GDE6 promotes progenitor identity in the vertebrate neural tube

The generation of neurons in the central nervous system is a complex, stepwise process necessitating the coordinated activity of mitotic progenitors known as radial glia. Following neural tube closure, radial glia undergo a period of active proliferation to rapidly expand their population, creating a densely packed neurepithelium. Simultaneously, radial glia positioned across the neural tube are uniquely specified to produce diverse neuronal sub-types. Although these cellular dynamics are well studied, the molecular mechanisms governing them are poorly understood. The six-transmembrane Glycerophosphodiester Phosphodiesterase proteins (GDE2, GDE3, and GDE6) comprise a family of cell-surface enzymes expressed in the embryonic nervous system. GDE proteins can release Glycosylphosphatidylinositol-anchored proteins from the cell surface via cleavage of their lipid anchor. GDE2 has established roles in motor neuron differentiation and oligodendrocyte maturation, and GDE3 regulates oligodendrocyte precursor cell proliferation. Here, we describe a role for GDE6 in early neural tube development. Using RNAscope, we show that Gde6 mRNA is expressed by ventricular zone progenitors in the caudal neural tube. Utilizing in-ovo electroporation, we show that GDE6 overexpression promotes neural tube hyperplasia and ectopic growths of the neurepithelium. At later stages, electroporated embryos exhibit an expansion of the ventral patterning domains accompanied by reduced cross-repression. Ultimately, electroporated embryos fail to produce the full complement of post-mitotic motor neurons. Our findings indicate that GDE6 overexpression significantly affects radial glia function and positions GDE6 as a complementary factor to GDE2 during neurogenesis

INVESTIGATING THE POSTNATAL FUNCTIONS OF GDE2 IN THE MAMMALIAN NERVOUS SYSTEM

Glycerophosphodiester phosphodiesterase 2 (GDE2) is a six-transmembrane protein that cleaves glycosylphosphatidylinositol (GPI) anchors to regulate GPI-anchored protein activity at the cell surface. In the developing spinal cord, GDE2 utilizes its enzymatic domain to regulate the production of specific classes of motor neurons and interneurons; however, GDE2’s roles beyond embryonic development have yet to be defined. In light of the growing appreciation for GPI-anchor regulation in neurodegenerative disease, we investigated whether GDE2 might promote neuronal survival in the adult nervous system. Focusing on the spinal cord, we show that GDE2 expression continues postnatally and adult mice lacking GDE2 exhibit a slow, progressive neuronal degeneration with pathologies similar to human neurodegenerative disease. Early phenotypes include vacuolization, cytoskeletal accumulation, altered ceramide homeostasis, and lipofuscin deposits followed by deficits in clearance mechanisms resulting in cytoplasmic inclusions, gliosis and cell death. Remaining motor neurons exhibit peripheral motor unit restructuring causing behavioral sensorimotor deficits. Conditional ablation of Gde2 using temporal and cell-type specific Cre lines demonstrates GDE2 expression is needed postnatally within oligodendrocytes to promote neuronal health. In addition, we have found that conditions in the SOD1G93A mouse model of familial Amyotrophic Lateral Sclerosis antagonize GDE2 function in-vivo, raising the possibility of GDE2 hypofunctionality as a component of human neurodegenerative disease. This work also establishes GDE2 involvement in preventing neuropathology within the adult brain and ensuring proper myelin formation. This thesis greatly expands our understanding of GDE2 in the postnatal nervous system and implicates deregulated GPI-anchored protein activity in pathways mediating neurodegeneration

INVESTIGATING THE POSTNATAL FUNCTIONS OF GDE2 IN THE MAMMALIAN NERVOUS SYSTEM

Glycerophosphodiester phosphodiesterase 2 (GDE2) is a six-transmembrane protein that cleaves glycosylphosphatidylinositol (GPI) anchors to regulate GPI-anchored protein activity at the cell surface. In the developing spinal cord, GDE2 utilizes its enzymatic domain to regulate the production of specific classes of motor neurons and interneurons; however, GDE2’s roles beyond embryonic development have yet to be defined. In light of the growing appreciation for GPI-anchor regulation in neurodegenerative disease, we investigated whether GDE2 might promote neuronal survival in the adult nervous system. Focusing on the spinal cord, we show that GDE2 expression continues postnatally and adult mice lacking GDE2 exhibit a slow, progressive neuronal degeneration with pathologies similar to human neurodegenerative disease. Early phenotypes include vacuolization, cytoskeletal accumulation, altered ceramide homeostasis, and lipofuscin deposits followed by deficits in clearance mechanisms resulting in cytoplasmic inclusions, gliosis and cell death. Remaining motor neurons exhibit peripheral motor unit restructuring causing behavioral sensorimotor deficits. Conditional ablation of Gde2 using temporal and cell-type specific Cre lines demonstrates GDE2 expression is needed postnatally within oligodendrocytes to promote neuronal health. In addition, we have found that conditions in the SOD1G93A mouse model of familial Amyotrophic Lateral Sclerosis antagonize GDE2 function in-vivo, raising the possibility of GDE2 hypofunctionality as a component of human neurodegenerative disease. This work also establishes GDE2 involvement in preventing neuropathology within the adult brain and ensuring proper myelin formation. This thesis greatly expands our understanding of GDE2 in the postnatal nervous system and implicates deregulated GPI-anchored protein activity in pathways mediating neurodegeneration

Additional file 1: Figure S1. of GDE2 is essential for neuronal survival in the postnatal mammalian spinal cord

Sensory neurons exhibit neurodegenerative pathology without impaired nerve conduction in the absence of Gde2. This figure illustrates the presence of neuropathology in the primary sensory neurons of the Gde2 KO, including vacuolization, lipid accrual, and cytoskeletal accumulation; and the maintenance of peripheral sensory nerve conduction. Figure S2-related to Fig. 9. Conditional ablation of Gde2 in the postnatal spinal cord prevents the developmental loss of motor neurons. This figure confirms the effective conditional ablation of Gde2 following neurogenesis. In the constitutive KO, GDE2’s absence during embryonic neurogenesis causes a reduction of alpha motor neurons in the lateral motor column; however, in the Gde2lox/-; ROSA:CreER animals, this loss is avoided by injecting 4-OHT at E17.5. Further, competitive PCR analysis shows a near complete deletion of the conditional Gde2 allele following 4-OHT delivery. Figure S3. Gde2 deletion does not perturb neuromuscular junction morphology. This figure uses wholemount immunohistochemistry to assess the integrity of the neuromuscular junction (NMJ) in aged Gde2 KO hindlimb muscle. At 19 months, no discernible pathology is present in the Gde2 KO NMJ. (DOCX 6586 kb

Presentation_1_GDE6 promotes progenitor identity in the vertebrate neural tube.pdf

The generation of neurons in the central nervous system is a complex, stepwise process necessitating the coordinated activity of mitotic progenitors known as radial glia. Following neural tube closure, radial glia undergo a period of active proliferation to rapidly expand their population, creating a densely packed neurepithelium. Simultaneously, radial glia positioned across the neural tube are uniquely specified to produce diverse neuronal sub-types. Although these cellular dynamics are well studied, the molecular mechanisms governing them are poorly understood. The six-transmembrane Glycerophosphodiester Phosphodiesterase proteins (GDE2, GDE3, and GDE6) comprise a family of cell-surface enzymes expressed in the embryonic nervous system. GDE proteins can release Glycosylphosphatidylinositol-anchored proteins from the cell surface via cleavage of their lipid anchor. GDE2 has established roles in motor neuron differentiation and oligodendrocyte maturation, and GDE3 regulates oligodendrocyte precursor cell proliferation. Here, we describe a role for GDE6 in early neural tube development. Using RNAscope, we show that Gde6 mRNA is expressed by ventricular zone progenitors in the caudal neural tube. Utilizing in-ovo electroporation, we show that GDE6 overexpression promotes neural tube hyperplasia and ectopic growths of the neurepithelium. At later stages, electroporated embryos exhibit an expansion of the ventral patterning domains accompanied by reduced cross-repression. Ultimately, electroporated embryos fail to produce the full complement of post-mitotic motor neurons. Our findings indicate that GDE6 overexpression significantly affects radial glia function and positions GDE6 as a complementary factor to GDE2 during neurogenesis.</p