The amyloidogenic A␤ peptide is liberated from the amyloid precursor protein (APP) by two proteolytic activities, ␤-secretase and ␥-secretase. Recently, a type I membrane protein termed BACE (␤-site APP cleaving enzyme) with characteristics of an aspartyl protease has been identified as the ␤-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 Nlinked 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 t1 ⁄2 of ϳ16 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.

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.

The amyloid ␤ (A␤) peptide that accumulates in Alzheimer's disease brain is derived from the proteolytic processing of the amyloid precursor protein by ␤-and ␥-secretase activities. The ␤-secretase enzyme ␤-site amyloid precursor protein-cleaving enzyme (BACE) generates the N terminus of A␤ by cleavage at either Asp 1 (␤-site) or Glu 11 (␤-site), ultimately leading to the production of full-length A␤1-40/42 or truncated A␤11-40/42. The functional significance of this variable cleavage site specificity as well as the relative pathological impact of full-length versus N-terminally truncated A␤ remains largely unknown. In our analysis of BACE reactivity in cell culture, we found that the preference of the protease for either ␤-or ␤-cleavage was strongly dependent on intracellular localization. Within the endoplasmic reticulum, ␤-site proteolysis predominated, whereas in the trans-Golgi network, ␤-cleavage was favored. Furthermore, the contrasting cleavage site specificities of BACE were not simply due to differences in organelle pH or the oligosaccharide composition of the glycoproteins involved. Examination of post-mortem brain specimens revealed significant levels of A␤11-40/42 within insoluble amyloid pools. Taken together, these data support an important role for ␤-cleavage in the process of cerebral amyloid deposition and localize the processing event to the trans-Golgi network.Senile plaques, lesions composed largely of aggregated amyloid ␤ (A␤) 1 protein, are a pathologic hallmark of Alzheimer's disease (AD) (1, 2). A␤ is derived from proteolytic processing of the type 1 membrane glycoprotein APP (3, 4), and its deposition most likely represents a crucial causative event in AD pathogenesis (5). The membrane-anchored aspartyl protease BACE acts on APP first at its ␤-cleavage site (6 -10), generating a membrane-bound C-terminal stub (C99) whose subsequent proteolysis by a second enzyme, ␥-secretase, yields A␤. In an alternative cellular pathway precluding A␤ production, APP is initially cleaved by ␣-secretase activity, ultimately leading to the release of a shorter peptide known as p3 (Fig. 1A) (11).Full-length A␤ encompasses a well-defined 40-or 42-amino acid residue stretch within the APP backbone (A␤1-40 and A␤1-42). However, in cerebral amyloid deposits, numerous N-terminally truncated variants of A␤40 and A␤42 (NtA␤), frequently harboring additional structural modifications, have been isolated (2,12,13). Whereas the functional significance of this N-terminal heterogeneity remains unclear, a variety of NtA␤ species aggregate more quickly in vitro than their fulllength counterparts (14). Whereas most types of NtA␤ are assumed to arise from the proteolysis of full-length peptides after their release from cells in the central nervous system, two such variants, A␤11-40 and A␤11-42, are generated directly from APP by BACE proteolysis at an alternative site, termed ␤Ј, between Tyr 10 and Glu 11 of A␤ (8,15,16). This event initially produces a shorter C-terminal stub (C89), which then acts as a substrate for ␥-se...

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.

PEN-2 is an integral membrane protein that is a necessary component of the ␥-secretase complex, which is central in the pathogenesis of Alzheimer's disease and is also required for Notch signaling. In the absence of PEN-2, Notch signaling fails to guide normal development in Caenorhabditis elegans, and amyloid ␤ peptide is not generated from the amyloid precursor protein.Human PEN-2 is a 101-amino acid protein containing two putative transmembrane domains. To understand its interaction with other ␥-secretase components, it is important to know the membrane topology of each member of the complex. To characterize the membrane topology of PEN-2, we introduced single amino acid changes in each of the three hydrophilic regions of PEN-2 to generate N-linked glycosylation sites. We found that the N-linked glycosylation sites present in the N-and C-terminal domains of PEN-2 were utilized, whereas a site in the hydrophilic "loop" region connecting the two transmembrane domains was not. The addition of a carbohydrate structure in the N-terminal domain of PEN-2 prevented association with presenilin 1, whereas glycosylation in the C-terminal region of PEN-2 did not, suggesting that the N-terminal domain is important for interactions with presenilin 1. Immunofluorescence microscopy with selective permeabilization of the plasma membrane of cells expressing epitope-tagged forms of PEN-2 confirmed the lumenal location of both the N and C termini. A protease protection assay also demonstrated that the loop domain of PEN-2 is cytosolic. Thus, PEN-2 spans the membrane twice, with the N and C termini facing the lumen of the endoplasmic reticulum.