Neural stem cell transplantation at critical period improves learning and memory through restoring synaptic impairment in Alzheimer's disease mouse model.

Alzheimer's disease (AD) is characterized by neuronal loss in several regions of the brain. Recent studies have suggested that stem cell transplantation could serve as a potential therapeutic strategy to halt or ameliorate the inexorable disease progression. However, the optimal stage of the disease for stem cell transplantation to have a therapeutic effect has yet to be determined. Here, we demonstrated that transplantation of neural stem cells into 12-month-old Tg2576 brains markedly improved both cognitive impairments and neuropathological features by reducing ß-amyloid processing and upregulating clearance of ß-amyloid, secretion of anti-inflammatory cytokines, endogenous neurogenesis, as well as synapse formation. In contrast, the stem cell transplantation did not recover cognitive dysfunction and ß-amyloid neuropathology in Tg2576 mice aged 15 months when the memory loss is manifest. Overall, this study underscores that stem cell therapy at optimal time frame is crucial to obtain maximal therapeutic effects that can restore functional deficits or stop the progression of AD.

Modified lateral orbitotomy for intact removal of orbital dumbbell dermoid cyst.

: We describe a modified lateral orbitotomy for intact removal of a dumbbell dermoid cyst involving the frontozygomatic suture. The clinical features and surgical treatment of the case are discussed.

Megakaryothrombopoiesis during ex vivo expansion of human cord blood CD34+ cells using thrombopoietin.

Thrombopoietin (TPO) is one of the most promising stimulants for ex vivo expansion of haematopoietic stem cells. Previously, we have found that TPO induces a characteristic pattern of apoptosis during ex vivo expansion of human cord blood (CB) CD34+ cells and that the TPO-induced apoptotic cells belong to megakaryocyte (MK) lineage. In this study, we have examined the maturation of MK and platelet production in association with the TPO-induced apoptosis. CD34+ cells, purified from human CB, were expanded in serum-free conditions stimulated with TPO. Apoptosis was confirmed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labelling (TUNEL) assay and electron microscopy (EM). Simultaneous measurement of DNA content and immunophenotyping revealed that the cells with higher DNA content (>8 N) constituted less than 5% of the CD41+ fractions until day 14, implying premature apoptosis of MKs before full polyploidization. Nevertheless, EM observation showed not only platelet territories but also newly produced platelets in which granules and microfilaments could be identified. Furthermore, flow cytometry demonstrated that the platelet fraction expressed P-selectin and an activation motif on GPIIb/IIIa recognized by monoclonal antibody PAC-1 upon stimulation with adenosine diphosphate (ADP). In addition, periodic acid-Schiff (PAS)-positive materials and nonspecific esterase activities could be demonstrated. Therefore, it is suggested that platelet production and the accompanying processes, rather than apoptosis only, be hastened during the ex vivo expansion of CB CD34+ cells when using TPO.

Protective effect of glutathione in HIV-1 lytic peptide 1-induced cell death in human neuronal cells.

To elucidate the pathogenic mechanisms involved in neurodegeneration in AIDS patients with cognitive deficits, we have examined the toxic effect of the lentivirus lytic peptide 1 (LLP-1) corresponding to the carboxyl terminus of HIV-1 transmembrane glycoprotein gp41 on human neuronal and glial cell lines. LLP-1 induced a significant lactate dehydrogenase (LDH, a marker of cell death) release from these cells in a concentration- and time-dependent manner, while the noncytolytic LLP-1 analog 2 had little effect. Application of LLP-1 to SH-SY5Y, a well-characterized human neuronal cell line, caused the decline of intracellular glutathione (GSH) content that appeared to occur before a significant LDH release. Furthermore, LLP-1 elicited a significant loss of mitochondrial function as measured by mitochondrial transmembrane potential (MTP). Among the reducing agents and antioxidants tested, GSH and a GSH prodrug N-acetylcysteine (NAC) provided protection against LLP-1-induced neuronal cell death, evidently by restoring the intracellular GSH levels and blocking the disruption of mitochondrial integrity. Thus, gp41-derived LLP-1 may be a potential neurotoxic agent capable of causing the intracellular GSH depletion and disturbing the mitochondrial function, possibly contributing to the neurodegenerative cascade as seen in HIV-1-associated dementia. Our data indicate that restoring both GSH concentration and mitochondrial function may hold promise as possible therapeutic strategies for slowing disease progression of dementia in AIDS patients.

Optimal concentration of human epidermal growth factor (hEGF) for epithelial healing in experimental corneal alkali wounds.

PURPOSE: By using both in vivo and in vitro (organ-cultured) systems, the optimal concentrations of hEGF to enhance epithelial healing after alkali wounds were evaluated in the rabbit cornea. METHODS: Alkali-injured corneas (pi = 5.5 mm, 1 N NaOH, 60 s) were treated with 0.01, 0.1, 1.0, 10 and 100 ng/ml hEGF for the in vitro study. The healing of epithelium and endothelium was determined at 1, 2, 3, 4, and 6 days after treatment. For the in vivo experiment, the eyes were treated with 2, 5, 10, and 50 microg/ml hEGF 3 times per day. The measurement of epithelial healing rate, transmission electron microscopy and immunohistochemical observation were performed after 7 days treatment. RESULTS: In in vitro tests, hEGF enhanced the epithelial healing rates, showing a maximum enhancement at the concentration of 1.0 ng/ml, and endothelial healing was increased at 100 ng/ml. In in vivo studies, no significant difference was observed in the rates of epithelial healing between control and each hEGF-treated group. Among the tested concentrations, 5 microg/ml hEGF induced the most active proliferation of basal cells and 50 microg/ml hEGF remarkably produced a vascular ingrowth to the central wound area. The thickness of re-surfaced epithelium was increased by hEGF in a concentration-dependent manner. CONCLUSIONS: The results of the present study indicate that a low concentration of hEGF may selectively enhance epithelial healing without affecting endothelial healing. The optimal concentration of hEGF for the stimulation of epithelial healing appears to be 5 microg/ml in rabbit corneal alkali wounds.

Effects of the beta-amyloid and carboxyl-terminal fragment of Alzheimer's amyloid precursor protein on the production of the tumor necrosis factor-alpha and matrix metalloproteinase-9 by human monocytic THP-1.

To explore the direct role of beta-amyloid (Abeta) and carboxyl-terminal fragments of amyloid precursor protein in the inflammatory processes possibly linked to neurodegeneration associated with Alzheimer's disease, the effects of the 105-amino acid carboxyl-terminal fragment (CT(105)) of amyloid precursor protein on the production of tumor necrosis factor-alpha (TNF-alpha) and matrix metalloproteinase-9 (MMP-9) were examined in a human monocytic THP-1 cell line and compared with that of Abeta. CT(105) elicited a marked increase in TNF-alpha and MMP-9 production in the presence of interferon-gamma in a dose- and time-dependent manner. Similar patterns were obtained with Abeta despite its low magnitude of induction. Autocrine TNF-alpha is likely to be a main mediator of the induction of MMP-9 because the neutralizing antibody to TNF-alpha inhibits MMP-9 production. Genistein, a specific inhibitor of tyrosine kinase, dramatically diminished both TNF-alpha secretion and subsequent MMP-9 release in response to CT(105) or Abeta. Furthermore, PD98059 and SB202190, specific inhibitors of ERK or p38 MAPK respectively, efficiently suppressed CT(105)-induced effects whereas only PD98059 was effective at reducing Abeta-induced effects. Our results suggest that CT(105) in combination with interferon-gamma might serve as a more potent activator than Abeta in triggering inflammatory processes and that both tyrosine kinase and MAPK signaling pathways may represent potential therapeutic targets for the control of Alzheimer's disease progression.

Expression of complement inhibitor protein CD59 in human neuronal and glial cell lines treated with HIV-1 gp41 peptides.

In attempts to elucidate the pathogenic mechanisms involved in neurodegeneration in AIDS patients with cognitive deficits, the possible effect of HIV-1 transmembrane envelope protein gp41 on expression of the membrane inhibitor of complement mediated cytolysis (CD59) was assessed in human neuronal (SK-N-SH) and astroglial (T98G) cell lines. Western blotting analyses demonstrated that an immunodominant (ID, aa 598-613) gp41 peptide as well as the recombinant gp41 protein encompassing this domain markedly reduced CD59 level in a dose dependent manner whereas p24 and control peptide had little effect. RT-PCR showed that ID peptide also elicited a reduction in the expressed CD59 mRNA level. This gp41 peptide apparently down-regulated phorbol 12,13-dibutyrate induced elevation of CD59 at the protein and mRNA levels in a manner similar to that conferred by protein kinase C inhibitor, H-7 or staurosporine in SK-N-SH. Interestingly, proinflammatory cytokines such as IL-1beta or IFN-gamma as well as LPS greatly decreased CD59 in SK-N-SH and to a lesser extent in T98G whereas TNF-alpha did not significantly alter it. In contrast, antioxidants and anti-inflammatory agents enhanced CD59 expression reversing gp41 peptide mediated inhibitory effect in SK-N-SH. Our data suggest that high level of gp41 or its metabolites as well as impaired protein kinase response, chronic inflammation or antioxidant depletion within HIV-1 infected brains may be associated with a diminished expression of CD59 which would render neuronal cells to susceptible to indirect bystander lysis in the presence of autologous complement.

Effect of HIV-1 gp41 peptides on secretion of beta-amyloid precursor protein in human astroglial cell line, T98G.

To understand the mechanism underlying cognitive deficits in AIDS patients, we examined the influence of gp41 peptides on the expression and the secretion of Alzheimer's amyloid precursor protein (APP) in human astroglial cell line T98G. Western blotting analyses demonstrated that treatment of glial cells with a putative immunosuppressive domain (aa 583-599) of gp41 remarkably downregulated the interleukin 1beta- (IL-1beta) induced elevation of the secreted form of APP (sAPP alpha) containing Kunitz-type protease inhibitor (KPI) domain without significant changes of the expression pattern of APP mRNAs as revealed by reverse transcriptase polymerase chain reaction (RT-PCR) analysis. Recombinant gp41 protein encoding for ectodomain, including aa 583-599 residues, also elicited a similar dose-dependent inhibitory effect, whereas the control peptides resulted in little change. The molecular mechanism underlying this gp41-mediated reduction of sAPP alpha secretion appears not to be owing to the difference in the function of extracellular proteases based on the finding of similar proteolytic activities responsible for APP metabolism in vitro present in the conditioned media from the cultures treated with or without gp41 peptide. However, the known PKC inhibitors such as H-7 or staurosporine, partially inhibited the elevation of sAPP alpha secretion in response to protein kinase C (PKC) agonist phorbol 12,13-dibutyrate (PdBu) as well as to IL-1beta, mimicking the immunosuppressive gp41 peptide. These observations implicate that part of the neurodegenerative cascade in AIDS brains may involve the inhibitory effect of gp41 on secretion of sAPP alpha, a potent glial neurotrophic factor, through impaired PKC response.

Increased activity of matrix metalloproteinase-2 in human glial and neuronal cell lines treated with HIV-1 gp41 peptides.

Part of the neurodegenerative cascade in AIDS dementia may involve overexpression of matrix metalloproteinases (MMPs). Here, we examined the possible effect of HIV-1 gp41, which has been shown as a key determinant associated with pathogenesis of AIDS dementia, on the activity of MMPs using human neuronal and glial cell lines. Zymographic analysis revealed that treatment with the gp41 peptide (aa 583-599) for 24 h markedly elevated the activity of MMP with Mr 66 kDa in the cultured media of glioblastoma cell line T98G in a concentration-dependent manner as well as of neuroblastoma cell line SK-N-SH despite of lower magnitude of the activity. In contrast, the immediately adjacent gp41 peptide (aa 598-613) as well as the reverse peptide (aa 598-583) had a little effect. Recombinant gp41 protein containing extracellular domain also elicited a similar effect, although with a lesser extent. This 66 kDa MMP was confirmed as gelatinase A (MMP-2) based on the results of its activity dependent on Ca2+ and inhibited in the presence of 1,10-phenanthroline or EDTA, as well as its specific immunoreactivity on the Western blot. N-acetyl cysteine (NAC) downregulated this gp41 peptide-induced MMP-2 activity in T98G. The soluble form of amyloid precursor protein (sAPP), which is synthesized in the Escherichia coli system, also inhibited the MMP-2 activity in vitro. Taken together, these results implicate that high production of HIV-1 gp41 or its metabolites containing aa 583-599 within central nervous system (CNS) could result in the increased activity of MMP-2 and that the extracellular deficiency of reducing agent or decreased level of sAPP within CNS could exacerbate this gp41-induced MMP-2 activity.

Expression of Alzheimer's amyloid precursor protein in human lymphocyte.

Beta-amyloid precursor protein (betaAPP) has been shown to be involved in cell growth regulation. In spleen, the majority of cells showing betaAPP like immunoreactivity was found in the T cell-dependent zone. In Northern blot, the expression of betaAPP was increased to reach the peak at 72 h after the treatment of phytohemagglutinin (PHA). But, in cytofluorometry, almost all CD4(+) T helper/inducer cells and the majority of CD(8+) T suppressor/cytotoxic cells show betaAPP immunoreactivity which remained constant during the stimulation with PHA. These results suggest that betaAPP is a surface molecule of T lymphocyte and the turnover or release of APP might be increased with the treatment of T cell mitogen.

Molecular physiology, biochemistry, and pharmacology of Alzheimer's amyloid precursor protein (APP).

The function of APP is not yet known in detail but growing evidence exists that APP may mediate cell interactions with the cell surface or soluble glycoproteins and defense mechanisms in the CNS involving the immune system. We describe here the finding that almost all CD4+ lymphocytes and the majority of CD8+ lymphocytes were positive for A beta and the antibodies against A beta or APP did not inhibit the [3H]-thymidine uptake of mitogen-treated lymphocytes significantly. There were no differences in the A beta immunoreactivity on the cell surface of lymphocytes between Alzheimer's disease (AD) and control samples. Excessive amyloidogenic pathway of APP processing may be the final common pathway involved in the pathogenesis of AD. Thus, the identification of proteases or factors leading to aberrant proteolysis which process APP to yield a variety of potentially amyloidogenic fragments would promise pharmacological targets to develop anti-AD drugs. In attempts to define the proteases or factors which alter the balance between nonamyloidogenic and amyloidogenic processing pathways, our study indicates that thrombin or acetylcholinesterase(AChE)-associated protease may be involved in the amyloidogenic processing pathway of APP in vivo to generate amyloidogenic intermediates linked to amyloid deposition. Highly specific and dose-dependent direct modulation of APP processing by biologically available metal ions including Ca2+, Zn2+, Fe2+/Fe3+ and Al3+ suggest the disrupted metal homeostasis as factors leading to overaccumulation of APP and subsequent aberrant proteolysis utilizing excessive amyloidogenic processing pathway. There is mounting evidence that at least some of the neurotoxicity associated with AD is due to fragments from APP. Most research has focused on the toxic effect and the ion channel activity of A beta in causation of the disease. The possible role of other cleaved products of APP is less clear. We investigated the channel-forming ability of various products of APP when applied to Xenopus oocytes and their neurotoxicity in vitro. CT105 peptide was found to be exceedingly potent at 500 nM concentration in forming nonselective ion channels during application from either outside or inside the oocyte and more toxic than either of the A beta fragments, A beta 25-35, or A beta 1-40. Taken together, these results suggest the possible involvement of CT peptide in inducing the neurotoxicity characteristic of AD through the direct damage on the cell membrane. Therefore, we hypothesize that amyloidogenic CT may make nonselective ion channels or pores in the membrane and may cause neuronal death in the early stage of AD and then further metabolized to more stable and less toxic A beta which may be finally deposited in the brain where it could inflict further toxicity to neurons. Here we report successful inhibition of APP gene expression by antisense oligodeoxynucleotides at the mRNA or the protein level in in vitro and cell culture systems.

Membrane currents induced in Xenopus oocytes by the C-terminal fragment of the beta-amyloid precursor protein.

There is mounting evidence that at least some of the neurotoxicity associated with Alzheimer's disease (AD) is due to proteolytic fragments of the beta-amyloid precursor protein (beta APP). Most research has focused on the amyloid beta protein (A beta), which has been shown to possess ion channel activity. However, the possible role of other cleaved products of the beta APP is less clear. We have investigated the ability of various products of beta APP to induce membrane ion currents by applying them to Xenopus oocytes, a model system used extensively for investigating electrophysiological aspects of cellular, including neuronal, signalling. We focussed on the 105-amino-acid C-terminal fragment (CT105) (containing the full sequence A beta), which has previously been found to be toxic to cells, although little is known about its mode of action. We have found that CT105 is exceedingly potent, with a threshold concentration of 100-200 nM, in inducing nonselective ion currents when applied from either outside or inside the oocyte and is more effective than either beta APP or the A beta fragments, beta 25-35 or beta 1-40. The ion channel activity of CT105 was concentration dependent and blocked by a monoclonal antibody to A beta. These results suggest the possible involvement of CT105 in inducing the neural toxicity characteristic of AD.

Amyloidogenic processing of Alzheimer's amyloid precursor protein in vitro and its modulation by metal ions and tacrine.

Recent studies implicate that excessive amyloidogenic pathway of amyloid precursor protein (APP) processing may be the final common pathway involved in the pathogensis of AD. In attempts to identify the proteases or factors leading to excessive amyloid deposition, we evaluated the potential role of acethylcholinesterase (AChE) and its associated protease for amyloidogenic processing of APP in vitro. Prolonged incubation of a recombinant APP770 with AChE produced several amyloidogenic fragments accumulating a relatively stable a 18 kDa A beta (amyloid beta-protein) bearing carboxy terminal peptide, which was further degraded by an increased concentration of AChE. Protease inhibitory profiles confirmed the trypsin-like serine protease activity present in AChE preparation. This observed APP processing was significantly enhanced by Ca2+, Mg2+, or Mn2+ at 1 mM concentration and modulated in concentration dependent manners by metal ions such as Ca2+, Zn2+, Fe2+/Fe3+, Al3+, or a tacrine, a centrally active cholinesterase inhibitor. Our data imply that AChE and its associated protease may be involved in the generation a 18 kDa amyloidogenic peptide under certain physiological condition in vivo and that the gradual changes in their proteolytic activities or locations and the locally disturbed metal homeostasis could be factors associated with abnormal accumulation of APP, eventually leading to amyloid deposition in AD brain. In addition, zinc or tacrine treatment of AD patients with high dosage or in the long term may have effects on the process of amyloidogensis.

Changes following the use of protraction headgear for early correction of Class III malocclusion.

The purpose of this study was to evaluate treatment effects and posttreatment changes following the application of elastic forces from a facemask to the dentition for the early correction of Class III malocclusion. Cephalograms made before and after treatment and at a minimum of 1 year follow-up (mean 3.57 years, S.D. 2.07) of 16 patients were compared with those of 13 untreated matched controls. Pretreatment age ranged from 4.58 to 8.25 years (mean 6.80 years, S.D. 1.13), and mean active treatment time was 0.61 years (S.D. 0.15). Available study models at the three time periods were also analyzed. Treatment resulted in a significant improvement in the maxillomandibular relationship and overjet (P < 0.001). The major treatment effect was a downward and backward movement of the mandible and retroclination of the mandibular incisors. However, any skeletal and dentoalveolar advancement of the maxilla contributed to the clinically significant improvement. No differences were observed between the patients and the controls during the posttreatment follow-up. Despite some relapse, the patients demonstrated a net improvement in maxillomandibular relationship and overjet at the end of follow-up relative to the controls. Overcorrection of the overjet during treatment may be important for maintaining a successful correction.

Age-related changes of mRNA expression of amyloid precursor protein in the brain of senescence-accelerated mouse.

APP695 mRNA is only expressed in the brains of SAM. The expression of APP mRNA in SAM P1 mice brains is more marked than that in SAM R1 mice brain. APP mRNA expression was increased with advancing age in all brain regions of SAM P1 mice compared with SAM R1. Especially, the changes of the amount of APP mRNA in the prosencephalon and the mesencephalon are significant at P value of 0.05. We suggest that overexpression of APP mRNA may be related to accelerated aging phenomenon in the SAM brain. This is the first report of age-related increase in the amount of APP mRNA in the SAM brain.

Aggregation of amyloid precursor proteins by aluminum in vitro.

The effect of various metal ions on aggregation of human recombinant amyloid precursor protein (APP) in vitro was investigated based on characterizations of altered migration on SDS-PAGE or immunoblots. Most biological metal ions tested had no significant effect on aggregation of APP. In contrast, AlCl3 in particular promoted aggregation of APP or APP-CT105 in a dose dependent manner. This effect of AlCl3 on APP mobility shift was prevented or reversed by the metal chelator, EDTA. Amorphous aggregates were observed in AlCl3 treated APP when examined by EM. These results suggest that aluminum may play a role in the pathogenesis of AD by directly promoting aggregation of APP.

Complete nucleotide sequence and tissue-specific expression of the rat phenylethanolamine N-methyltransferase gene.

The rat phenylethanolamine N-methyltransferase (PNMT) gene was isolated from a genomic library by cross-hybridization with a bovine PNMT cDNA probe. Complete nucleotide sequence analysis of a genomic clone showed that this gene contained three exons and spanned about 2.8 kb in length. There were the acute-phase response element, TATA, SP1, and GRE sequences. The physicochemical properties of rat adrenal PNMT were different from those of the brainstem PNMT. However, northern blot and reverse transcription-polymerase chain reaction analysis showed that the rat PNMT gene may not express the multiple forms of mRNA. These results suggest that the rat PNMT gene might produce a single enzyme protein, whose activity may be differentially modulated by tissue-specific environment in the central and peripheral systems.

Overexpression and simple purification of human immunodeficiency virus-1 gag epitope derived from a recombinant antigen in E. coli and its use in ELISA.

To develop a test for diagnosis of human immunodeficiency virus-1 (HIV-1) exposure sensitivity, a part of the gag gene was cloned and expressed in Escherichia coli, using expression vectors containing a trp promoter. The immunoreactivity of recombinant protein was determined using HIV-1 specific antibodies in a Western blot analysis. The recombinant plasmid, pYHCgag3, gag gene was fused to the trpE' gene linked to the hydroxylamine (HA) cleavage recognition sequence which was induced to overexpress a core antigen (gag a.a. 121-398 from plasmid BH10) as fusion protein in the form of insoluble inclusion body. Recombinant gag was purified by a simple single step purification procedure. After partial purification of inclusion bodies and subject to the HA-cleavage treatment, gag protein was further purified to homogeneity using DEAE-Sepharose chromatography. The purified core antigen offered reliable results with high sensitivity and specificity for identification of HIV-1 antibodies when tested in the enzyme-linked immunosorbent assay (ELISA). These results suggest that mass production of recombinant core antigen will provide a valuable resource to HIV-1 serodiagnostics for the screening of large groups of blood donors to prevent HIV-1 infection.

Enhancement of EIAV replication and disease by immunization with a baculovirus-expressed recombinant envelope surface glycoprotein.

The potential for antibody-dependent enhancement of replication of macrophage/monocyte tropic viruses has posed a significant problem in the development of vaccines for several animal and human viruses and has raised significant concern in the design of potential AIDS vaccines. Using the previously described equine infectious anemia virus/Shetland pony system as a model for HIV-1 vaccine development, we have evaluated the efficacy of a recombinant subunit vaccine containing a baculovirus-expressed envelope surface glycoprotein (gp90) of EIAV. The results of these trials demonstrate not only that the recombinant vaccine failed to protect against infection by standard homologous and heterologous EIAV challenge strains, but that it resulted in a marked enhancement of virus replication and exacerbation of disease in immunized ponies exposed to the heterologous virus strain. Thus, the recombinant EIAV gp90 vaccine provides a novel in vivo model for examining in detail the mechanisms of immune enhancement of a lentivirus infection and for evaluating strategies to avoid the production of deleterious immune responses in AIDS vaccine design.

Bacterial expression, purification of full length and carboxyl terminal fragment of Alzheimer amyloid precursor protein and their proteolytic processing by thrombin.

Human amyloid protein precursor(APP770) and its carboxyl terminal portion (CT105) including beta/A4 domain were highly expressed using strong expression systems in E. coli. These recombinant APP peptides were purified with a combination of urea solubilization and ion-exchange chromatography and used for proteolytic processing by thrombin. Three thrombin cleavage sites were predicted by the decrease of APP770 and the appearance of M(r) 56, 27 and 18 kDa fragments containing beta/A4 domain on SDS-PAGE gel and on the immunoblot. A similar but limited proteolysis of platelet APPs exposed to thrombin resulted in the stimulated production of 60 and 27 KDa carboxyl terminal peptides containing the intact beta/A4. This thrombin mediated proteolysis was completely blocked by hirudin, the specific thrombin inhibitor. These results suggest that thrombin may play a role in altered processing of APP to generate potentially amyloidogenic intermediates in vivo leading to amyloid deposition.