Amyloid plaques are a neuropathological hallmark of Alzheimer's disease (AD), but their relationship to neurodegeneration and dementia remains controversial. In contrast, there is a good correlation in AD between cognitive decline and loss of synaptophysin-immunoreactive (SYN-IR) presynaptic terminals in specific brain regions. We used expression-matched transgenic mouse lines to compare the effects of different human amyloid protein precursors (hAPP) and their products on plaque formation and SYN-IR presynaptic terminals. Four distinct minigenes were generated encoding wild-type hAPP or hAPP carrying mutations that alter the production of amyloidogenic A␤ peptides. The platelet-derived growth factor ␤ chain promoter was used to express these constructs in neurons. hAPP mutations associated with familial AD (FAD) increased cerebral A␤ 1-42 levels, whereas an experimental mutation of the ␤-secretase cleavage site (671 M3I ) eliminated production of human A␤.High levels of A␤ 1-42 resulted in age-dependent formation of amyloid plaques in FAD-mutant hAPP mice but not in expression-matched wild-type hAPP mice. Yet, significant decreases in the density of SYN-IR presynaptic terminals were found in both groups of mice. Across mice from different transgenic lines, the density of SYN-IR presynaptic terminals correlated inversely with A␤ levels but not with hAPP levels or plaque load. We conclude that A␤ is synaptotoxic even in the absence of plaques and that high levels of A␤ 1-42 are insufficient to induce plaque formation in mice expressing wild-type hAPP. Our results support the emerging view that plaque-independent A␤ toxicity plays an important role in the development of synaptic deficits in AD and related conditions.

Proteolytic processing of the amyloid precursor protein (APP) generates amyloid beta (Abeta) peptide, which is thought to be causal for the pathology and subsequent cognitive decline in Alzheimer's disease. Cleavage by beta-secretase at the amino terminus of the Abeta peptide sequence, between residues 671 and 672 of APP, leads to the generation and extracellular release of beta-cleaved soluble APP, and a corresponding cell-associated carboxy-terminal fragment. Cleavage of the C-terminal fragment by gamma-secretase(s) leads to the formation of Abeta. The pathogenic mutation K670M671-->N670L671 at the beta-secretase cleavage site in APP, which was discovered in a Swedish family with familial Alzheimer's disease, leads to increased beta-secretase cleavage of the mutant substrate. Here we describe a membrane-bound enzyme activity that cleaves full-length APP at the beta-secretase cleavage site, and find it to be the predominant beta-cleavage activity in human brain. We have purified this enzyme activity to homogeneity from human brain using a new substrate analogue inhibitor of the enzyme activity, and show that the purified enzyme has all the properties predicted for beta-secretase. Cloning and expression of the enzyme reveals that human brain beta-secretase is a new membrane-bound aspartic proteinase.

Autosomal dominant forms of familial Alzheimer's disease (FAD) are associated with increased production of the amyloid ␤ peptide, A␤42, which is derived from the amyloid protein precursor (APP). In FAD, as well as in sporadic forms of the illness, A␤ peptides accumulate abnormally in the brain in the form of amyloid plaques. Here, we show that overexpression of FAD(717 V3 F )-mutant human APP in neurons of transgenic mice decreases the density of presynaptic terminals and neurons well before these mice develop amyloid plaques. Electrophysiological recordings from the hippocampus revealed prominent deficits in synaptic transmission, which also preceded amyloid deposition by several months. Although in young mice, functional and structural neuronal deficits were of similar magnitude, functional deficits became predominant with advancing age. Increased A␤ production in the context of decreased overall APP expression, achieved by addition of the Swedish FAD mutation to the APP transgene in a second line of mice, further increased synaptic transmission deficits in young APP mice without plaques. These results suggest a neurotoxic effect of A␤ that is independent of plaque formation.Alzheimer's disease (AD) is a progressive dementing illness in which the brain becomes littered with neuritic amyloid plaques. These plaques are associated with degenerating neuronal processes and consist primarily of fibrillar aggregates of the amyloid ␤ peptide, A␤. A␤ is derived from the amyloid protein precursor (APP), presumably via proteolytic cleavage of APP by ␤-and ␥-secretases (1). The predominant forms of A␤ are 40 (A␤40) or 42 (A␤42) amino acids in length (2). A␤42 and A␤40 appear to be generated in different intracellular compartments, and A␤42 has a greater propensity to selfaggregate into insoluble fibrils than A␤40 (3, 4). Various point mutations in three distinct genes (APP, presenilin 1, presenilin 2) have been linked to autosomal dominant forms of familial AD (FAD). Notably, all of these mutations increase the production of A␤42 (5).Although A␤ has been shown to be neurotoxic in cell culture (6-8), a causal role for A␤ in widespread neuronal degeneration in vivo remains speculative. A particularly controversial question concerns whether A␤-induced neurotoxicity requires deposition of aggregated A␤ into plaques (9-13). Transgenic mice in which full-length FAD-mutant APPs and A␤ are coexpressed at high levels develop typical neuritic amyloid plaques (14-17). However, loss of neurons so far has been identified in only one of these models (18) whereas two others showed no significant neuronal loss despite extensive cerebral A␤ deposition (19,20). No electrophysiological studies have been reported in these models.In the current study, we investigated in transgenic mice what early effects neuronal expression of full-length, FAD-mutant human APP has on the anatomy and physiology of the hippocampus, a central nervous system structure considered crucial for learning and memory. Our study demonstrates that the development ...

Mutations were made at 64 positions on the external surface of the adeno-associated virus type 2 (AAV-2) capsid in regions expected to bind antibodies. The 127 mutations included 57 single alanine substitutions, 41 single nonalanine substitutions, 27 multiple mutations, and 2 insertions. Mutants were assayed for capsid synthesis, heparin binding, in vitro transduction, and binding and neutralization by murine monoclonal and human polyclonal antibodies. All mutants made capsid proteins within a level about 20-fold of that made by the wild type. All but seven mutants bound heparin as well as the wild type. Forty-two mutants transduced human cells at least as well as the wild type, and 10 mutants increased transducing activity up to ninefold more than the wild type. Eighteen adjacent alanine substitutions diminished transduction from 10-to 100,000-fold but had no effect on heparin binding and define an area (dead zone) required for transduction that is distinct from the previously characterized heparin receptor binding site. Mutations that reduced binding and neutralization by a murine monoclonal antibody (A20) were localized, while mutations that reduced neutralization by individual human sera or by pooled human, intravenous immunoglobulin G (IVIG) were dispersed over a larger area. Mutations that reduced binding by A20 also reduced neutralization. However, a mutation that reduced the binding of IVIG by 90% did not reduce neutralization, and mutations that reduced neutralization by IVIG did not reduce its binding. Combinations of mutations did not significantly increase transduction or resistance to neutralization by IVIG. These mutations define areas on the surface of the AAV-2 capsid that are important determinants of transduction and antibody neutralization.Adeno-associated virus type 2 (AAV-2) is a parvovirus belonging to the Dependovirus genus (16). AAV-2 has a linear, single-stranded DNA genome containing 4,679 nucleotides that encodes two genes called rep and cap. The rep gene encodes four proteins that play roles in viral DNA replication, DNA packaging, and the regulation of transcription and splicing. The cap gene encodes three proteins (VP1, VP2, and VP3) that package viral DNA and determine viral tropism. The external surface of the capsid is also the target for neutralizing antibodies. To date, about 180 different AAV capsids have been identified. The three capsid proteins are produced from the same translational reading frame by means of alternative splicing and translation initiation sites.The C termini, each of which is typically about 530 amino acids long, are identical and represent VP3. VP3 is the most abundant capsid protein, representing about 80% of the protein in an assembled capsid. VP2 is typically about 65 amino acids longer than VP3 at the amino terminus and is dispensable for most AAV-2 functions (51). VP1 is typically about 135 amino acids longer than VP2 at the amino terminus. This N-terminal domain is located inside the capsid (26) and encodes the phospholipase A2, which is required f...

Using polymerase chain reactions and genome walking strategies, adeno-associated virus (AAV)-like capsid genes were isolated from rat and mouse liver genomic DNA, where they are present at <5 copies per cell. These genes define two new species of AAVs since their amino acid sequences are <60% identical to each other or to any other AAV capsid. They are most similar to the AAV-5 and goat AAV capsids. A recombinant vector with the mouse AAV capsid and a lacZ transgene (rAAV-mo.1 lacZ) was able to transduce rodent cell lines in vitro. However, it was not able to transduce eight human cell lines or primary human fibroblasts in vitro. It did not bind heparin and its ability to transduce cells in vitro was not inhibited by heparin, mucin, or sialic acid suggesting it uses a novel entry receptor. rAAV-mo.1 lacZ was 29 times more resistant to in vitro neutralization by pooled, purified human IgG than AAV-2. In vivo, rAAV-mo.1 lacZ efficiently transduced murine ocular cells after a subretinal injection. Intramuscular injection of a rAAV-mo.1 human factor IX (hFIX) vector into mice resulted in no detectable hFIX in plasma, but intravenous injection resulted in high plasma levels of hFIX, equivalent to that obtained from a rAAV-8 hFIX vector. Biodistribution analysis showed that rAAV-mo.1 primarily transduced liver after an intravenous injection. These AAV capsids may be useful for gene transfer in rodents.