Maturation and endosomal targeting of beta-site amyloid precursor protein-cleaving enzyme. The Alzheimer's disease beta-secretase.

The amyloidogenic Abeta peptide is liberated from the amyloid precursor protein (APP) by two proteolytic activities, beta-secretase and gamma-secretase. Recently, a type I membrane protein termed BACE (beta-site APP cleaving enzyme) with characteristics of an aspartyl protease has been identified as the beta-secretase. We undertook a series of biochemical and morphological investigations designed to characterize the basic properties of this protein. Initial studies indicated that BACE undergoes N-linked glycosylation at three of four potential sites. Metabolic pulse-chase experiments revealed that after core glycosylation, BACE is rapidly and efficiently transported to the Golgi apparatus and distal secretory pathway. BACE was also found to be quite stable, being turned over with a t(12) of approximately 16 h. Retention of BACE in the endoplasmic reticulum by introduction of a C-terminal dilysine motif prevented complex carbohydrate processing and demonstrated that propeptide cleavage occurs after exit from this organelle. BACE exhibited intramolecular disulfide bonding but did not form oligomeric structures by standard SDS-polyacrylamide gel electrophoresis analysis and sedimented as a monomer in sucrose velocity gradients. Immunofluorescence studies showed a largely vesicular staining pattern for BACE that colocalized well with endosomal, but not lysosomal, markers. Measurable levels of BACE were also detected on the plasma membrane by both immunostaining and cell surface biotinylation, and cycling of the protein between the cell membrane and the endosomes was documented. A cytoplasmic dileucine motif was found to be necessary for normal targeting of BACE to the endosomal system and accumulation of the protein in this intracellular site.

A distinct ER/IC gamma-secretase competes with the proteasome for cleavage of APP.

The deposition of amyloid-beta peptides (Abeta) in senile plaques (SPs) is a central pathological feature of Alzheimer's disease (AD). Since SPs are composed predominantly of Abeta1-42, which is more amyloidogenic in vitro, the enzymes involved in generating Abeta1-42 may be particularly important to the pathogenesis of AD. In contrast to Abeta1-40, which is generated in the trans-Golgi network and other cytoplasmic organelles, intracellular Abeta1-42 is produced in the endoplasmic reticulum/intermediate compartment (ER/IC), where it accumulates in a stable insoluble pool. Since this pool of insoluble Abeta1-42 may play a critical role in AD amyloidogenesis, we sought to determine how the production of intracellular Abeta is regulated. Surprisingly, the production of insoluble intracellular Abeta1-42 was increased by a putative gamma-secretase inhibitor as well as by an inhibitor of the proteasome. We further demonstrate that this increased generation of Abeta1-42 in the ER/IC is due to a reduction in the turnover of Abeta-containing APP C-terminal fragments. We conclude that the proteasome is a novel site for degradation of ER/IC-generated APP fragments. Proteasome inhibitors may augment the availability of APP C-terminal fragments for gamma-secretase cleavage and thereby increase production of Abeta1-42 in the ER/IC. Based on the organelle-specific differences in the generation of Abeta by gamma-secretase, we conclude that intracellular ER/IC-generated Abeta1-42 and secreted Abeta1-40 are produced by different gamma-secretases. Further, the fact that a putative gamma-secretase inhibitor had opposite effects on the production of secreted and intracellular Abeta may have important implications for AD drug design.

Recovery of neurofilament expression selectively in regenerating reticulospinal neurons.

During regeneration of lamprey spinal axons, growth cones lack filopodia and lamellipodia, contain little actin, and elongate much more slowly than do typical growth cones of embryonic neurons. Moreover, these regenerating growth cones are densely packed with neurofilaments (NFs). Therefore, after spinal hemisection the time course of changes in NF mRNA expression was correlated with the probability of regeneration for each of 18 identified pairs of reticulospinal neurons and 12 cytoarchitectonic groups of spinal projecting neurons. During the first 4 weeks after operation, NF message levels were reduced dramatically in all axotomized reticulospinal neurons, on the basis of semiquantitative in situ hybridization for the single lamprey NF subunit (NF-180). Thereafter, NF expression returned toward normal in neurons whose axons normally regenerate beyond the transection but remained depressed in poorly regenerating neurons. The recovery of NF expression in good regenerators was independent of axon growth across the lesion, because excision of a segment of spinal cord caudal to the transection site blocked regeneration but did not prevent the return of NF-180 mRNA. The early decrease in NF mRNA expression was not accompanied by a reduction in NF protein content. Thus the axotomy-induced loss of most of the axonal volume resulted in a reduced demand for NF rather than a reduction in volume-specific NF synthesis. We conclude that the secondary upregulation of NF message during axonal regeneration in the lamprey CNS may be part of an intrinsic growth program executed only in neurons with a strong propensity for regeneration.

Neurofilament spacing, phosphorylation, and axon diameter in regenerating and uninjured lamprey axons.

It has been postulated that phosphorylation of the carboxy terminus sidearms of neurofilaments (NFs) increases axon diameter through repulsive electrostatic forces that increase sidearm extension and interfilament spacing. To evaluate this hypothesis, the relationships among NF phosphorylation, NF spacing, and axon diameter were examined in uninjured and spinal cord-transected larval sea lampreys (Petromyzon marinus). In untransected animals, axon diameters in the spinal cord varied from 0.5 to 50 microns. Antibodies specific for highly phosphorylated NFs labeled only large axons (> 10 microns), whereas antibodies for lightly phosphorylated NFs labeled medium-sized and small axons more darkly than large axons. For most axons in untransected animals, diameter was inversely related to NF packing density, but the interfilament distances of the largest axons were only 1.5 times those of the smallest axons. In addition, the lightly phosphorylated NFs of the small axons in the dorsal columns were widely spaced, suggesting that phosphorylation of NFs does not rigidly determine their spacing and that NF spacing does not rigidly determine axon diameter. Regenerating neurites of giant reticulospinal axons (GRAs) have diameters only 5-10% of those of their parent axons. If axon caliber is controlled by NF phosphorylation via mutual electrostatic repulsion, then NFs in the slender regenerating neurites should be lightly phosphorylated and densely packed (similar to NFs in uninjured small caliber axons), whereas NFs in the parent GRAs should be highly phosphorylated and loosely packed. However, although linear density of NFs (the number of NFs per micrometer) in these slender regenerating neurites was twice that in their parent axons, they were highly phosphorylated. Following sectioning of these same axons close to the cell body, axon-like neurites regenerated ectopically from dendritic tips. These ectopically regenerating neurites had NF linear densities 2.5 times those of uncut GRAs but were also highly phosphorylated. Thus, in the lamprey, NF phosphorylation may not control axon diameter directly through electrorepulsive charges that increase NF sidearm extension and NF spacing. It is possible that phosphorylation of NFs normally influences axon diameter through indirect mechanisms, such as the slowing of NF transport and the formation of a stationary cytoskeletal lattice, as has been proposed by others. Such a mechanism could be overridden during regeneration, when a more compact, phosphorylated NF backbone might add mechanical stiffness that promotes the advance of the neurite tip within a restricted central nervous system environment.

Glial cells of the lamprey nervous system contain keratin-like proteins.

Lamprey axons regenerate following spinal cord transection despite the formation of a glial scar. As we were unable to detect a lamprey homologue of glial fibrillary acidic protein (GFAP), a major constituent of astrocytes, we studied the composition of intermediate filament (IF) proteins of lamprey glia. Monoclonal antibodies (mAbs) were raised to lamprey spinal cord cytoskeletal extracts and these mAbs were characterized by using Western blotting and immunocytochemistry. On two-dimensional (2-D) Western blots, five of the mAbs detected three major IF polypeptides in the molecular weight (MW) range of 45-56 kD. Further studies were conducted to determine the relationship between the lamprey glial-specific antigen and other mammalian IF proteins. Antikeratin 8 antibody recognized two of the three polypeptides. Several of the glial-specific mAbs reacted with human keratins 8 and 18 on Western blots. Keratin-like immunoreactivity was found in all parts of the central and peripheral nervous systems in both larval and adult lampreys. The immunocytochemical staining patterns of glial-specific mAbs were indistinguishable on lamprey spinal cord sections. However, on brain sections, two distinct patterns were observed. A subset of mAbs stained only a few glial fibers in the brain, whereas others stained many more brain glia, particularly the ependymal cells. The former group of mAbs recognized only the two lower MW polypeptides on 2-D Western blots, but the latter group of mAbs recognized all three major IF polypeptides. This correlation is supported by the observation that the highest MW IF polypeptide has an increased level of expression in the brain relative to the spinal cord. Thus, in the lamprey, the glial cells of both spinal cord and brain express molecules similar to simple epithelial cytokeratins, but their IFs may contain these keratins in different stoichiometric proportions. The widespread presence in the lamprey of primitive glial cells containing keratin-like intermediate filaments may have significance for the extraordinary ability of lamprey spinal axons to regenerate.

Structure of reticulospinal axon growth cones and their cellular environment during regeneration in the lamprey spinal cord.

The large larval sea lamprey is a primitive vertebrate that recovers coordinated swimming following complete spinal transection. An ultrastructural study was performed in order to determine whether morphologic features of regenerating axons and their cellular environment would provide clues to their successful regeneration compared to their mammalian counterparts. Three larval sea lampreys were studied at 3, 4 and 11 weeks following complete spinal transection and compared with an untransected control. Müller and Mauthner cells or their giant reticulospinal axons (GRAs) were impaled and injected with horseradish peroxidase (HRP). Alternating thick and thin sections were collected for light and electron microscopy. A total of 9 neurites were examined. At all times, growth cones of GRAs differed from those of cultured mammalian neurons in being packed with neurofilaments and in lacking long filopodia, suggesting possible differences in the mechanisms of axon outgrowth. Morphometric analysis suggested that GRA growth cones contact glial fibers disproportionately compared to the representation of glial surface membranes in the immediate environment of these growth cones. No differences were found between glial cells in regenerating spinal cords and those of untransected control animals with regard to the size of the cell body and nucleus and the packing density of their intermediate filaments. Glial fibers in control animals and glial fibers located far from a transection were oriented transversely. Glial cells adjacent to the transection site sent thickened, longitudinally oriented processes into the blood clot at the transection site. These longitudinal glial processes preceded the regenerating axons. Desmosomes were observed on glia adjacent to the lesion but were scarce in the lesion during the first four weeks post-transection. These findings suggest that longitudinally oriented glial fibers may serve as a bridge along which axons can regenerate across the lesion. The presence of desmosomes might prevent migration of astrocytes near the transection, thus stabilizing the glial bridge.