Hydrophilic anti-migraine triptans are substrates for OATP1A2, a transporter expressed at human blood-brain barrier.

OATP1A2 is expressed in the luminal membrane of human blood-brain barrier (BBB). The human tissue with the highest OATP1A2 mRNA expression is the brain. We have established a robust BacMam2-OATP1A2 transduced HEK293 system. Among the 36 central nervous system (CNS) marketed drugs tested, hydrophilic triptans, 5-HT(1B/1D) receptor agonists for the treatment of migraine attacks, were identified as OATP1A2 substrates. Kinetics (K(m) and V(max)) were determined for six marketed triptans. Structure-activity relationship (SAR) obtained from 18 triptan structural analogs revealed that the positively charged basic amine atom was essential for efficient OATP1A2-mediated triptan uptake and uptake rate was in the order of tertiary > secondary > primary. Preliminary quantitative SAR analysis of the triptan analogs demonstrated positive correlation between OATP1A2-mediated uptake rate and van der Waals volume (vdw_vol). OATP1A2 was specifically expressed on the apical side of MDCKII monolayer after BacMam2-OATP1A2 transduction and can facilitate transport of triptans across the MDCKII monolayer from apical to basolateral side. Involvement of OATP1A2 for brain penetration of triptans in human requires further investigation.

Leading tip drives soma translocation via forward F-actin flow during neuronal migration.

Neuronal migration involves coordinated extension of the leading process and translocation of the soma, but the relative contribution of different subcellular regions, including the leading process and cell rear, in driving soma translocation remains unclear. By local manipulation of cytoskeletal components in restricted regions of cultured neurons, we examined the molecular machinery underlying the generation of traction force for soma translocation during neuronal migration. In actively migrating cerebellar granule cells in culture, a growth cone (GC)-like structure at the leading tip exhibits high dynamics, and severing the tip or disrupting its dynamics suppressed soma translocation within minutes. Soma translocation was also suppressed by local disruption of F-actin along the leading process but not at the soma, whereas disrupting microtubules along the leading process or at the soma accelerated soma translocation. Fluorescent speckle microscopy using GFP-alpha-actinin showed that a forward F-actin flow along the leading process correlated with and was required for soma translocation, and such F-actin flow depended on myosin II activity. In migrating neurons, myosin II activity was high at the leading tip but low at the soma, and increasing or decreasing this front-to-rear difference accelerated or impeded soma advance. Thus, the tip of the leading process actively pulls the soma forward during neuronal migration through a myosin II-dependent forward F-actin flow along the leading process.

Long-range Ca2+ signaling from growth cone to soma mediates reversal of neuronal migration induced by slit-2.

Neuronal migration and growth-cone extension are both guided by extracellular factors in the developing brain, but whether these two forms of guidance are mechanistically linked is unclear. Application of a Slit-2 gradient in front of the leading process of cultured cerebellar granule cells led to the collapse of the growth cone and the reversal of neuronal migration, an event preceded by a propagating Ca(2+) wave from the growth cone to the soma. The Ca(2+) wave was required for the Slit-2 effect and was sufficient by itself to induce the reversal of migration. The Slit-2-induced reversal of migration required active RhoA, which was accumulated at the front of the migrating neuron, and this polarized RhoA distribution was reversed during the migration reversal induced by either the Slit-2 gradient or the Ca(2+) wave. Thus, long-range Ca(2+) signaling coordinates the Slit-2-induced changes in motility at two distant parts of migrating neurons by regulating RhoA distribution.

Ca2+-dependent regulation of rho GTPases triggers turning of nerve growth cones.

Cytoplasmic Ca2+ elevation and changes in Rho GTPase activity are both known to mediate axon guidance by extracellular factors, but the causal relationship between these two events has been unclear. Here we show that direct elevation of cytoplasmic Ca2+ by extracellular application of a low concentration of ryanodine, which activated Ca2+ release from intracellular stores, upregulated Cdc42/Rac, but downregulated RhoA, in cultured cerebellar granule cells and human embryonic kidney 293T cells. Chemoattractive turning of the growth cone triggered by a gradient of ryanodine was blocked by overexpression of mutant forms of Cdc42 but not of RhoA in Xenopus spinal cord neurons. Furthermore, Ca2+-induced GTPase activity correlated with activation of protein kinase C and required a basal activity of Ca2+/calmodulin-dependent protein kinase II. Thus, Rho GTPases may mediate axon guidance by linking upstream Ca2+ signals triggered by guidance factors to downstream cytoskeletal rearrangements.

Calcium signaling in chemorepellant Slit2-dependent regulation of neuronal migration.

Migration of neuronal precursor cells in the developing brain is guided by extracellular cues, but intracellular signaling processes underlying the guidance of neuronal migration are largely unknown. By examining the migration of cerebellar granule neurons along the surface of cocultured astroglial cells, we found that an extracellular gradient of Slit2, a chemorepellant for neuronal migration in vivo, caused a reversal in the direction of migration without affecting the migration speed. A Slit2 gradient elevated the intracellular concentration of Ca2+, probably due to calcium release from the internal store, led to a reversal of the preexisting asymmetric intracellular Ca2+ distribution in the soma of migrating neurons, and this reversal was closely related with its action of reversing the migrating direction. Asymmetric Ca2+ distribution in the soma was both necessary and sufficient for directing neuronal migration. These results have demonstrated an important role for Ca2+ in mediating neuronal responses to Slit2 and suggest a general mechanism for neuronal guidance.