Mechanisms of the Release of Anterogradely Transported Neurotrophin-3 from Axon Terminals

Abstract

Neurotrophins have profound effects on synaptic function and structure. They can be derived from presynaptic, as well as postsynaptic, sites. To date, it has not been possible to measure the release of neurotrophins from axon terminals in intact tissue. We implemented a novel, extremely sensitive assay for the release and transfer of anterogradely transported neurotrophin-3 (NT-3) from a presynaptic to a postsynaptic location that uses synaptosomal fractionation after introduction of radiolabeled NT-3 into the retinotectal projection of chick embryos. Release of the anterogradely transported NT-3 in intact tissue was assessed by measuring the amount remaining in synaptosomal preparations after treatment of whole tecta with pharmacological agents. Use of this assay reveals that release of NT-3 from axon terminals is increased by depolarization, calcium influx via N-type calcium channels, and cAMP analogs, and release is most profoundly increased by excitation with kainic acid or mobilization of calcium from intracellular stores. NT-3 release depends on extracellular sodium, CaM kinase II activity, and requires intact microtubules and microfilaments. Dantrolene inhibits the high potassium-induced release of NT-3, indicating that release of calcium from intracellular stores is required. Tetanus toxin also inhibits NT-3 release, suggesting that intact synaptobrevin or synaptobrevin-like molecules are required for exocytosis. Ultrastructural autoradiography and immunolabel indicate that NT-3 is packaged in presumptive large dense-core vesicles. These data show that release of NT-3 from axon terminals depends on multiple regulatory proteins and ions, including the mobilization of local calcium. The data provide insight in the mechanisms of anterograde neurotrophins as synaptic modulators. ; presynaptic terminals; calcium; axonal transport; neurotrophin; synapse; synaptic transmission Neurotrophins are important regulators of neuronal differentiation and synaptic plasticity (Lohof et al., 1993;Snider, 1994;Kang and Schuman, 1995;Thoenen, 1995;McAllister et al., 1999;Poo, 2001). Traditionally, neurotrophins were believed to be derived by retrograde axonal transport from target cells, but recent studies revealed that neurotrophins are also transported anterogradely along axons (von Bartheld et al Key words: neurotrophic factors; secretion

Cellular Mechanisms Regulating Activity-Dependent Release of Native Brain-Derived Neurotrophic Factor from Hippocampal Neurons

“…Our results demonstrate that activity-dependent release of native BDNF from newborn hippocampal neurons requires calcium influx specifically through N-type channels, regardless of the pattern of stimulation. A similar requirement for N-type channels in release of neurotrophin-3 from retinotectal axon terminals has been reported recently (Wang et al, 2002). Our findings are supported by observations that blockade of N-type, but not L-type, channels also leads to a significant reduction of synaptic vesicle recycling in newborn hippocampal neurons (Pravettoni et al, 2000).…”

The Secretory Granule-Associated Protein CAPS2 Regulates Neurotrophin Release and Cell Survival

“…However, little research into the molecules involved in the neurotrophin release from neurons has been reported (Canossa et al, 2001;Balkowiec and Katz, 2002;Wang et al, 2002). Thus, the present finding provides a clue to elucidating the underlying molecular basis.…”

“…This feature in the molecular composition and electron microscopic view is likely consistent with the fact that CGB does not play a direct role in dense-core SG biogenesis in PC12 cells (Corradi et al, 1996;Kim et al, 2001). On the contrary, the presence of NT-3 and BDNF in large dense-core SGs has been reported in the retinotectal terminals of chicken embryos (Wang et al, 2002) and the axonal terminals of rat dorsal root ganglions (Michael et al, 1997), respectively. Cerebellar granule cells, although they express BDNF and NT-3, do not express CGA mRNAs (Mahata et al, 1991).…”

“…Recent evidence indicates their modulatory roles in synaptic plasticity such as long-term potentiation relevant to learning and memory (Lu and Figurov, 1997;Kovalchuk et al, 2002;Egan et al, 2003) and in neuronal diseases, including Alzheimer's disease and Parkinson's disease (Murer et al, 2001). It was reported that increasing intracellular Ca 2ϩ regulates activity dependency on the neurotrophin release from neuronal processes (Canossa et al, 2001;Balkowiec and Katz, 2002;Wang et al, 2002). However, the underlying molecular mechanism is poorly understood.…”

Neurotrophin-4, Alone or Heterodimerized with Brain-derived Neurotrophic Factor, Is Sorted to the Constitutive Secretory Pathway

“…To our knowledge, neither receptor has been found expressed in AtT-20 cells. The regulated release of neurotrophins from PC12 cells may reflect release of recycled rather than freshly produced protein (40).…”