We have investigated the synthesis and transport of apoE, the major apolipoprotein of the central nervous system, in the retina of the living rabbit. Four hours after the injection of [ ]Met/ Cys-labeled apoE is rapidly transported into the optic nerve and its terminals in the lateral geniculate and superior colliculus within 3-6 h in two distinguishable vesicular compartments. Mü ller glia in cell culture also synthesize and secrete apoE. Taken together, these results suggest that apoE is synthesized by Mü ller glia and secreted into the vitreous. ApoE is also internalized by retinal ganglion cells and/or synthesized by these cells and rapidly transported into the optic nerve and brain as an intact molecule. We discuss the possible roles of retinal apoE in neuronal dynamics.ApoE, a 36-kDa glycoprotein, is a component of a number of circulating plasma lipoproteins, including very low density lipoproteins, high density lipoproteins, and chylomicron remnants (1). Its primary function appears to be that of a recognition ligand for the receptor-specific removal of cholesteryl ester-rich lipoproteins from the circulation (2, 3). It also plays a role in local transport of cholesterol, as seen in nerves of both peripheral (4, 5) and central nervous systems (reviewed in Ref. 6) and has been implicated in mediating immune responses and cell proliferation, processes that may not be related to its association with lipid (for review, see Ref. 1).In plasma, apoE transports lipoproteins containing cholesterol, triglycerides and other lipids to various cells via binding to the low density lipoprotein (LDL) 1 receptor (7) or the LDL receptor-related protein (LRP)/␣ 2 -macroglobulin receptor (8, 9). In the central nervous system, apoE is primarily synthesized by the major glial cell, the astrocyte, in rodents and humans (10, 11) and is found in significant quantities in the cerebrospinal fluid (10). Since apolipoprotein B, the other molecule that can mediate the internalization of lipoproteins via association with the LDL receptor, is not synthesized in the central nervous system (reviewed in Ref. 6), it is highly likely that apoE plays a major role in cholesterol and lipid transport in this compartment.We have employed the in vivo rabbit retina to probe the synthesis, intracellular transport, and metabolism of central nervous system proteins including kinesin, the motor for anterograde rapid transport (12, 13), and the ␤-amyloid precursor protein (APP) (14, 15). Since it has been shown recently that individuals having the ⑀4 allele of apoE are at high risk for Alzheimer's disease (16), we decided to use this widely accepted model of normal adult retinal protein synthesis and metabolism (e.g. Refs. 17 and 18) to examine the synthesis and transport of apoE. In this report, we present evidence, obtained by the injection of [ 35 S]methionine/cysteine into the vitreous chamber that overlies the retina, that apoE is synthesized in significant quantities in vivo by rabbit Mü ller cells, the predominant glial cell of the retina. ApoE ...

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We have investigated the synthesis, axonal transport, and processing of the β‐amyloid precursor protein (APP) in in vivo rabbit retinal ganglion cells. These CNS neurons connect the retina to the brain via axons that comprise the optic nerve. APP is synthesized in retinal ganglion cells and is rapidly transported into the optic nerve in small transport vesicles. It is then transferred to the axonal plasma membrane, as well as to the nerve terminals and metabolized with a f1/2 of less than 5 h. A significant accumulation of C‐terminal amyloidogenic or nonamyloidogenic fragments is seen in the optic nerve 5 h after [35S]‐ methionine, [35S]cysteine injection, which disappears by 24 h. The major molecular mass species of APP in the optic nerve is ∼110 kDa, and is an APP isoform that does not contain a Kunitz protease inhibitor domain. Higher molecular mass species containing this sequence are seen mostly in the retina. A protease(s) that can potentially cleave APP to generate an amyloidogenic fragment is present in the same optic nerve membrane compartment as APP.

The amyloid beta-protein (A beta) is a major component of extracellular deposits that are characteristic features of Alzheimer's disease. A beta is derived from the large transmembrane beta-amyloid precursor protein (beta APP). In the rabbit optic nerve/optic tract (ON), beta APP is synthesized in vivo in retinal ganglion cell perikarya, rapidly transported into the ON axons in small transport vesicles and is subsequently transferred to the axonal plasma membrane as well as to the presynaptic nerve terminals (Morin, P. J., Abraham, C. R., Amaratunga, A., Johnson, R.J., Huber, G., Sandell, J. H., and Fine, R. E. (1993) J. Neurochem. 61, 464-473). Present results indicate that there is rapid processing of beta APP in the ON to generate a 14-kDa C-terminal membrane-associated fragment that contains the A beta sequence. By using equilibrium sucrose density gradient fractionation, this fragment, as well as non-amyloidogenic C-terminal fragments and intact beta APP, are detected in at least two classes of transport vesicles destined for the plasma membrane and the presynaptic nerve terminal. The two classes of transported vesicles are distinguished by labeling kinetics as well as by density. In contrast to the ON, only nonamyloidogenic C-terminal fragments are generated in the retina, which contains the perikarya of retinal ganglion cells and glial (Muller) cells which also produce beta APP.

We have previously demonstrated that the in vivo vitreal injection of an antisense oligonucleotide directed to the kinesin heavy chain inhibits retinal kinesin synthesis by 82% and concomitantly inhibits rapid transport of total protein into the optic nerve by 70%. These results establish a major role for kinesin in rapid axonal transport in vivo. Recently, the cloning of a family of kinesin‐like molecules from the mammalian brain has been reported, and some of these proteins are also expressed in neurons. To assign a specific function to the kinesin heavy chain we inhibited the kinesin synthesis with an antisense kinesin oligonucleotide and assessed the axonal transport into the optic nerve of representative proteins from each of three vesicle classes that contain rapidly transported proteins. Marker proteins used were substance P for peptide‐containing synaptic vesicles, the amyloid precursor protein for plasma membrane precursor vesicles, and several integral synaptic vesicle proteins. Our results indicate that the major anterograde motor protein for all three vesicle classes utilizes kinesin heavy chain, although we discuss alternative explanations.