Robin J. Johnson

KDEL proteins are found on the surface of NG108-15 cells

Although KDEL proteins are primarily localized to the endoplasmic reticulum (ER), we have employed surface biotinylation method to demonstrate that the KDEL proteins calreticulin (Crt), protein disulfide isomerase (PDI) and the 78-kDa glucose regulated protein (GRP78) are found on the surface of the NG108-15 cell line. In contrast, the 94-kDa glucose regulated protein (GRP94), another KDEL protein, is not found on the cell surface. Calnexin (Cnx), a type-1 integral transmembrane ER protein which is partially homologous to Crt but lacks the KDEL sequence, is not detected on the cell surface either. While only small amounts of the total GRP78, PDI and Crt molecules exist on the cell surface at steady state, a significant fraction of the newly synthesized molecules are transported to the cell surface and transport of these proteins is inhibited by treatment with brefeldin A. The surface GRP78 contains the KDEL sequence. On the cell surface, GRP78, PDI and Crt associate with other proteins and form complexes of different sizes. Surface Crt is found to be essential for the neurite formation when NG108-15 cells are induced to differentiate using dibutyryl cAMP.

Amyloid precursor protein is synthesized by retinal ganglion cells, rapidly transported to the optic nerve plasma membrane and nerve terminals, and metabolized.

Abstract: 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.

Toxicity of various amyloid β peptide species in cultured human blood–brain barrier endothelial cells: Increased toxicity of Dutch‐type mutant

The amyloid β peptide (Aβ) is the major component of the neuritic and cerebrovascular amyloid plaques that are one of the characteristic features of Alzheimers disease (AD). This peptide has been shown to be toxic to several relevant cell types, including neurons, cerebrovascular smooth muscle cells, and endothelial cells. We have studied the toxic effects of both soluble and aggregated species of Aβ1–40 and the mutation Aβ1–40Glu→Gln22, which is the major species deposited in the cerebrovascular blood vessels of victims of hereditary cerebral hemorrhage with amyloidosis, Dutch type. We find that aggregates of both peptides, as well as of Aβ1–42 and Aβ25–35, are toxic to cultured human cerebrovascular endothelial cells (hBEC) obtained from the brain of a victim of AD (at doses lower than those that are toxic to CNS neurons or leptomeningeal smooth muscle cells). Soluble Aβ1–40 Gln22 is equally toxic to hBEC, whereas wild‐type Aβ1–40 is toxic only at higher doses. This toxicity is seen at the lowest dose of Aβ1–40 Gln 22 used, 20 nM. The soluble Aβ1–40Gln22 aggregates on the surface of the cells, in contrast to Aβ1–40, and its toxicity can be blocked both by an inhibitor of free radical formation and by Congo red, which inhibits amyloid fibril formation. We discuss the possibility that the enhanced toxicity of Aβ1–40Gln22 is mediated by a Aβ receptor on the endothelial cells. J. Neurosci. Res. 60:804–810, 2000.

Retinal Muller glia secrete apolipoproteins E and J which are efficiently assembled into lipoprotein particles

We have shown that apolipoprotein E (ApoE) is synthesized by Muller cells, the major glial cell of the rabbit retina, and secreted into the vitreous after which it is taken up by retinal ganglion cells and rapidly transported into the optic nerve [Amaratunga et al., J. Biol. Chem. 271 (1996) 5628-5632]. In this report we demonstrate that the ApoE secreted by Muller cells in vivo and in culture is efficiently assembled into lipoprotein particles. Apolipoprotein J (ApoJ) is also synthesized by these cells and assembled into lipoprotein particles. The lipoproteins are triglyceride-rich and contain cholesterol esters and free cholesterol. They are heterogeneous, with densities between 1.006 and 1.18 and diameters between 14 and 45 nm. We discuss the possible role of these lipoproteins in supplying the needs of neurons for lipids, especially long axonal projection neurons such as retinal ganglion cells, which are vulnerable to age-related neurodegenerative diseases including Alzheimers disease.

CALRETICULIN IS TRANSPORTED TO THE SURFACE OF NG108-15 CELLS WHERE IT FORMS SURFACE PATCHES AND IS PARTIALLY DEGRADED IN AN ACIDIC COMPARTMENT

Although calreticulin (Crt) is primarily localized to the endoplasmic reticulum (ER), our results using biotinylation and immunocytochemical methods indicate that a small but significant amount of Crt is present and forms large patches on the surface of NG108‐15 cells (a mouse neuroblastoma‐rat glioma hybrid cell line). 35S‐labelled Crt molecules begin to reach the cell surface after only 10 min of labelling and disappear slowly from the cell surface. After 4 hr of labelling, approximately half of the newly synthesized Crt molecules are on the cell surface. We believe that some Crt molecules may escape from the KDEL receptor‐mediated salvage pathway as they are synthesized and proceed through the secretory pathway to the cell surface. Immunoprecipitation from the culture medium shows that Crt is not released from the cell surface to the medium, suggesting tight binding to surface molecules. NH4Cl can block the degradation of Crt; therefore, Crt is presumably degraded in the lysosome pathway. However, blockage of the disappearance of surface Crt is less efficient than that of internal Crt. This suggests that the disappearance of Crt from the cell surface may not be due solely to its degradation, but may reflect transport into another cell compartment such as the ER. J. Neurosci. Res. 58:652–662, 1999.

Calreticulin functions as a molecular chaperone for the β-amyloid precursor protein1

Processing of the beta-amyloid precursor protein (APP) in the endoplasmic reticulum and the Golgi apparatus may be critical in generating the beta-amyloid molecules linked to the pathogenesis of Alzheimers disease. Since chaperone molecules such as calreticulin (Crt) have been shown to be important in the maturation of many glycoproteins, we investigated the interaction between Crt and APP. We show that APP binds transiently to Crt in a manner that is pH, divalent cation, and N-linked glycosylation-dependent. Both immature APP (containing only N-linked sugars) and mature APP (containing both N-linked and O-linked sugars) bind to Crt. Both proteins are part of a complex that appears to be large enough to accommodate other proteins as well. However, while most of the immature form is associated with the complexes, very little of the mature form is. The interaction between APP and Crt is likely to be of physiological significance with respect to APP maturation since Crt is involved in quality control of nascent glycoproteins in the secretory pathway.

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.

Serum amyloid A in Alzheimer's disease brain is predominantly localized to myelin sheaths and axonal membrane*

Immunohistochemical localization of the injury specific apolipoprotein, acute phase serum amyloid A (A-apoSAA), was compared in brains of patients with neuropathologically confirmed Alzheimers disease (AD), multiple sclerosis (MS), Parkinsons disease (PD), Picks disease (Picks), dementia with Lewy bodies (DLB), coronary artery disease (CAD), and schizophrenia. Affected regions of both AD and MS brains showed intense staining for A-apoSAA in comparison to an unaffected region and non-AD/MS brains. The major site of A-apoSAA staining in both diseases was the myelin sheaths of axons in layers V and VI of affected cortex. A-apoSAA contains a cholesterol binding site near its amino terminus and is likely to have a high affinity for cholesterol-rich myelin. These findings, along with our recent evidence that A-apoSAA can inhibit lipid synthesis in vascular smooth muscle cells suggest that A-apoSAA plays a role in the neuronal loss and white matter damage occurring in AD and MS.

Isolation and characterization of substance P-containing dense core vesicles from rabbit optic nerve and termini

In neurons, neuropeptides and other synaptic components are transported down the axon to the synapse in vesicles using molecular motors of the kinesin family. In the synapse, these neuropeptides are found in dense core vesicles (DCVs), and, following calcium‐mediated exocytosis, they interact with receptors on the target cell. We have developed a rapid, large‐scale technique for purifying peptide‐containing DCVs from specific nuclei in the central nervous system. By using differential velocity gradient and equilibrium gradient centrifugation, neuropeptide‐containing DCVs can be separated by size and density from optic nerve (ON) and its termini, the lateral geniculate nuclei and the superior colliculi. Isolated DCVs contain neuropeptides (substance P and brain‐derived neurotrophic factor), synaptic vesicle (SV) membrane proteins (SV2, synaptotagmins, synaptophysin, Rab3 and synaptobrevin), SV‐associated proteins (α‐synuclein), secretory markers for DCVs previously isolated (secretogranin II), and β‐amyloid precursor protein. By using electron microscopic techniques, DCV were also visualized and shown to be immunoreactive for neuropeptides, neurotrophins, and SV membrane proteins. Because of the interesting group of physiological and potentially pathophysiological proteins associated with these vesicles; this isolation procedure, applicable to other CNS nuclei, should represent an important research tool. J. Neurosci. Res. 62:830–839, 2000.

Increased calreticulin stability in differentiated NG-108-15 cells correlates with resistance to apoptosis induced by antisense treatment

Since its first identification as a high-affinity calcium-binding protein over two decades ago [T.J. Ostwald and D.H. MacLennan, Isolation of a high-affinity calcium-binding protein from sarcoplasmic reticulum, J. Biol. Chem., 249 (1974) 974-979], calreticulin has become recognized as a multifunctional protein involved in a wide variety of cellular processes. We have previously shown that it has a protective function in Ca2+-mediated cell death [N. Liu, R.E. Fine, E. Simons and R.J. Johnson, Decreasing calreticulin expression lowers the Ca2+ response to bradykinin and increases sensitivity to ionomycin in NG-108-15 cells, J. Biol. Chem. , 269 (1994) 28635-28639]. We report here that in NG-108-15 neuroblastomaxglioma hybrid cells, calreticulin protein levels increase markedly when these cells are induced to differentiate by treating them with N,N-dibutyryl cAMP (db-cAMP). We demonstrate that the reason for this increase is mostly due to a large increase in the turnover time of calreticulin in differentiated cells. We also show that a calreticulin antisense oligonucleotide, CrtAS1, previously described by Liu and co-workers [N. Liu, R.E. Fine, E. Simons and R.J. Johnson, Decreasing calreticulin expression lowers the Ca2+ response to bradykinin and increases sensitivity to ionomycin in NG-108-15 cells, J. Biol. Chem., 269 (1994) 28635-28639] causes cell death in undifferentiated NG-108-15 cells when antisense treatment is extended for more than 24 h. This effect is not seen in NG-108-15 cells that have been induced to differentiate with db-cAMP until the cells have been treated with antisense for more than 4 days, due to the increased stability of Crt in these cells. Our results indicate that the mechanism by which these cells die is likely to be apoptosis.