Xue-Jun Li - US grants

[unreadable] DESCRIPTION (provided by applicant): motoneurons in the cerebral cortex, also known as upper motoneurons, control our muscle movement through lower motoneurons in the brain stem and spinal cord. Degeneration of upper motoneurons results in progressive spastic and weakness in muscles, which underlies some debilitating neurological disorders, such as amyotrophic lateral sclerosis (ALS), hereditary spastic hemiplegia and primary lateral sclerosis. There is no effective treatment for these motoneuron disorders, for the most part due to the lack of understanding how cortical motoneurons are born, functionally mature and degenerate. This application, based on our success in producing spinal motoneurons from human embryonic stem cells (ESCs), will explore the feasibility of generating upper motoneurons from self-renewing human ESCs (H1 and H9 lines, NIH Registry WA01 and WA09). We have established a chemically defined culture system to direct human ESCs to neuroepithelial cells and discovered a critical primitive neuroepithelial stage for specification of region-specific neuronal subtypes. We will first induce differentiation of dorsal telencephalic neural progenitors by examining the effect of such morphogens as Wnts, Wnt antagonists, and fibroblast growth factors (FGFs), individually, in combination, or sequentially. These progenitors will then be further differentiated to motoneurons in the presence of neurotrophic factors and/or target cells (lower motoneuron) and/or local environment. The function of in vitro generated upper motoneurons will be assessed by their interaction with lower motoneurons in culture and following transplantation to dorsal telencephalon of a chick embryo. This application will thus produce the currently unavailable upper motoneurons from limitless ESCs and offer an otherwise inaccessible tool for studying how human upper motoneurons are born, functionally mature, and become sick. These cells will also provide a target for screening pharmaceuticals that stop the process of motoneuron degeneration and a source for potential future cell therapy for upper motoneuron related diseases. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Impaired axonal development and degeneration are implicated in many debilitating disorders, such as hereditary spastic paraplegia (HSP), amyotrophic lateral sclerosis, and periphery neuropathy. HSP is caused by distal axonopathy involving the longest corticospinal tract axons, leading to spasticity and weakness of the lower extremities. The most common early-onset form of HSP, SPG3A, is caused by mutations in the atlastin-1 gene. This gene encodes atlastin-1 protein, which is a member of the dynamin-related large GTPase superfamily. Knockdown of atlastin-1 in rat cortical neuron in vitro cultures inhibits the axonal outgrowth and elongation. However, how altered atlastin-1 activity leads to axonal defects and why specific axons degenerate in HSP patients are largely unclear. The goal of this proposed study is to establish human neuronal models of SPG3A to delineate the mechanisms underlying the axonal defects in HSP. This study's hypothesis is that atlastin mutations result in axonal defects selectively in cortical projection neurons (cortical PNs), and this effect is mediated mainly by dysregulated bone morphogenetic protein (BMP) signaling. This hypothesis will be tested by pursuing the following two aims: 1) to examine the axonal outgrowth and transport in cortical PNs derived from iPSCs that are generated from SPG3A patients and normal individuals (as controls); 2) to delineate the role of BMP signaling in the axonal defects in SPG3A. By comparing the axonal defects, atlastin-1 activity, and BMP signaling alterations in cortical PNs, cortical interneurons, and spinal motor neurons derived from control and SPG3A iPSCs, this study will be able to delineate the cell type-specific defects in HSP and the underlying mechanisms. The cause-effect relationship between loss of atlastin function and axonal phenotypes will be confirmed by knocking down atlastin-1 in wild-type (WT) neurons and by expressing WT atlastin-1 in SPG3A iPSCs. Moreover, rescue experiments will be performed to identify the potential approaches for rescuing the axonal pathology, such as overexpression of atlastin or treatment with BMP antagonists. Together, this study will provide valuable insights into the roles of atlastin-1 and BMP signaling in HSP pathology and developing new therapeutics for rescuing the axonal degeneration in HSP.

SUMMARY Impaired axonal development and axonal degeneration underlie many debilitating neurodegenerative disorders including hereditary spastic paraplegia (HSP), amyotrophic lateral sclerosis, and periphery neuropathy. HSPs are a heterogeneous group of more than 70 genetic disorders characterized by progressive lower limb spasticity due to a length-dependent degeneration of corticospinal motor neuron axons. SPG11 and SPG15, the most common autosomal recessive forms of HSP, are caused by mutations in KIAA1840 and ZFYVE26 that encode spatacsin and spastizin protein, respectively. Knockdown of spatacsin and spastizin in zebrafish led to abnormal axonal outgrowth and locomotor impairment. However, how altered spatacsin and spastizin activities lead to axonal defects and why specific axons degenerate in HSP patients are largely unclear. The goal of this proposed study is to establish human neuronal models of SPG11 and SPG15 to delineate the mechanisms underlying axonal defects in HSP. Based on strong preliminary data, this study?s hypothesis is that impaired autophagy and lysosomal function caused by perturbed spatacsin and spastizin levels result in axonal defects selectively in cortical PNs, which is mediated by mitochondrial dysfunction. This hypothesis will be tested by pursuing the following two aims: 1) to determine the effect of perturbed spatacsin and spastizin on axonal development and degeneration; 2) to determine the role of impaired mitochondrial dynamics in axonal defects in HSP. By comparing axonal defects, spatacsin and spastizin levels, mitophagy, and mitochondrial dynamics in cortical projection neurons, cortical interneurons, and spinal motor neurons derived from control and SPG11/SPG15 iPSCs, this study will delineate the cell type- specific defects in HSP and the underlying mechanisms. The cause-effect relationship between loss of spatacsin and spastizin function and axonal phenotypes will be confirmed by knocking out spatacsin and spastizin in wild-type neurons and by correcting spatacsin and spastizin mutations in SPG11 and SPG15 iPSCs, respectively. Moreover, rescue experiments will be performed to identify potential approaches for rescuing axonal pathology, such as correcting mutations of spatacsin and spastizin, treatment with fission/fusion targeting agents, and genetically regulating mitochondrial dynamics. Together, this study will provide valuable insights into understanding the role of mitochondrial dysfunction in HSP pathology and developing new therapeutics for rescuing axonal degeneration in HSP.

SUMMARY Hereditary spastic paraplegia (HSP) is a large heterogeneous group of neurogenetic disorders caused by the length-dependent degeneration of cortical motor neuron axons. Cortical motor neurons, a group of projection neurons located in motor cortex, control muscle movement through lower motor neurons in the brain stem and spinal cord. Degeneration of these neurons interrupts the signal transmission from brain to spinal cord and then muscles, resulting in progressive spasticity and weakness in muscles. Currently, there remains a lack of effective treatment to ameliorate, stop, or reverse axonal defects in HSPs. Recent studies show that several HSP proteins can regulate the size of lipid droplets, implying their roles in lipid metabolisms. Glial cells play an important for generating and regulating lipid metabolism in the brain. However, whether lipid metabolism is altered in HSP brain and what role glial cells play in the pathogenesis of HSP are largely unknown. The goal of this proposed study is to dissect the novel role of lipid metabolism and the interplay between glial cells and neurons in the pathogenesis of HSP using co-cultures of cortical neurons and glial cells derived from iPSCs of SPG3A patients. SPG3A is the most common early-onset form of HSP caused by mutations in the ATL-1 gene that encodes atlastin-1 protein. We will test our hypotheses by pursuing the following three aims: 1) to identify the contribution of glial cells to axonal and synaptic defects in SPG3A, 2) to determine the role of glial cells in impaired cholesterol homeostasis in SPG3A, and 3) to rescue axonal and synaptic defects in SPG3A by targeting the impaired glia-neuron interaction. By comparing co-cultures of cortical neurons with normal or SPG3A glial cells, our study will provide insights into the role of glial cells in HSP. The cause-effect relationship between atlastin-1 mutations and axonal phenotypes will be confirmed by rescuing the mutations in SPG3A iPSCs and by knocking in mutations to normal human pluripotent stem cells. Moreover, rescue experiments will be performed to identify potential approaches for mitigating axonal and synaptic defects in HSP through regulating lipid metabolism in glial cells. Together, our study is expected to reveal novel roles of glial cells in the pathogenesis of HSP and identify new targets for therapeutic intervention in HSP.