Targeted perturbation of perineuronal nets in specific neuron types to investigate adaptive plasticity after spinal cord injury

Spinal cord injury typically causes permanent loss of sensory and motor functions. But some limited functional recovery can occur through a process called adaptive plasticity, and much current research is focused on how to promote this. A key issue is the molecular basis of adaptive plasticity, how it is triggered and how and why it is limited. In this study, we aim to investigate the role of a particular molecular structure, the perineuronal nets (PNNs), which surround many nerve cells and are believed to limit a nerve cell's ability to make or change signalling contacts with other nerve cells. An obvious hypothesis is that nerve cells with PNNs are less capable of making new contacts than nerve cells that do not have PNNs. To test this hypothesis, we have used genetic modification to create mice that lack the PNNs, either throughout the spinal cord or in specific types of nerve cells in the spinal cord. We then use these mice determine whether the lack of PNNs promotes adaptive plasticity. Since the start of the project we have mapped the expression of PNN proteins in the mouse spinal cord, with a particular focus on key proteins ("link proteins") that play a crucial role in the molecular anchoring of PNNs. We are currently working on a revised manuscript and a new manuscript based on this mapping which we expect to see published in 2021. We have also used our transgenic mouse line (transgenic conditional knockout mouse) in which one of these "link proteins" (CRTL1) can be knocked out conditionally in specific spinal neuron populations. This mouse line is an important tool moving forward in our studies of the role of PNNs in regulating plasticity in the spinal cord. During 2020 we have conducted experiments in which CRTL1 has been knocked out in spinal cord neurons, and we are analyzing results from these experiments. These analyses will be the subject of an additional manuscript which we plan to publish in 2021. We have further established methods through which we can selectively knock out CRTL1 in specific populations of motor nerve cells (which innervate muscle), and we will continue with experiments using this approach in 2021.

Generated new transgenic mouse model that enable CRTL1 to be conditionally knocked out in selected spinal neuron types Mapped the expression of PNNs in the mouse spinal according to different types of motoneurons. Charted the development of different molecular components of PNNs in the mouse spinal cord, from the first week of birth through adult. Established a method for selectively knocking out CRTL1 in specific populations of motoneurons, by injection of a Cre-expressing virus into limb muscles. Currently working on two manuscripts with another planned, all for publication in 2021.

In this proposal we address a major issue in the field of mammalian brain and spinal cord injury: how adaptive plasticity works at the molecular level and whether it can be manipulated at the molecular level to direct reorganization of neuronal circuits. This issue is relevant not only for spinal cord injury (SCI), which we focus on here, but for injuries anywhere in the brain. To address this issue, we will use transgenic technology to perturb the synapse-stabilizing perineuronal nets (PNNs) in selected subpopulations of spinal neurons. We will then use combinations of cutting edge anatomical and physiological technologies to assess how such manipulation influences the targeting of post-SCI synaptic remodelling to different neurons and pathways. These experiments will test the role of PNNs in regulating adaptive plasticity in specific neuron subtypes and provide novel avenues for selectively targeting synaptic plasticity to specific neural circuit components. This would open new opportunities for directing the formation of new synaptic connections after an injury, thereby allowing the selective engineering of neural circuits to accomplish desired functional recovery. The project will therefore provide novel insight with relevance both to the basic science of spinal cord plasticity, as well as to the potential harnessing of adaptive plasticity in a future clinical setting. The proposal builds on an already established experimental mouse model of spinal cord injury. The project will utilize transgenic technology that attenuates the expression of key molecules involved in PNN formation, either through a CNS-restricted knockout, or through the Cre/loxP system to target shRNA inhibition to specific spinal neuron subpopulations. Our proposal thus provides an innovative approach to a major biological problem of clinical relevance.