Krüppel-like Factor 7 engineered for transcriptional activation promotes axon regeneration in the adult corticospinal tract

Abstract

Axon regeneration in the central nervous system normally fails, in part because of a developmental decline in the intrinsic ability of CNS projection neurons to extend axons. Members of the KLF family of transcription factors regulate regenerative potential in developing CNS neurons. Expression of one family member, KLF7, is down-regulated developmentally, and overexpression of KLF7 in cortical neurons in vitro promotes axonal growth. To circumvent difficulties in achieving high neuronal expression of exogenous KLF7, we created a chimera with the VP16 transactivation domain, which displayed enhanced neuronal expression compared with the native protein while maintaining transcriptional activation and growth promotion in vitro. Overexpression of VP16-KLF7 overcame the developmental loss of regenerative ability in cortical slice cultures. Adult corticospinal tract (CST) neurons failed to upregulate KLF7 in response to axon injury, and overexpression of VP16-KLF7 in vivo promoted both sprouting and regenerative axon growth in the CST of adult mice. These findings identify a unique means of promoting CST axon regeneration in vivo by reengineering a developmentally down-regulated, growth-promoting transcription factor. A xon regeneration generally fails after injury to the CNS, preventing substantial recovery. CNS regeneration is limited in part by the presence of extrinsic inhibitory molecules at the injury site, and also because adult CNS neurons possess an intrinsically low capacity for axon growth compared with embryonic or peripheral nervous system (PNS) neurons (1-3). Intrinsic regenerative capacity appears to be particularly low in the corticospinal tract (CST), an essential motor control pathway and important therapeutic target in humans (4). For instance, CST axons have shown mixed responses when inhibitory signals are neutralized (5, 6), and regenerate minimally or not at all into growth permissive tissue grafts that support some regeneration from propriospinal and brainstem neurons (7-10).The intrinsic molecular mechanisms that limit CNS axon growth remain unclear, but likely reflect a suboptimal pattern of regenerative gene expression (1, 11). In sensory neurons, overexpression of GAP43 and CAP23 or transcription factors, including ATF3, STAT3, or ID2, have produced modest gains in spinal regeneration (12-15). In the visual system, overexpression of p300 modestly increases growth (16), and knockdown of PTEN and SOCS3 results in substantial regeneration (17,18). In the CST, overexpression of the neurotrophin receptor TrkB enables regeneration into subcortical BDNF-secreting tissue grafts, and, notably, knockout of PTEN evokes regeneration in the spinal cord (19,20). The regenerative response evoked by these gene manipulations, including PTEN knockout, remains incomplete in terms of both the distance traveled by regenerating axons and the percent of neurons that respond to treatment.These findings illustrate the critical need to develop additional tools to enhance the intrinsic growth state of CNS neurons.We...

KLF7-transfected Schwann cell graft transplantation promotes sciatic nerve regeneration

“…These results are consistent with former results from similar studies performed previously (Veldman et al, 2007). KLF7 can regulate several target genes, including the NGF (Caiazzo et al, 2010), TrkA (Lei et al, 2001;Lei et al, 2006), TrkB (Kingsbury and Krueger, 2007) and GAP43 (Blackmore et al, 2012;Kajimura et al, 2007). As stated earlier, KLF7 overexpression in SCs likely functions therapeutically through the onset of downstream signaling through its various growth and repair targets.…”

“…KLF7 has been found to be strongly activated in response to nerve injury, mediating axonal response to the injury (Veldman et al, 2007;Zou et al, 2009). KLF7 can promote both sprouting and regenerative axon growth in the corticospinal tract (CST) of adult mice (Blackmore et al, 2012). Our previous studies demonstrated that KLF7 promotes the axonal regeneration of the peripheral motor and sensory nerve (Wang et al, 2016), which suggests that KLF7 may play a role in enhancing the neuronal intrinsic growth capacity (Moore et al, 2009).…”

“…Our previous studies demonstrated that KLF7 promotes the axonal regeneration of the peripheral motor and sensory nerve (Wang et al, 2016), which suggests that KLF7 may play a role in enhancing the neuronal intrinsic growth capacity (Moore et al, 2009). Additionally, it has been noted that NGF (Caiazzo et al, 2010), TrkA (Lei et al, 2001; 6 Lei et al, 2006), TrkB (Kingsbury and Krueger, 2007) and GAP43 (Blackmore et al, 2012;Kajimura et al, 2007) are direct KLF7 target genes.…”

B-RAF kinase drives developmental axon growth and promotes axon regeneration in the injured mature CNS

“…Note that the mice in these experiments were from a Bax WT background to enable direct comparison with the original PTEN deletion data (Park et al, 2008); however, this means that possible survivalpromoting effects of the mutant alleles cannot be excluded. The extent of regeneration achieved by direct genetic activation of specific intracellular signaling pathways, including B-RAF, DLK, PI3-kinase-mTOR, KLF, and JAK-STAT pathways Yan et al, 2009;Park et al, 2010;Blackmore et al, 2012;Shin et al, 2012;Lang et al, 2013), compares favorably with what has been reported for application of growth factors such as NGF, BDNF, GDNF, and CNTF (Lykissas et al, 2007;Zhang et al, 2009;Allen et al, 2013;Leibinger et al, 2013). Growth factors as promoters of regeneration are hobbled by two major issues.…”

Krüppel-like factors in mammalian stem cells and development

“…For instance, studies in rats have shown that KLF4 and KLF9 are repressors of axon growth in retinal ganglion cells (RGCs), whereas KLF6 and KLF7 are capable of stimulating neurite growth in these cells (Moore et al, 2009). Furthermore, KLF7 induction in the adult rat corticospinal tract improves axon regeneration in conjunction with upregulation of Trk neurotrophin receptors (Blackmore et al, 2012), and KLF9 was revealed as a factor that contributes to myelin regeneration in the mouse cortex following cuprizone-mediated demyelination, acting via thyroid hormone pathway induction (Dugas et al, 2012). Overall, these various studies highlight the therapeutic potential of KLFs in regenerative medicine, although further studies are needed to dissect their precise roles and mechanisms of action.…”