The tumor suppressor HHEX inhibits axon growth when prematurely expressed in developing central nervous system neurons

Abstract

Neurons in the embryonic and peripheral nervous system respond to injury by activating transcriptional programs supportive of axon growth, ultimately resulting in functional recovery. In contrast, neurons in the adult central nervous system (CNS) possess a limited capacity to regenerate axons after injury, fundamentally constraining repair. Activating pro-regenerative gene expression in CNS neurons is a promising therapeutic approach, but progress is hampered by incomplete knowledge of the relevant transcription factors. An emerging hypothesis is that factors implicated in cellular growth and motility outside the nervous system may also control axon growth in neurons. We therefore tested sixty-nine transcription factors, previously identified as possessing tumor suppressive or oncogenic properties in non-neuronal cells, in assays of neurite outgrowth. This screen identified YAP1 and E2F1 as enhancers of neurite outgrowth, and PITX1, RBM14, ZBTB16, and HHEX as inhibitors. Follow-up experiments focused on the tumor suppressor HHEX, one of the strongest growth inhibitors. HHEX is widely expressed in adult CNS neurons, including corticospinal tract neurons after spinal injury, but is present in only trace amounts in immature cortical neurons and adult peripheral neurons. HHEX overexpression in early postnatal cortical neurons reduced both initial axonogenesis and the rate of axon elongation, and domain deletion analysis strongly implicated transcriptional repression as the underlying mechanism. These findings suggest a role for HHEX in restricting axon growth in the developing CNS, and substantiate the hypothesis that previously identified oncogenes and tumor suppressors can play conserved roles in axon extension.

Intrinsic mechanisms of neuronal axon regeneration

“…Although overexpression of ATF3 can promote peripheral nerve regeneration 109 , it fails to do so in several models of CNS injury 110,111 . Conversely, JUN overexpression can promote regeneration in cultured cortical 110,112,113 and DRG neurons 114 ; however, as described above, its activation downstream of DLK also promotes cell death in RGCs 66 . Whereas neuronal JUN is clearly necessary for peripheral axon regeneration 115 , JNK-mediated phosphorylation of JUN appears to not be required for its full function in facial nerve regeneration 116 .…”

“…In addition to the previously highlighted transcription factors, several others — namely Myc proto-oncogene protein (MYC) 137 , hypoxia-inducible factor 1α (HIF1α) 138 , CREB1 (REFS 36,113 ), SOX11 (REFS 139–143 ), TP53 (REFS 46,144 ), SRF48,145 and XBP1 (REFS 90,91 ) — have been identified to have roles in axon regeneration (see TABLE 1 and REFS 146,147 ). As little is known about the mechanisms by which these transcription factors are activated after injury, their regulation during development or their downstream targets, they will not be further discussed in detail here.…”

Epigenetic profiling reveals a developmental decrease in promoter accessibility during cortical maturation in vivo

“…To create nuclear-localized reporter constructs, the open reading frame of AP-1 factors (JUN aa 1-334; ATF3 aa 1-181; FOS aa1-380) was PCR-amplified and used to replace EBFP in expression vector CMV-EBFP-2A-H2B-EGFP as previously described (20), creating CMV-JUN/ATF3/FOS-2A-H2B-EGFP. DNA was prepared by EndoFree Plasmid Maxi Kit (Qiagen 12362) and diluted to 1μg/μl in endotoxin-free TE buffer.…”

“…Cortical neurons were prepared from Sprague Dawley rat pups (Harlan), and procedures for dissociation, transfection, and neurite outgrowth were performed as in (20). Briefly, early postnatal (P3-P5) frontal cortices were dissected, and neurons dissociated.…”

“…Viability assays were modified from (20). Neurons were transfected with EBFP-2A-mCherry or EBFP-2A-H2B-EGFP, plated at a density of 10,000 cells/well, and grown in culture for one day before addition of HAT and HDAC inhibitors, as described above.…”