Maunze, B. | bioRxiv Science

bioRxiv2018

Global connectivity and function of descending spinal input revealed by 3D microscopy and retrograde transduction

The brain communicates with the spinal cord through numerous axon tracts that arise from discrete nuclei, transmit distinct functions, and often collateralize to facilitate the coordination of descending commands. In efforts to restore supraspinal connectivity after injury or disease, this complexity presents a major challenge to interpreting functional outcomes, while the wide distribution of supraspinal nuclei complicates the delivery of therapeutics. Here we harness retrograde viral vectors to overcome these challenges. We demonstrate highly efficient retrograde transduction by AAV2-Retro in adult female mice, and employ tissue clearing to visualize descending tracts and create a mesoscopic projectome for the spinal cord. Moreover, chemogenetic silencing of supraspinal neurons with retrograde vectors results in complete and reversible forelimb paralysis, illustrating effective modulation of supraspinal function. Retrograde efficacy persists even after spinal injury, highlighting therapeutic potential. These data provide a global view of supraspinal connectivity and illustrate the potential of retrograde vectors to parse the functional contributions of supraspinal inputs.

neuroscience↗

bioRxiv2018

KLF6 activates complementary gene modules relevant to axon growth and promotes corticospinal tract regeneration after spinal injury

Members of the KLF family of transcription factors can exert both positive and negative effects on axon regeneration in the central nervous system, but the underlying mechanisms are unclear. KLF6 and -7 share nearly identical DNA binding domains and stand out as the only known growth-promoting family members. Here we confirm that similar to KLF7, expression of KLF6 declines during postnatal cortical development and that forced re-expression of KLF6 in corticospinal tract neurons of adult female mice enhances axon regeneration after cervical spinal injury. Unlike KLF7, however, these effects were achieved with wildtype KLF6, as opposed constitutively active mutants, thus simplifying the interpretation of mechanistic studies. To clarify the molecular basis of growth promotion, RNA sequencing identified 454 genes whose expression changed upon forced KLF6 expression in cortical neurons. Network analysis of these genes revealed sub-networks of downregulated genes that were highly enriched for synaptic functions, and sub-networks of upregulated genes with functions relevant to axon extension including cytoskeleton remodeling, lipid synthesis and transport, and bioenergetics. The promoter regions of KLF6-sensitive genes showed enrichment for the binding sequence of STAT3, a previously identified regeneration-associated gene. Notably, co-expression of constitutively active STAT3 along with KLF6 in cortical neurons produced synergistic increases in neurite length. Finally, genome-wide ATAC-seq footprinting detected frequent co-binding by the two factors in pro-growth gene networks, indicating co-occupancy as an underlying mechanism for the observed synergy. These findings advance understanding of KLF-stimulated axon growth and indicate functional synergy of KLF6 transcriptional effects with those of STAT3.\n\nSIGNIFICANCE STATEMENTThe failure of axon regeneration in the CNS limits recovery from damage and disease. These findings show the transcription factor KLF6 to be a potent promoter of axon growth after spinal injury, and more importantly clarify the underlying transcriptional changes. In addition, bioinformatics analysis predicted a functional interaction between KLF6 and a second transcription factor, STAT3, and genome-wide footprinting confirmed frequent co-occupancy. Co-expression of the two factors yielded synergistic elevation of neurite growth in primary neurons. These data point the way toward novel transcriptional interventions to promote CNS regeneration.

neuroscience↗