KIF1A is the primary anterograde motor protein required for the axonal transport of dense-core vesicles in cultured hippocampal neurons
Research highlights
► Dense core vesicles and KIF1A co-transport in a bidirectional manner in axons. ► Knockdown of KIF1A reduces bidirectional axonal transport of dense-core vesicles. ► Dense-core vesicles likely contain a coordinated motor complex.
Introduction
Secreted signaling molecules, such as chemical neurotransmitters and signaling peptides, are released into the synaptic cleft from either small, clear synaptic vesicles (SVs) or large, dense-core vesicles (DCVs). SVs are filled with classical neurotransmitters locally at presynaptic release sites. By contrast, DCVs are formed and filled with signaling peptide cargos in the cell body, and then travel long distances into the axon and dendrites to pre- and postsynaptic sites. During transit, DCV neuropeptide contents are processed and then released upon stimulation [28]. A wide range of biological processes is facilitated by DCV cargos, including neuronal survival, development, learning and memory [5]. For example, brain-derived neurotrophic factor (BDNF), a neuropeptide transported in DCVs, is required for neuronal development, both at the level of circuit development in the brain and synaptic maturation and function [36]. In the axon, DCVs must travel extremely long distances, emphasizing the need for active cytoskeletal transport and microtubule-based motors such as kinesin family members and dynein [37], [4], [21]. Despite the importance of DCVs in neuronal physiology, little is known regarding the motors required for their axonal transport.
Over 45 kinesins have been identified in the mammalian genome. As motor-cargo interactions are very specific [13], these motors must distinguish among a multitude of cellular cargos (or vice versa) for efficient transport. Previous work has shown that in Drosophila and C. elegans, the homolog of mammalian KIF1A, unc-104, is required for axonal transport of DCVs. Mutant animals lacking UNC-104 showed transport and localization defects of the DCV cargos, including atrial natriuretic factor (ANF), IDA-1, and critical neuropeptide processing enzymes [37], [4], [16]. Co-immunoprecipitation studies in mouse brain suggest a conserved role of KIF1A in the transport of DCVs in mammals [24], [3]. However, until now, no functional data exists demonstrating the reliance of DCV transport on KIF1A in mammalian neurons. Through the use of live-cell, two-color imaging of fluorescently tagged KIF1A and DCV marker proteins in axons and shRNA technology, we demonstrate that KIF1A is the primary motor responsible for fast axonal transport of DCVs in primary cultured hippocampal neurons. Additionally, we show that KIF1A, an anterograde motor, co-transports with DCVs that move in the retrograde direction, indicating that KIF1A can remain associated with DCVs as cargo. More generally, these data contribute to the understanding of molecular mechanisms of transport in neurons, an area of intensive investigation in the context of basic nerve cell biology, as well as synaptic plasticity and neurodegeneration [29].
Section snippets
Hippocampal cell culture and expression of transgenes
Primary cultures of dissociated neurons from rat embryonic day 18 (E18) hippocampi were prepared as previously described [17]. Plasmids were transfected into neurons after 7 DIV and allowed to express for 24–48 h before imaging. Lipofectamine 2000 (Invitrogen) was used to transfect cells with 1 μg of each plasmid, as described by Sampo et al. [27]. For immunoblot analysis of KIF1A protein levels, freshly dissociated E18 hippocampal neurons were transfected with shRNA plasmids by electroporation,
Results
If the location and movements of two labeled proteins are identical – that is, they start together, travel at the same rates, and stop together – it is likely they reside on or in the same organelle. To determine if KIF1A co-transports with DCVs, KIF1A-GFP was co-expressed with either BDNF-RFP or ChrA-RFP in primary cultured hippocampal neurons. Axonal transport of each DCV marker and KIF1A-GFP was then examined by two-color, live-cell imaging in cells where the orientation of the axon in
Discussion
Previous studies have implicated KIF1A in the transport of DCVs in mammalian neurons [24], [3]. Here, using a combination of live-cell imaging and RNAi technology, we have shown a direct role for KIF1A in the microtubule-based transport of DCVs in axons of cultured rat hippocampal neurons. In addition, we provide evidence that KIF1A remains associated with DCVs during retrograde axonal transport, demonstrating that the DCVs retain molecular machinery readily competent of transport in either
Acknowledgements
This research was supported by grants from the Natural Science and Engineering Research Council, (NSERC; #327100-06), the Canadian Foundation for Innovation (CFI; #12793), and the Canadian Institutes of Health Research (CIHR; #90396). We thank David Kwinter for initiating these studies, Diana Hunter for her expert technical assistance, and Drs. Nick Inglis and Elisa Ramser for their critical reading of this manuscript. We also thank the Simon Fraser University Animal Care Services staff for
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These authors contributed equally to this work.
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Present address: Centre National de la Recherche Scientifique UMR 5091, Physiologie Cellulaire de la Synapse, Université Bordeaux, Bordeaux, France.