , 1 mM MgCl 2 , 1 mM SO 4 CH 3 , and 10 mM glucose (pH 7)., placed in oxygenated Ringer solution at 4°C, and coronal sections (400 μm thick) of the cerebellum were prepared.…”

“…The brains were homogenized using a manual homogenizer (pestle B). The homogenates were centrifuged at 500 × g at 4°C for 7 min and the pellets were subjected to nuclear and cytoplasmic extractions, as previously described [40–42]. Briefly, for the cytoplasmic extract, cells were re-suspended in buffer A (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 1 mM EDTA) containing a mixture of protease inhibitors [final concentrations: 2 μg/ml aprotinin, 2 μg/ml antipain, 2 μg/ml leupeptin, 1 μg/ml pepstatin A, 2 μg/ml PMSF (phenylmethylsulfonyl fluoride)], and kept on ice for 15 min.…”

Dissociation between telomerase activity and expression during mice cortical development due to a DNA-bound telomerase inhibitor 

“…The cytoplasmic extract was prepared as previously described [12]. Briefly, cells were resuspended in buffer A (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 1 mM EDTA) containing a mixture of protease inhibitors [final concentrations: 2 μg/ml aprotinin, 2 μg/ml antipain, 2 μg/ml leupeptin, 1 μg/ml pepstatin A, 2 μg/ml PMSF (phenylmethylsulfonyl fluoride)], and kept on ice for 15 min.…”