Abstract
ABSTRACTNeurofilaments (NFs) are thought to provide structural support to mature axons via crosslinking of cytoskeletal elements mediated by the C-terminal region of the high molecular weight NF subunit (NF-H). Herein, we inhibited NF-H expression in differentiating mouse NB2a/d1 cells with shRNA directed against murine NF-H without affecting other NF subunits, microtubules or actin. shRNA-mediated NF-H knockdown not only in compromised of late-stage axonal neurite stabilization but also compromised early stages of axonal neurite elongation. Expression of exogenous rat NF-H was able to compensate for knockdown of endogenous NF-H and restored the development and stabilization of axonal neurites. This rescue was prevented by simultaneous treatment with shRNA that inhibited both rat and murine NF-H, or by expression of exogenous rat NF-H lacking the C-terminal sidearm during knockdown of endogenous NF-H. Demonstration of a role for NF-H in the early stages of axonal elaboration suggests that axonal stabilization is not delayed until synaptogenesis, but rather that the developing axon undergoes sequential NF-H-mediated stabilization along its length in a proximal–distal manner, which supports continued pathfinding in distal, unstabilized regions.
Highlights
Establishment of appropriate connections among neurons in the developing nervous system is accomplished by detection and response to a complex series of guidance cues and ultimate target recognition, collectively referred to as ‘‘axonal pathfinding’’
Knockdown of NF-H compromised axonal outgrowth and stabilization Since treatment with this shRNA cocktail reduced NF-H levels, we examined the impact of transfection with
Phospho-mediated interactions mediated by NF-H are thought to provide stability to the axonal cytoskeleton
Summary
Establishment of appropriate connections among neurons in the developing nervous system is accomplished by detection and response to a complex series of guidance cues and ultimate target recognition, collectively referred to as ‘‘axonal pathfinding’’ (for a review, see Raper and Mason, 2010) During this process, a neuron must modify its axonal cytoskeleton from a relatively plastic scaffold that accommodates rapid elongation into a stabilized structure that essentially locks into place in order to maintain synaptic connections for the lifetime of the individual. Initial elaboration of putative axonal neurites requires localized depolymerization of subcortical actin filaments and Center for Cellular Neurobiology and Neurodegeneration Research, Department of Biological Sciences, University of Massachusetts at Lowell, Lowell, MA 01854, USA Continued elongation of putative axonal neurites is no longer dependent upon vimentin which is downregulated within 24 hr following axonal initiation (Shea et al, 1993; Yabe et al, 2003), but instead requires neurofilaments (NFs), which are the major intermediate filament of mature neurons (Shea and Beermann, 1999)
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