The long-term goal of our research is to understand the mechanisms that generate neuronal networks during development and then apply this knowledge to regenerating the neural circuitry lost after injury or neurodegenerative diseases. An important step towards this goal is to identify the molecular cues that permit axons to navigate towards their synaptic targets. However, although many of the extrinsic factors that orient axons to project in a particular direction are well-described, the mechanism(s) that control the rate of axon outgrowth remain unresolved. Our recent studies have shed light on this issue;we showed that cofilin, and its negative regulator Lim kinase 1 (Limk1), control the speed of growth for a population of dorsal commissural interneurons in the spinal cord. Thus, axons are also instructed by extrinsic signals to grow at a particular rate. Such """"""""temporal"""""""" cues have the potential to control the rate and/or time at which directional information is interpreted and are a important mechanism that ensures that axonal circuits develop in concert with the rest of the developing embryo. Moreover, this finding raises the possibility that the signaling pathways that control the rate of axon growth during development could be manipulated to accelerate axonal outgrowth in a regenerative or neuroprotective context and thereby speed up the lengthy process of regrowing neural circuits in a human patient. To work towards this goal, we will determine whether the signals that regulate the rate of axon growth are important for the establishment of spinal motor circuits during development and in an embryonic model that tests the functionality of stem-cell derived MNs.
In Aim 1 of this proposal we will test the hypothesis that the balance between the activation states of cofilin and Limk1 controls the rate of endogenous motor axon extension during development. We will utilize in ovo electroporation of chicken embryos and mouse loss-of-function genetics to increase the activity levels of cofilin in developing embryos and assess the effect of elevated cofilin activity on the rate and trajectory of spinal motor axon extension.
In Aim 2 of this proposal we will test the hypothesis that elevating levels of cofilin in embryonic stem cell (ESC)-derived motor neurons (MN) results in their generating functional motor circuits more rapidly. We will use lentivirus transfection methods to intrinsically increase cofilin activity in ESC-derived MNs and then will assess the rate of motor axon extension as well as their ability to form functional neural circuits in culture. ESC-derived MNs are a promising candidate to replacing MNs that are damaged or lost after injury or disease. The ability to intrinsically accelerate axon extension from ESC-derived MNs may lead to more efficient recovery times when paired with other axon regeneration therapies.
Understanding the mechanisms that establish neural circuitry during development will directly inform studies seeking to regenerate neural circuitry damaged by injury or neurodegenerative diseases. In addition to the well-known challenge in axon regeneration of overcoming the inhibitory environment of the CNS, a less obvious but equally important problem is the considerable distances that axons must grow to reestablish functional circuits in human patients. By understanding the developmental mechanisms that control the rate of axon outgrowth, intrinsic signaling pathways could be manipulated to accelerate axon extension in regenerative therapies, leading to more efficient recovery times.
