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...

To investigate the basis for envelope (Env) determinants influencing simian immunodeficiency virus (SIV) tropism, we studied a number of Envs that are closely related to that of SIVmac239, a pathogenic, T-tropic virus that is neutralization resistant. The Envs from macrophage-tropic (M-tropic) virus strains SIVmac316, 1A11, 17E-Fr, and 1100 facilitated infection of CCR5-positive, CD4-negative cells. In contrast, the SIVmac239 Env was strictly dependent upon the presence of CD4 for membrane fusion. We also found that the Envs from M-tropic virus strains, which are less pathogenic in vivo, were very sensitive to antibody-mediated neutralization. Antibodies to the V3-loop, as well as antibodies that block SIV gp120 binding to CCR5, efficiently neutralized CD4-independent, M-tropic Envs but not the 239 Env. However, triggering the 239 Env with soluble CD4, presumably resulting in exposure of the CCR5 binding site, made it as neutralization sensitive as the M-tropic Envs. In addition, mutations of N-linked glycosylation sites in the V1/V2 region, previously shown to enhance antigenicity and immunogenicity, made the 239 Env partially CD4 independent. These findings indicate that Env-based determinants of M tropism of these strains are generally associated with decreased dependence on CD4 for entry into cells. Furthermore, CD4 independence and M tropism are also associated with neutralization sensitivity and reduced pathogenicity, suggesting that the humoral immune response may exert strong selective pressure against CD4-independent M-tropic SIVmac strains. Finally, genetic modification of viral Envs to enhance CD4 independence may also result in improved humoral immune responses.

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.

Schwann cells (SCs) are essential for proper peripheral nerve development and repair, although the mechanisms regulating these processes are incompletely understood. We previously showed that the adhesion G protein-coupled receptor Gpr126/Adgrg6 is essential for SC development and myelination. Interestingly, the expression of Gpr126 is maintained in adult SCs, suggestive of a function in the mature nerve. We therefore investigated the role of Gpr126 in nerve repair by studying an inducible SC-specific Gpr126 knock-out mouse model. Here, we show that remyelination is severely delayed after nerve-crush injury. Moreover, we also observe noncell-autonomous defects in macrophage recruitment and axon regeneration in injured nerves following loss of Gpr126 in SCs. This work demonstrates that Gpr126 has critical SC-autonomous and SC-nonautonomous functions in remyelination and peripheral nerve repair.

Nicastrin (NCT) is a type I integral membrane protein that is one of the four essential components of the ␥-secretase complex, a protein assembly that catalyzes the intramembranous cleavage of the amyloid precursor protein and Notch. Other ␥-secretase components include presenilin-1 (PS1), APH-1, and PEN-2, all of which span the membrane multiple times. The mechanism by which NCT associates with the ␥-secretase complex and regulates its activity is unclear. To avoid the misfolding phenotype often associated with introducing deletions or mutations into heavily glycosylated and disulfidebonded proteins such as NCT, we produced chimeras between human (hNCT) and Caenorhabditis elegans NCT (ceNCT). Although ceNCT did not associate with human ␥-secretase components, all of the ceNCT/hNCT chimeras interacted with ␥-secretase components from human, C. elegans, or both, indicating that they folded correctly. A region at the C-terminal end of hNCT, encompassing the last 50 residues of its ectodomain, the transmembrane domain, and the cytoplasmic domain was important for mediating interactions with human PS1, APH-1, and PEN-2. This finding is consistent with the fact that the bulk of the ␥-secretase complex proteins resides within the membrane, with relatively small extramembranous domains. Finally, hNCT associated with hAPH-1 in the absence of PS, consistent with NCT and APH-1 forming a subcomplex prior to association with PS1 and PEN-2 and indicating that the interactions between NCT with PS1 may be indirect or stabilized by the presence of APH-1.

Neuronal hyperexcitability is one of the major characteristics of fragile X syndrome (FXS), yet the molecular mechanisms of this critical dysfunction remain poorly understood. Here we report a major role of voltage-independent potassium (K ϩ)-channel dysfunction in hyperexcitability of CA3 pyramidal neurons in Fmr1 knockout (KO) mice. We observed a reduction of voltage-independent small conductance calcium (Ca 2ϩ)-activated K ϩ (SK) currents in both male and female mice, leading to decreased action potential (AP) threshold and reduced medium afterhyperpolarization. These SK-channel-dependent deficits led to markedly increased AP firing and abnormal input-output signal transmission of CA3 pyramidal neurons. The SK-current defect was mediated, at least in part, by loss of FMRP interaction with the SK channels (specifically the SK2 isoform), without changes in channel expression. Intracellular application of selective SK-channel openers or a genetic reintroduction of an N-terminal FMRP fragment lacking the ability to associate with polyribosomes normalized all observed excitability defects in CA3 pyramidal neurons of Fmr1 KO mice. These results suggest that dysfunction of voltage-independent SK channels is the primary cause of CA3 neuronal hyperexcitability in Fmr1 KO mice and support the critical translation-independent role for the fragile X mental retardation protein as a regulator of neural excitability. Our findings may thus provide a new avenue to ameliorate hippocampal excitability defects in FXS.

SUMMARYSix3 exerts multiple functions in the development of anterior neural tissue of vertebrate embryos. Whereas complete loss of Six3 function in the mouse results in failure of forebrain formation, its hypomorphic mutations in human and mouse can promote holoprosencephaly (HPE), a forebrain malformation that results, at least in part, from abnormal telencephalon development. However, the roles of Six3 in telencephalon patterning and differentiation are not well understood. To address the role of Six3 in telencephalon development, we analyzed zebrafish embryos deficient in two out of three Six3-related genes, six3b and six7, representing a partial loss of Six3 function. We found that telencephalon forms in six3b;six7-deficient embryos; however, ventral telencephalic domains are smaller and dorsal domains are larger. Decreased cell proliferation or excess apoptosis cannot account for the ventral deficiency. Instead, six3b and six7 are required during early segmentation for specification of ventral progenitors, similar to the role of Hedgehog (Hh) signaling in telencephalon development. Unlike in mice, we observe that Hh signaling is not disrupted in embryos with reduced Six3 function. Furthermore, six3b overexpression is sufficient to compensate for loss of Hh signaling in isl1-but not nkx2.1b-positive cells, suggesting a novel Hh-independent role for Six3 in telencephalon patterning. We further find that Six3 promotes ventral telencephalic fates through transient regulation of foxg1a expression and repression of the Wnt/b-catenin pathway.

The gamma-secretase complex functions to cleave several type I transmembrane proteins within their transmembrane domains. These include the amyloid precursor protein, which is central to Alzheimer's disease pathogenesis, as well as N-cadherin and Notch, which regulate transcription. This complex is composed of four requisite integral membrane proteins: presenilin 1 (PS1) or presenilin 2 (PS2), nicastrin, Pen-2, and Aph-1. How these proteins coordinately regulate one another and assemble to form a functional complex is not well understood. In this report we demonstrate that PS1 selectively enhances the stability of Pen-2 protein but not that of nicastrin or Aph-1. In the absence of PS1, Pen-2 was rapidly degraded by the proteasome. As PS1 levels increased, so too did the half-life of Pen-2 and therefore its steady-state levels. In addition, Pen-2 protein levels correlated with PS1 levels not only in cell culture but in transgenic mouse models as well. The genetic absence of PS1 and PS2, and therefore of gamma-secretase-dependent mediation of transcriptional activity, did not affect Pen-2 mRNA levels. Rather, presenilin (PS) regulates Pen-2 levels posttranslationally by preventing its degradation by the proteasome. Thus, the amount of Pen-2 protein is effectively titrated by its PS binding partner, and the rapidity with which Pen-2 is degraded in the absence of PS interactions could provide a mechanism to tightly regulate gamma-secretase complex assembly.