Yutaka Yoshida - US grants

Abstract (Summary): Spinal cord injury (SCI) often causes permanent paralysis due to failure of injured axons to regenerate. Although injured CS axons do not re-grow beyond the lesion after SCI, they form new connections with propriospinal neurons above (rostral to) the lesion which contributes to the limited recovery seen in these injury models. In this study, we propose two-pronged strategies to simultaneously induce re- growth of injured corticospinal (CS) axons beyond the lesion site and enhance remodeling of connectivity between injured CS axons and propriospinal neurons after SCI. CS axonal growth can be promoted by reducing phosphatase and tensin homolog (Pten) activity in the sensorimotor cortex. Our preliminary data reveal increased CS connectivity after SC in mice lacking PlexA1 encoding a semaphorin6D receptor. Since injured CS axons in PlexA1 mutants do not pass beyond the lesion site, our preliminary data strongly suggest that new connections between injured CS axons and propriospinal neurons are enhanced through the uninjured ventral regions after SCI to compensate for the loss of CS inputs. Based on these preliminary data, the central hypothesis of this proposal is that a combined modulation of Sema6D-PlexA1 and Pten signaling will facilitate rewiring of CS connectivity with propriospinal neurons as well as re-growth of injured CS axons after SCI at cervical levels. Therefore, these interventions will improve functional CS connectivity and motor recovery. In Aim 1, we will determine whether deletion of both PlexA1 and Pten in the sensorimotor cortex will enhance connectivity between injured CS axons and propriospinal neurons to a greater degree than the loss of PlexA1 or Pten alone. In Aim 2, we will determine whether PlexA1, Pten, or PlexA1/Pten deletion increases circuits between injured CS neurons and muscles. Finally, in Aim 3, we will examine whether motor recovery is enhanced in mice lacking PlexA1 and Pten in the sensorimotor cortex.

? DESCRIPTION (provided by applicant): Corticospinal neurons, the key conveyers of motor instructions controlling voluntary movement, originate in layer V of the motor cortex and are the major efferent source of descending motor pathways. The overall goal of this proposal is to understand the role of synapse elimination in establishment of corticospinal motor circuits and voluntary movement control. During brain development there is an overabundance of synapse number. However the brain must eliminate excess synapses so that different brain areas can develop specific functions, and avoid stimuli overload. We are just beginning to recognize that improper synapse elimination contributes to neurological disorders such as epilepsy, autism and schizophrenia1-7. However, there are large gaps in our knowledge of the role played by synapse elimination in normal circuit formation and how deficiencies in synapse elimination cause aberrant neural circuit formation and function in vivo. We have recently established unique animal models harboring synapse elimination defects by selectively manipulating genes in specific neural populations during development. Our Preliminary Data implicate regulation of activity-dependent corticospinal synapse elimination by interaction between the transmembrane semaphorin Sema6D and its plexinA1 (PlexA1) receptor. We found that during early postnatal development, CS axons transiently form synapses with spinal neurons. However, these synapses are not eliminated in mice lacking the receptor PlexA1. Importantly, PlexA1 mutants exhibit disrupted skilled movements. Thus we hypothesize that Sema6D-PlexA1-mediated synapse elimination of required for proper patterns of muscle activity during skilled movements. The first aim will determine whether Sema6D-PlexA1 signaling controls synapse elimination via RhoA in an activity-dependent manner. The second aim will examine whether synapses between corticospinal neurons and specific classes of spinal neurons are eliminated by Sema6D-PlexA1 signaling. Finally the third aim will determine whether the Sema6D-PlexA1-mediated CSN synapse elimination is required for co//rrect patterns of muscle activity for skilled movements.

The highly orchestrated muscle activation sequences during motor behaviors are achieved directly through the fine-tuned firing of motor neurons in the ventral spinal cord. These motor neurons are mainly regulated by spinal interneurons present in all mammals, which are, in turn, connected to other spinal neurons as well as various types of descending neurons from the brain, such as corticospinal (CS), reticulospinal and rubrospinal neurons. Until recently, the identities and functioning of the interneuron subtypes and descending neurons participating in individual circuits had remained elusive. What remains lacking is knowledge of the arrangement and functional role of the spinal interneuron subtypes in individual circuits. There is, therefore, a critical need to determine the anatomical and functional connectivity of these spinal interneuron subtypes and how they regulate motor behaviors. Our overall objectives in this application are to (i) map anatomical and functional connectivity of different classes of spinal interneurons (Aims 1 & 2), and (ii) elucidate how those interneurons effect motor behaviors (Aim 3). Our central hypothesis is that each interneuron subtype will exhibit preferential connections with distinct descending neurons to control discrete forms of locomotor and skilled movements.

The highly orchestrated muscle activation sequences during motor behaviors are achieved directly through the fine-tuned firing of motor neurons in the ventral spinal cord. These motor neurons are mainly regulated by spinal interneurons present in all mammals, which are, in turn, connected to other spinal neurons as well as various types of descending neurons from the brain including corticospinal (CS) neurons (CSNs). CSNs located in the motor cortex connect to spinal interneurons to control motor neuron activity in all species, and thereby coordinate the activity of flexor and extensor limb muscles to control skilled movements. Although we and others mainly focused on outputs of CSNs through their axons, CSNs also receive inputs from their presynaptic neurons through their dendrites. However, the identification and understanding of the function of presynaptic neurons of CSNs (pre-CSNs) remains limited. We developed rabies virus-based assays to identify pre-CSNs. We hypothesize that each population of pre-CSNs will be distinctly activated to control discrete phases of skilled movements and muscle activation. To test our hypothesis, in Aim 1 we will map presynaptic partners of CSNs in the brain. We will further determine whether those connections are functional (Aim 2). Finally, we will determine how pre-CSNs control forelimb skilled movements and muscle activity (Aim 3). These results will provide the necessary framework for not only defining spinal motor circuitry, but also subsequent development of novel targeted interventions to treat motor disabilities.

Abstract (Summary): In patients with amyotrophic lateral sclerosis (ALS) and the related motor neuron disease (MND) primary lateral sclerosis (PLS), deficits in motor control occur as a consequence of the degeneration of corticospinal neurons (CSNs). ALS is more common than PLS, and genetically more complex, with familial forms associated with causal mutations in over 30 ALS-related genes. In these ALS mice, however, dysfunction and degeneration of CSNs have not been carefully examined, and data implicating corticospinal (CS) circuits in these model systems of ALS is surprisingly limited. One reason for this may be the very different pattern of connectivity between CSNs and spinal MNs in humans vs. mice. In humans, CS axons located in the ventral and lateral funiculi form direct connections with both MNs (cortico-motoneuronal (CM) connections) and interneurons. In contrast, CS axons in mice are located mainly in the dorsal funiculus and only form indirect connections with MNs through pre-motor interneurons. Therefore, we will use PlexinA1 mutant mice which have CM connections together with ALS mouse models to analyze CS circuits. Our central hypothesis is that progressive defects in CS circuitry in ALS mice will be exacerbated by the establishment of CM connections. In Aim 1, we will determine formation of CS circuits in ALS mouse models with CM connections. In Aim 2, we will determine function of CS circuits in ALS mouse models with CM connections. In Aim 3, we will examine skilled movements in ALS mouse models with CM connections. These studies will provide a model system to study mechanisms of CS degeneration in ALS/PLS, and to test novel therapeutics targeting upper motor neuron dysfunction in these disorders.