Glial Lipoproteins Stimulate Axon Growth of Central Nervous System Neurons in Compartmented Cultures

Abstract

The role of lipoproteins secreted by cortical glial cells in axon growth of central nervous system (CNS) neurons was investigated. We first established compartmented cultures of CNS neurons (retinal ganglion cells). Addition of glial cell-conditioned medium (GCM) to distal axons increased the rate of axon extension by ϳ50%. Inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase in glial cells diminished the secretion of cholesterol and apolipoprotein E, and prevented the growth stimulatory effect of GCM. When glia-derived lipoproteins containing apolipoprotein E were provided to distal axons, axon extension was stimulated to the same extent as by GCM. In contrast, addition of lipoproteins to cell bodies failed to enhance growth. The growth stimulatory effect of glial lipoproteins was abrogated in the presence of receptor-associated protein, RAP, indicating involvement of receptor(s) of the low density lipoprotein receptor family in stimulation of axonal extension. These observations suggest that glial cells stimulate axon growth of CNS neurons by providing lipoproteins containing cholesterol and apolipoprotein E to distal axons.Identification of factors that regulate axon growth and regeneration of CNS 1 neurons has been hampered by the lack of a model that recapitulates the in vivo environment. In intact animals, neuronal cell bodies and axon terminals often reside far apart in distinct environments. The issue of spatial separation of cell bodies and axons was addressed for peripheral neurons by the establishment of sympathetic neuron cultures in which cell bodies were plated in one compartment and axons extended into adjacent compartments (1). Consequently, cell bodies and distal axons could be exposed to independent fluid environments and the rate of axon extension accurately measured. Our objective was to culture CNS neurons in compartmented cultures and identify factors that stimulated axon growth.The most numerous cell types in the brain are glial cells: oligodendrocytes, astrocytes, and microglia. Glial cells interact with neurons and are associated with neuronal excitability and synaptic transmission (2) (3-7). Glial cell-conditioned medium (GCM) stimulates synapse development of CNS neurons (8, 9), and cholesterol, associated with apolipoprotein (apo) E, was identified as the factor that promotes synaptogenesis (10).ApoE is synthesized in the brain primarily by astrocytes and microglia (11-13) and is the major CNS apolipoprotein. ApoJ is also abundant in the brain (14). Cerebrospinal fluid, which is separated from the plasma compartment by the blood-brain barrier, contains a population of lipoproteins distinct from those in plasma (12,(15)(16)(17)(18). Several observations imply that apoE plays an important role in lipid metabolism in the nervous system (19). For example, apoE synthesis in glial cells dramatically increases after injury to the central (20 -22) and peripheral nervous systems (21). ApoE is also a ligand for receptors of the low density lipoprotein receptor (LDLr) superfamily, several of w...

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“…Compartmented Culture of RGCs-RGCs in compartmented cultures were prepared as described previously (12,20). Distal axons and cell bodies/dendrites/proximal axons of the primary neurons can be separately maintained with different media in compartmented cultures.…”

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