Antisense Morpholino Oligonucleotides Reduce Neurofilament Synthesis and Inhibit Axon Regeneration in Lamprey Reticulospinal Neurons

Abstract

The sea lamprey has been used as a model for the study of axonal regeneration after spinal cord injury. Previous studies have suggested that, unlike developing axons in mammal, the tips of regenerating axons in lamprey spinal cord are simple in shape, packed with neurofilaments (NFs), and contain very little F-actin. Thus it has been proposed that regeneration of axons in the central nervous system of mature vertebrates is not based on the canonical actin-dependent pulling mechanism of growth cones, but involves an internal protrusive force, perhaps generated by the transport or assembly of NFs in the distal axon. In order to assess this hypothesis, expression of NFs was manipulated by antisense morpholino oligonucleotides (MO). A standard, company-supplied MO was used as control. Axon retraction and regeneration were assessed at 2, 4 and 9 weeks after MOs were applied to a spinal cord transection (TX) site. Antisense MO inhibited NF180 expression compared to control MO. The effect of inhibiting NF expression on axon retraction and regeneration was studied by measuring the distance of axon tips from the TX site at 2 and 4 weeks post-TX, and counting the number of reticulospinal neurons (RNs) retrogradely labeled by fluorescently-tagged dextran injected caudal to the injury at 9 weeks post-TX. There was no statistically significant effect of MO on axon retraction at 2 weeks post-TX. However, at both 4 and 9 weeks post-TX, inhibition of NF expression inhibited axon regeneration.

The role of RhoA in retrograde neuronal death and axon regeneration after spinal cord injury

“…S1A), whose regeneration probabilities have been determined previously (Jacobs et al ., 1997; Busch & Morgan, 2012). Interestingly, the large RS axons do not demonstrate collateral sprouting after spinal cord hemi-TX, but only true regeneration (Zhang et al ., 2015), and this, together with the ready regeneration after complete TX, allows us to study mechanisms of regeneration unambiguously. Moreover, after spinal cord TX many injured axon tips can be observed in vivo (Fig.…”

“…Previous studies from our lab and others’ showed that a very delayed form of retrograde neuronal death occurs in spinal-projecting neurons of the brain after SCI in lampreys (Shifman et al ., 2008; Busch & Morgan, 2012; Hu et al ., 2013; Zhang et al ., 2015). Similar phenomena have been reported in mammals (Mori et al ., 1997; Houle & Ye, 1999; Novikova et al ., 2000; Hains et al ., 2003).…”

“…Unlike the situation in mammalian SCI studies, because our SCI model is a complete TX, the growth of RS axons caudal to the lesion represents true regeneration of injured axons, not collateral sprouting of spared axons. Indeed, after spinal cord hemi-section in lamprey, collateral sprouting was not observed (Zhang et al ., 2015). Thus the present findings add evidence to support therapeutic use of RhoA inhibitors to promote true axon regeneration after SCI.…”

“…This allows us to test the role of inhibiting RhoA expression on retrograde neuronal death unambiguously. In the lamprey, fluorescently-labeled morpholino antisense oligonucleotides (MOs) are efficiently transported retrogradely from the site of axotomy, labeling the RS neurons (Zhang et al ., 2015). MOs bind to complementary RNA to knock down gene expression specifically (Summerton, 2007).…”

Reducing synuclein accumulation improves neuronal survival after spinal cord injury

“…As a control, we used a 5-base pair mismatch synuclein morpholino (MM MO). The lissamine-labeled morpholinos were loaded at the time and site of spinal transection, as is standard in zebrafish and lamprey SCI models (Becker et al, 2004; Zhang et al, 2015). Afterwards, fluorescence in the cell body indicated that the morpholinos were retrogradely transported to the RS neuron cell bodies, where they remained up to 11 weeks post-transection (Fig.…”