Murray Blackmore, Ph.D. - US grants

DESCRIPTION (provided by applicant): The failure of axon regeneration in the central nervous system (CNS) prevents treatment of a wide range of CNS afflictions, including spinal cord injury, stroke, and diseases like Parkinson's. One major reason that regeneration fails is that many CNS undergo a developmental loss in their intrinsic capacity for axon growth. To restore function after CNS injury or disease it is essential that we devise means to enhance the intrinsic growth state of CNS neurons. We recently identified the Kr¿ppel-like family of transcription factors (KLFs) as key regulators of intrinsic regenerative capacity in CNS neurons. Critically, a transcriptionally active mutant, VP16-KLF7, promotes axon regeneration by injured corticospinal tract (CST) neurons, an important therapeutic target. Robust regenerative responses were achieved using viral delivery to adult wild-type animals, raising potential clinical relevance for this novel pro-regenerative tool. To further explore the potential of KLF7 activity t promote CNS regeneration, three key questions must be addressed, corresponding to our aims. First because recovery from SCI depends on multiple functional modalities carried by diverse fiber tracts, we will use viral-mediated gene delivery to test the ability of VP16-KLF7 to promote regeneration in additional neuronal populations. Targeting additional neuronal subtypes has the potential to broaden the range of behavioral improvement after SCI. Second, we will examine the expression of pro-regenerative genes in neurons stimulated by VP16-KLF7 in order to determine the potential relationship between with known pro-regenerative (e.g. mTOR, CNTF) or anti-regenerative (e.g. chondroitin sulfate proteoglycan (CSPG)) pathways and signals. Based on this information we will rationally combine VP16/KLF7 with viral particles that target complementary pathways, potentially inducing additive or synergistic improvements in axon regeneration. Finally, we will examine the extent to which KLF7-stimulated axons succeed in forming functional synapses on appropriate target cells and ultimately contribute to functional recovery. Using viral gene delivery we will co-express VP16- KLF7 with optogenetic constructs that enable the treated neurons to be reversibly activated or silenced. This technique will allow us to determine the specific contribution of the treated neurons to electrophysiological and behavioral output as regeneration proceeds. Taken together, these experiments have a strong potential to extend the use of a promising new pro-regenerative tool and lead to the development of novel combinatorial methods to promote axon regeneration and functional recovery in the injured CNS, leading to novel treatment options for people suffering from CNS afflictions.

? DESCRIPTION (provided by applicant): Here we seek to clarify the molecular mechanisms by which a transcription factor called HHEX restricts the ability of neurons to extend axons, and explore the potential of interfering with HHEX function to promote axon growth in the central nervous system. This work is significant because the low regenerative ability of neurons in the central nervous system prevents full recovery from stroke, neurodegenerative disease, or injury to the brain or spinal cord. A major therapeutic goal is to enhance regenerative ability in CNS neurons, but the molecular mechanisms that restrict growth remain incompletely understood. HHEX, which has been linked to cellular growth in cells outside the nervous system but is largely unstudied in the brain, emerged unexpectedly from a large-scale screening experiment that examined the effect of various transcription factors on the morphology of cortical neurons. Expression of HHEX strongly decreases axon growth. Furthermore, HHEX is expressed in adult CNS neurons that regenerate poorly, but not in regeneration-competent neurons in the peripheral nervous system. Finally, a structure-function analysis revealed that HHEX blocks axon growth by suppressing target genes, and that an artificial construct that activates HHEX target genes reverses the normal activity, that is, enhances axon growth. Combined, these data suggest that HHEX acts as a novel factor that suppresses axon growth ability in CNS neurons, and that manipulating HHEX function can promote axon growth. In this grant we will 1) clarify the set of genes that are regulated by HHEX in order to clarify the mechanism of growth suppression and 2) test the ability of HHEX-based manipulations to promote axonal sprouting and regeneration in an animal model of spinal cord injury. Ultimately, these studies will fill a critical gap in knowledge in the field through the identification of novel transcriptional components that regulate CNS regeneration, and explore the potential of this new target to improve regenerative capacity.

? DESCRIPTION (provided by applicant): Damage to the central nervous system (CNS) due to spinal cord injury (SCI), stroke, or neurodegenerative disorders such as Alzheimer's or Parkinson's disease result in failed axon regeneration and permanent loss of function. Some types of neurons (e.g. peripheral or embryonic) do succeed in regenerating axons by engaging pro-growth transcriptional programs. Thus it is increasingly clear that in addition to overcoming inhibitory external factors, re-activation of a pro-regenerative transcriptional program is crucial for successful CNS regeneration. The specific genes and associated molecular mechanisms that are required to initiate a successful regenerative response remain largely unknown, but growing evidence indicates that core molecular mechanisms driving cellular growth are well conserved across cell types. Specifically, it appears that genes that regulate growth and motility in the context of proliferating, cancerous cells may be important regulators of regenerative abilit in post- mitotic neurons. Consistent with this overall hypothesis, bioinformatics analysis has revealed a remarkable degree of overlap between transcription factors (TFs) that are implicated in cancer growth and TFs known to modulate axon regeneration. Moreover, new data indicates that many TFs previously identified as oncogenic or tumor suppressive have proven to regulate axon growth when expressed in neurons. In three Specific Aims, we will (1) Combine our expertise in high content screening technology along with highly efficient CRISPR based knockdown strategies to identify novel genes involved in regulation of axon growth by testing requirements for transcription factors implicated in cancer biology in promoting axon growth in vitro; (2) Engineer transcriptionally active and inactive forms of candidate genes to identify specific transcriptional domains of novel genes that are required for promoting regenerative axon growth; (3) Use integrated bioinformatics and high content screening to define functional networks of interacting transcription factors and identify optimal combinations for growth promotion. Ultimately, these studies will fill a critical gap in knowledge in the field through the identification of novel genes and concomitant transcriptional components that aid in CNS regeneration and thereby reveal therapeutic targets to improve regenerative capacity in humans.

PROJECT SUMMARY A major effort in regenerative neuroscience is to improve axon growth after injury to the central nervous system (CNS). Once growth is achieved, however, a second hurdle to improving function is that regenerated axons must succeed in forming synaptic contacts with appropriate sets of post-synaptic neurons. The challenge of restoring effective circuitry is especially acute after spinal injuries that damage the corticospinal tract (CST), a pathway critical for fine motor control. The CST mediates descending motor control by synapsing on specific subsets of spinal neurons, which in humans and rodents alike include a diverse set of interneurons in addition to the direct CST-motor-neuron contacts that characterize primates. The field has achieved increasing success in promoting CST axon growth, yet gains in behavioral recovery have lagged. This work will address the need to monitor the connectivity of regenerated CST axons, and to optimize their behavioral output. To do so we will employ rodent models of spinal injury and capitalize on combined stem cell bridging and viral expression of a pro-regenerative gene called KLF6, which we recently found to evoke robust regenerative CST growth. In addition, we will leverage a recently developed trans-synaptic viral labeling technique that enables an unprecedented ability to visualize post-synaptic target selection. First, we will render KLF6 expression controllable and reversible, in order to silence KLF6 after regeneration occurs in order to determine whether prolonged KLF6 expression itself interferes with behavioral recovery. This will address the pressing question of the degree to which pro-regenerative growth mechanisms may come at the expense of effective synaptic refinement or target selection. Next, we will test the ability of rehabilitative training to sculpt target selection by regenerating CSTs and improve their behavioral output. Finally, we will employ both electrical and chemogenetic means to chronically elevate activity in regenerating CST axons, which we hypothesize will both enhance CST sprouting and improve competition for synaptic territory. These complementary approaches will create optimal strategies to maximize the behavioral benefit that can be extracted from regenerated CST axons.

PROJECT SUMMARY In adults, axons in the central nervous system (CNS) generally fail to regenerate after they are lost to injury or disease, leading to permanent and incurable disability. Axon growth is prevented by a hostile growth environment, as well as a developmental loss in the intrinsic capacity for axon growth as CNS neurons age. Transcription factors (TFs) interact with DNA and coordinate the production of broad sets of cellular materials, and have emerged as important therapeutic targets to boost regenerative ability within injured neurons. For example, forced re-expression of a pro-regenerative TF called KLF6 in adult neurons can improve their capacity for axon growth after spinal injury. We will now test three complementary and mutually supportive strategies to enhance the promising pro-regenerative properties of KLF6. First, using a novel bioinformatics pipeline, we have predicted additional TFs that functionally interact with KLF6 and verified their ability to synergistically enhance axon growth when combined with KLF6 in cell culture models of axon growth. We will therefore perform in vivo tests of three selected factors, EOMES, NR5A2, and RARB, for the ability to enhance KLF6?s pro-regenerative properties in animal models of spinal cord injury. Second, we will supplement these TF interventions with transplants of growth- permissive stem cells into sites of spinal injury. These grafts will alleviate persistent growth inhibition in the spinal cord environment, and thus unmask the pro-regenerative effects of TF treatments. Finally, we will harness a newly developed gene therapy vector that enables retrograde delivery of genes with unprecedented efficiency. Injection of this vector to the spinal cord results in widespread gene expression in injured neurons throughout the brainstem, midbrain, and motor cortex. This delivery system engages a larger number and a wider diversity of cell types than the current practice of direct brain injection, thus maximizing the chance of achieving functional gains after spinal injury. Throughout these aims, tissue clearing and 3D microscopy will reveal new anatomical details of the evoked regeneration. Bringing together these cutting-edge improvements to a TF-centered strategy will move the field toward novel and effective treatments for individuals suffering from the debilitating consequences of CNS injury.

The overall objective of this project is to identify combinatorial sets of transcription factors that can act in central nervous system (CNS) neurons to improve their regenerative abilities. To do so we will take advantage of a well- characterized transition in nervous system development, in which maturing neurons abruptly switch from an intrinsic state that allows rapid axon growth to one in which axon growth is slow or abortive. It is clear that this transition involves changes in gene expression, and we have had partial success in improving axon growth in older neurons by supplying pro-regenerative transcription factors. Here our central hypothesis is that in addition to transcriptional changes, underlying changes in chromatin structure also act to limit regenerative ability. It is well established in other fields that during cellular maturation, regions of chromatin can adopt a closed conformation that prevents access of transcription factors to specific genomic loci. Thus similar restriction events are likely to limit the expression of regeneration-associated genes in CNS neurons, and to prevent their re- expression even when pro-growth transcription factors are supplied. A special class of transcription factors, termed pioneer factors, can ameliorate this constraint by binding to closed chromatin and re-opening it, thus allowing subsequent waves of transcription factors to drive transcription. Our objective here is to use a newly developed technique called ATACseq to create genome-wide maps of chromatin accessibility in corticospinal tract neurons as they age and lose regenerative ability, and as they respond to axon injury. This will create a detailed picture of the relationship between regenerative ability and chromatin structure. Then, by examining regions of the genome that specifically open and close across time and in response to injury, we will use a bioinformatic approach to identify transcription factors that interact with these regions, with particular attention paid to potential pioneer factors. Finally we will perform a first-pass test of combined expression of predicted pioneers with known pro-regenerative factors in two cell culture models of axon growth. Ultimately these studies will fill a critical gap in knowledge in the field regarding chromatin accessibility and regenerative ability, and will use this information to identify transcription factors that act through a novel chromatin-based mechanism to improve axon growth.