Abstract
BackgroundRecently, we and others proposed plasticity-related gene 3 (PRG3) as a novel molecule in neuritogenesis based on PRG3 overexpression experiments in neuronal and non-neuronal cell lines. However, direct information on PRG3 effects in neuronal development and, in particular, its putative spatio-temporal distribution and conditions of action, is sparse.ResultsWe demonstrate here that PRG3 induces filopodia formation in HEK293 cells depending on its N-glycosylation status. The PRG3 protein was strongly expressed during mouse brain development in vivo from embryonic day 16 to postnatal day 5 (E16 – P5). From P5 on, expression declined. Furthermore, in early, not yet polarized hippocampal cultured neurons, PRG3 was expressed along the neurite shaft. Knock-down of PRG3 in these neurons led to a decreased number of neurites. This phenotype is rescued by expression of an shRNA-resistant PRG3 construct in PRG3 knock-down neurons. After polarization, endogenous PRG3 expression shifted mainly to axons, specifically to the plasma membrane along the neurite shaft. These PRG3 pattern changes appeared temporally and spatially related to ongoing synaptogenesis. Therefore we tested (i) whether dendritic PRG3 re-enhancement influences synaptic currents and (ii) whether synaptic inputs contribute to the PRG3 shift. Our results rendered both scenarios unlikely: (i) PRG3 over-expression had no influence on miniature excitatory postsynaptic currents (mEPSC) and (ii) blocking of incoming signals did not alter PRG3 distribution dynamics. In addition, PRG3 levels did not interfere with intrinsic neuronal properties.ConclusionTaken together, our data indicate that endogenous PRG3 promotes neurite shaft protrusion and therefore contributes to regulating filopodia formation in immature neurons. PRG3 expression in more mature neurons, however, is predominantly localized in the axon. Changes in PRG3 levels did not influence intrinsic or synaptic neuronal properties.
Highlights
We and others proposed plasticity-related gene 3 (PRG3) as a novel molecule in neuritogenesis based on PRG3 overexpression experiments in neuronal and non-neuronal cell lines
Our results indicate that i) PRG3 in immature neurons is located in all neurite shafts, where it determines neurite protrusions; ii) PRG3 expression in more mature neurons is predominantly axonal; iii) PRG3 expression patterns are not influenced by and do not influence synaptic currents; and iv) glycosylation of PRG3 is a prerequisite for membrane insertion
Subcellular distribution of endogenous PRG3 in primary hippocampal neurons changes during development Motivated by the prominent neuronal expression of endogenous PRG3 and its role in filopodia formation, we investigated the subcellular distribution of PRG3 in primary neurons at different developmental stages
Summary
We and others proposed plasticity-related gene 3 (PRG3) as a novel molecule in neuritogenesis based on PRG3 overexpression experiments in neuronal and non-neuronal cell lines. Neurite elongation and branching are governed by coordinated rearrangements of the cytoskeletal dynamics of actin and microtubules [1,2,3]. The actin cytoskeleton provides protrusive and contractile forces and microtubules form a polarized network allowing organelle and protein movement throughout the cell. Neurite protrusion requires a protrusive force to push the plasma membrane forward, as well as an addition of membrane materials. This includes microtubule fragmentation and local accumulation of F-actin for the formation of filopodia and lamellipodia and subsequent neurite branching, called sprouting [4]. Calpain, which is localized along the neurite shaft, prevents branching through the proteolysis of cortactin [10]
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