Richard E. Fine

Apolipoprotein E Is Synthesized in the Retina by Müller Glial Cells, Secreted into the Vitreous, and Rapidly Transported into the Optic Nerve by Retinal Ganglion Cells

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 [S]methionine/cysteine into the vitreous, 44% of [S]Met/Cys-labeled apoE is in soluble and membrane-enclosed retinal fractions, while 50% is in the vitreous. A significant amount of intact [S]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. Müller glia in cell culture also synthesize and secrete apoE. Taken together, these results suggest that apoE is synthesized by Müller 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.

ISOLATION AND CHARACTERIZATION OF RAPID TRANSPORT VESICLE SUBTYPES FROM RABBIT OPTIC NERVE

Abstract: Subcellular fractionation of rabbit optic nerve resolves three populations of membranes that are rapidly labelled in the axon. The lightest membranes are >200 nm and are relatively immobile. The intermediate density membranes consist of 84 nm vesicles which disappear from the nerve with kinetics identical to those of the rapid component. A third population of membranes, displaying a distinct protein profile, is present in the most dense region of the gradient. Immunological characterization of these membranes suggests the following. (1) The lightest peak contains rapidly transported glucose transporter and most of the total glucose transporters present in the nerve; this peak is therefore enriched in axolemma. (2) The intermediate peak contains rapidly transported glucose transporters and synaptophysin, an integral synaptic vesicle protein, and about half of the total synaptophysin; this peak therefore contains transport vesicles bound for both the axolemma and the nerve terminal, and these subpopulations can be separated by immunoadsorption with specific antibodies against the aforementioned proteins. (3) The heaviest peak contains rapidly transported synaptophysin and tachykinin neuromodulators and about half of the total synaptophysin, and 80% of the total tachykinins present in the nerve; this peak appears to represent a class of synaptic vesicle precursor bound for the nerve terminal exclusively. (4) Synaptophysin is present in the membranes of vesicles carrying tachykinins. (5) Both the intermediate and the heaviest peaks are enriched in kinesin heavy chain, suggesting that both vesicle classes may be transported by the same mechanism.

Pyroglutamate-Aβ 3 and 11 colocalize in amyloid plaques in Alzheimer's disease cerebral cortex with pyroglutamate-Aβ 11 forming the central core.

N-terminal truncated amyloid beta (Aβ) derivatives, especially the forms having pyroglutamate at the 3 position (AβpE3) or at the 11 position (AβpE11) have become the topic of considerable study. AβpE3 is known to make up a substantial portion of the Aβ species in senile plaques while AβpE11 has received less attention. We have generated very specific polyclonal antibodies against both species. Each antibody recognizes only the antigen against which it was generated on Western blots and neither recognizes full length Aβ. Both anti-AβpE3 and anti-AβpE11 stain senile plaques specifically in Alzheimers disease cerebral cortex and colocalize with Aβ, as shown by confocal microscopy. In a majority of plaques examined, AβpE11 was observed to be the dominant form in the innermost core. These data suggest that AβpE11 may serve as a generating site for senile plaque formation.

Differences in the subcellular localization of calreticulin and organellar Ca2+-ATPase in neurons

It has become clear that calcium is an important mediator in the transduction of signals due to ligand binding to cell surface receptors. Cytosolic calcium is typically maintained at low levels in both muscle and non-muscle cells and intracellular sequestering of calcium appears to be important in this process. The identification of intracellular calcium pools has been the subject of much recent study, and it has been proposed that such pools would contain three components: a calcium-activated pump or Ca(2+)-ATPase, a calcium channel such as the inositol trisphosphate receptor or ryanodine receptor, and a high-capacity calcium-binding protein such as calsequestrin or calreticulin. We report here on the localization of two components, the organellar Ca(2+)-ATPase (SERCA) and calreticulin, in neuronal tissues. Using immunofluorescence and subcellular fractionation, we have found that for the most part, these two proteins do not co-localize in neuron cell bodies, dendrites, or axons; but may co-localize at the axon terminal.

Comparison of cDNAs from bovine brain coding for two isoforms of calreticulin

Calreticulin is a major calcium-binding protein of the endoplasmic reticulum of non-muscle cells. In addition to a 1.9-kb calreticulin mRNA, some evidence has suggested the existence of another transcript of 3.75 kb, which is very similar to calreticulin. We report here the isolation and sequencing of cDNA clones from a bovine brain lambda gt11 cDNA library, two of which appear to code for calreticulin and a third for a novel isoform of calreticulin. The deduced amino-acid sequence of the novel clone shares high similarity with mouse calreticulin in the C-terminal 318 amino acids. However, its N-terminal sequence is completely divergent. Northern blot analysis of bovine cerebral cortex RNA indicates that the conserved region of the clone hybridizes to two messages of 1.9 kb and 3.75 kb. The divergent region of this clone hybridizes to the 3.75-kb message, but not to the 1.9-kb message. We believe that this novel clone corresponds to an alternate form of calreticulin which is identical to calreticulin toward the C-terminus, but completely different at the N-terminal region, and that this isoform is encoded by a much larger message.

Brain endothelial cell enzymes cleave platelet-retained amyloid precursor protein

We have previously demonstrated that thrombin-activated platelets from patients with advanced Alzheimers disease (AD) retain significantly more surface membrane-bound amyloid precursor protein (mAPP) than platelets from non-demented age-matched individuals (AM). We have studied interactions between these platelets and the cerebrovascular endothelium to which activated platelets adhere in a model system, investigating their involvement in the formation of amyloid beta peptide (Abeta) deposits in AD patients. We report here that there appear to be alpha and beta secretase-like activities in primary human blood brain barrier endothelial cell (BEC) cultures from both AD patients and AM control subjects (AD-BEC and AM-BEC, respectively) as well as a gamma secretase-like activity that appears only in AD-BEC. No such activities were observed in human umbilical vein endothelial cells (HUVECs). Furthermore, there is more penetration of the platelet-released products platelet factor 4 and soluble APP through the BEC layer grown from AD patients than that grown from AM individuals, whereas none penetrate through a HUVEC layer. Thus the interaction between platelets, the APP they have retained or released, and cerebral vascular endothelial cells may be at least partially responsible for amyloidogenic deposits around the cerebral vasculature of AD patients.

Kinesin is rapidly transported in the optic nerve as a membrane associated protein

We have investigated the membrane vs. cytosolic distribution of newly synthesized and total kinesin in rabbit retinal ganglion cell axons which comprise the optic nerve. We find that kinesin is rapidly transported into the axon and that this newly synthesized protein is completely membrane-associated while approximately two third of the total kinesin in the optic nerve is membrane associated. Of this membrane associated kinesin about half is resistant to removal by treatment with 100 mM Na2CO3 (pH 11.3) and none can be stripped by 1 M NaCl. The newly synthesized axonal kinesin is completely resistant to removal by Na2CO3 treatment. By these criteria, at least one third of the total and essentially all of the rapidly transported axonal kinesin appears to exist as an integral membrane protein, consistent with it functioning as the anterograde motor for rapid vesicle transport from the cell body through the axon.