A comparative analysis of leucine-rich repeat kinase 2 (Lrrk2) expression in mouse brain and Lewy body disease.

Pathogenic substitutions in leucine-rich repeat kinase 2 (LRRK2, Lrrk2) have been genetically linked to familial, late-onset Parkinsonism. End-stage disease is predominantly associated with nigral neuronal loss and Lewy body pathology, but patients may have gliosis, tau or ubiquitin inclusions (pleomorphic pathology). The anatomical distribution of Lrrk2 protein may provide insight into its function in health and neurodegeneration, thus we performed a comparative study with 'in-house' and commercially available Lrrk2 antibodies using brain tissue from wild type and human Lrrk2 transgenic bacterial artificial chromosome (BAC) mice, and from diffuse Lewy body disease (DLBD) patients. Lrrk2 protein was ubiquitously expressed and relatively abundant in most brain regions, including the substantia nigra, thalamus and striatum. Lrrk2 was not a major component of Lewy body or neuritic pathology associated with Parkinson's disease. However, selective loss of dopaminergic neurons in Lrrk2-associated Parkinsonism argues the protein may have regional-specific interactions. Lrrk2 immunohistochemical staining was present in the subventricular zone, a region containing stem cells that give rise to both neurons and glia. A role for Lrrk2 in neurogenesis might provide further insight into the aberrant role of mutant protein in age-associated neurodegeneration with pleomorphic pathology.

alpha-Synuclein fibrils constitute the central core of oligodendroglial inclusion filaments in multiple system atrophy.

Multiple system atrophy (MSA) belongs to synucleinopathies and is characterized pathologically by oligodendroglial inclusions (GCIs) composed of 20- to 30-nm tubular filaments. alpha-Synuclein fibrils formed in vitro, however, range between 10 and 12 nm in diameter. To understand the relationship between alpha-synuclein and GCI filaments, we conducted structural analyses of GCIs in fixed brain sections and isolated from fresh-frozen MSA brains. In fixed brain sections, GCIs were composed of amorphous material-coated filaments up to 30 nm in size. The filaments were often organized in parallel bundles extending into oligodendroglial processes. In freshly isolated GCIs, progressive buffer washes removed amorphous material and revealed that GCI filaments consisted of 10-nm-sized central core fibrils that were strongly alpha-synuclein immunoreactive. Image analysis revealed that each core fibril was made of two subfibrils, and each subfibril was made of a string of 3- to 6-nm-sized particles probably alpha-synuclein oligomers. Immunogold labeling demonstrated that epitopes encompassing entire alpha-synuclein molecule were represented in the core fibrils, with the N-terminal 11-26 and C-terminal 108-131 amino acid residues most accessible to antibodies, probably exposed on the surface of the fibril. Our study indicates that GCI filaments are multilayered in structure, with alpha-synuclein oligomers forming the central core fibrils of the filaments.

Aspartyl protease inhibitor pepstatin binds to the presenilins of Alzheimer's disease.

Mutations in the presenilin genes PS1 and PS2 cause early-onset Alzheimer's disease by altering gamma-secretase cleavage of the amyloid precursor protein, the last step in the generation of Abeta peptide. Ablation of presenilin (PS) genes, or mutation of two critical aspartates, abolishes gamma-secretase cleavage, suggesting that PS may be the gamma-secretases. Independently, inhibition experiments indicate that gamma-secretase is an aspartyl protease. To characterize the putative gamma-secretase activity associated with presenilins, lysates from human neuroblastoma SH-SY5Y and human brain homogenates were incubated with biotin derivatives of pepstatin, followed by immunoprecipitation of PS and associated proteins, and biotin detection by Western blotting. Precipitation with PS1 antibodies, directed to either N-terminal or loop regions, yielded the same 43 kDa band, of apparent molecular mass consistent with that of full-length PS1, although it may represent an aspartyl protease complexed with PS1. Incubation of cell lysates with pepstatin-biotin, followed by streptavidin precipitation and PS1 Western blotting, revealed PS1 fragments and full-length protein, indicating that pepstatin-biotin bound to both cleaved and uncleaved PS1. Binding could be competed by gamma-secretase inhibitor L-685,458 and could not be achieved with a PS1 mutant lacking the two transmembrane aspartates. Pepstatin-biotin was also shown to bind to PS2. PS1 was specifically absorbed to pepstatin-agarose, with an optimal pH of 6. Binding of pepstatin-biotin to PS1 from lymphocytes of a heterozygous carrier of pathologic exon 9 deletion was markedly decreased as compared to control lymphocytes, suggesting that this PS1 mutation altered the pepstatin binding site.

Duck hepatitis B virus replication in primary bile duct epithelial cells.

Primary cultures of intrahepatic bile duct epithelial (IBDE) cells isolated from duckling livers were successfully grown for studies of duck hepatitis B virus (DHBV). The primary IBDE cells were characterized by immunohistochemistry using CAM 5.2, a cytokeratin marker which was shown to react specifically to IBDE cells in duck liver tissue sections and in primary cultures of total duck liver cells. Immunofluorescence assay using anti-duck albumin, a marker for hepatocytes, revealed that these IBDE cultures did not appear to contain hepatocytes. A striking feature of these cultures was the duct-like structures present within each cell colony of multilayered IBDE cells. Normal duck serum in the growth medium was found to be essential for the development of these cells into duct-like structures. When the primary cultures of duck IBDE cells were acutely infected with DHBV, dual-labeled confocal microscopy using a combination of anti-DHBV core proteins and CAM 5.2 or a combination of anti-pre-S1 proteins and CAM 5.2 revealed that the IBDE cell colonies contained DHBV proteins. Immunoblot analysis of these cells showed that the DHBV pre-S1 and core proteins were similar to their counterparts in infected primary duck hepatocyte cultures. Southern blot analysis of infected IBDE preparations using a digoxigenin-labeled positive-sense DHBV riboprobe revealed the presence of hepadnavirus covalently closed circular (CCC) DNA, minus-sense single-stranded (SS) DNA, double-stranded linear DNA, and relaxed circular DNA. The presence of minus-sense SS DNA in the acutely infected IBDE cultures is indicative of DHBV reverse transcriptase activity, while the establishment of a pool of viral CCC DNA reveals the ability of these cells to maintain persistent infection. Taken collectively, the results from this study demonstrated that primary duck IBDE cells supported hepadnavirus replication as shown by the de novo synthesis of DHBV proteins and DNA replicative intermediates.

The solubility of alpha-synuclein in multiple system atrophy differs from that of dementia with Lewy bodies and Parkinson's disease.

Intracellular inclusions containing alpha-synuclein (alpha SN) are pathognomonic features of several neurodegenerative disorders. Inclusions occur in oligodendrocytes in multiple system atrophy (MSA) and in neurons in dementia with Lewy bodies (DLB) and Parkinson's disease (PD). In order to identify disease-associated changes of alpha SN, this study compared the levels, solubility and molecular weight species of alpha SN in brain homogenates from MSA, DLB, PD and normal aged controls. In DLB and PD, substantial amounts of detergent-soluble and detergent-insoluble alpha SN were detected compared with controls in grey matter homogenate. Compared with controls, MSA cases had significantly higher levels of alpha SN in the detergent-soluble fraction of brain samples from pons and white matter but detergent-insoluble alpha SN was not detected. There was an inverse correlation between buffered saline-soluble and detergent-soluble levels of alpha SN in individual MSA cases suggesting a transition towards insolubility in disease. The differences in solubility of alpha SN between grey and white matter in disease may result from different processing of alpha SN in neurons compared with oligodendrocytes. Highly insoluble alpha SN is not involved in the pathogenesis of MSA. It is therefore possible that buffered saline-soluble or detergent-soluble forms of alpha SN are involved in the pathogenesis of other alpha SN-related diseases.

Accumulation of insoluble alpha-synuclein in dementia with Lewy bodies.

The alpha-synuclein (alpha SN) protein is thought to play a central role in the pathogenesis of neurodegenerative diseases where it aggregates to form intracellular inclusions. We have used Western blotting to examine the expression levels and solubility of alpha SN in brain homogenates from dementia with Lewy bodies (DLB), Parkinson's disease (PD), Alzheimer's disease (AD), and normal controls using samples from the parahippocampus/transentorhinal cortex. Compared to controls, DLB brains accumulate significantly greater amounts of sodium dodecyl sulfate (SDS)-soluble and SDS-insoluble alpha SN but levels of TBS-soluble alpha SN did not change. Levels of synaptophysin, a marker of synaptic integrity, were significantly lower in DLB cases than in normal aged controls regardless of whether concurrent changes of AD were present. This limbic synaptic dysfunction may contribute to cognitive impairment in DLB. Whether aggregated alpha SN is a cause or effect of the disease process in DLB and PD remains to be determined, but the presence of aggregated alpha SN is consistent with a pathogenesis similar to that associated with aggregates of Abeta amyloid in AD.

Targeted mutagenesis of Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3) disrupts cytoadherence of malaria-infected red blood cells.

Adhesion of parasite-infected red blood cells to the vascular endothelium is a critical event in the pathogenesis of malaria caused by Plasmodium falciparum. Adherence is mediated by the variant erythrocyte membrane protein 1 (PfEMP1). Another protein, erythrocyte membrane protein-3 (PfEMP3), is deposited under the membrane of the parasite-infected erythrocyte but its function is unknown. Here we show that mutation of PfEMP3 disrupts transfer of PfEMP1 to the outside of the P.FALCIPARUM:-infected cell. Truncation of the C-terminal end of PfEMP3 by transfection prevents distribution of this large (>300 kDa) protein around the membrane but does not disrupt trafficking of the protein from the parasite to the cytoplasmic face of the erythrocyte membrane. The truncated PfEMP3 accumulates in structures that appear to be associated with the erythrocyte membrane. We show that accumulation of mutated PfEMP3 blocks the transfer of PfEMP1 onto the outside of the parasitized cell surface and suggest that these proteins traffic through an erythrocyte membrane-associated compartment that is involved in the transfer of PfEMP1 to the surface of the parasite-infected red blood cell.

Presenilin 2 expression in neuronal cells: induction during differentiation of embryonic carcinoma cells.

Mutations in the presenilin 1 and 2 (PS1 and PS2) genes cause most cases of early onset Alzheimer's disease. The genes encode two homologous multipass membrane proteins. Since the endogenous expression of PS2 has been poorly analyzed to date, we studied PS2 expression and localization in cultured human neuroblastoma cells and mouse neuronal cells. PS2 was mainly detected as a full-length protein of about 52 kDa in these cells and in brain, in contrast to PS1 that is mainly detected as endoproteolytic N-terminal and C-terminal fragments. Using immunofluorescence we found that like PS1, PS2 colocalized with markers of the endoplasmic reticulum-Golgi intermediate compartment, ERGIC-53 and beta-COP. Double labeling for PS1 and PS2 indicated that both proteins are colocalized in neuroblastoma SH-SY5Y cells. To study PS2 expression during differentiation, mouse embryonic carcinoma P19 cells were treated with retinoic acid. We found minimal PS2 expression in undifferentiated cells, an increase from day 2, and a maximum at day 8 after treatment. PS1 expression remained constant during this period. The differential expression of PS1 and PS2 within the P19 cells following retinoic acid treatment indicates different utilization or temporal requirements for these proteins during neuronal differentiation.

Presenilin I expression in yeast lowers secretion of the amyloid precursor protein.

Presenilin (PS) mutations are associated with early-onset Alzheimer's disease and PS proteins are involved with gamma-secretase cleavage of the amyloid precursor protein, APP. We have shown previously that alpha-, beta- and gamma-secretase cleavages of APP are conserved in Pichia pastoris. Here, we report co-expression of APP and PSI in P. pastoris and show by immunoelectron microscopy colocalization of these two proteins in expanded endoplasmic reticulum. Western blot analysis indicates a drastic reduction of both alpha- and beta-secretase products. A relative increase in beta-secretase product derived from immature APP is also observed, pointing to a beta-secretase activity of P. pastoris associated with the early secretory pathway.

Non-Abeta component of Alzheimer's disease amyloid (NAC) revisited. NAC and alpha-synuclein are not associated with Abeta amyloid.

alpha-Synuclein (alphaSN), also termed the precursor of the non-Abeta component of Alzheimer's disease (AD) amyloid (NACP), is a major component of Lewy bodies and Lewy neurites pathognomonic of Parkinson's disease (PD) and dementia with Lewy bodies (DLB). A fragment of alphaSN termed the non-Abeta component of AD amyloid (NAC) had previously been identified as a constituent of AD amyloid plaques. To clarify the relationship of NAC and alphaSN with Abeta plaques, antibodies were raised to three domains of alphaSN. All antibodies produced punctate labeling of human cortex and strong labeling of Lewy bodies. Using antibodies to alphaSN(75-91) to label cortical and hippocampal sections of pathologically proven AD cases, we found no evidence for NAC in Abeta amyloid plaques. Double labeling of tissue sections in mixed DLB/AD cases revealed alphaSN in dystrophic neuritic processes, some of which were in close association with Abeta plaques restricted to the CA1 hippocampal region. In brain homogenates alphaSN was predominantly recovered in the cytosolic fraction as a 16-kd protein on Western analysis; however, significant amounts of aggregated and alphaSN fragments were also found in urea extracts of SDS-insoluble material from DLB and PD cases. NAC antibodies identified an endogenous fragment of 6 kd in the cytosolic and urea-soluble brain fractions. This fragment may be produced as a consequence of alphaSN aggregation or alternatively may accelerate aggregation of the full-length alphaSN.

Alpha-synuclein immunoisolation of glial inclusions from multiple system atrophy brain tissue reveals multiprotein components.

Immunohistochemical studies have shown that oligodendroglial inclusions in multiple system atrophy contain alpha-synuclein, a synaptic protein also found in Lewy bodies in Parkinson's disease. We have now used density gradient enrichment and an anti-alpha-synuclein immunomagnetic technique to isolate pure and morphologically intact oligodendroglial inclusions from brain white matter of patients dying with multiple system atrophy. Filamentous inclusion structures were obtained only from multiple system atrophy tissue, but not from normal brain tissues, or from multiple system atrophy tissue processed without anti-alpha-synuclein antibody. We confirmed the purity and morphology of isolated inclusions by electron microscopy. The inclusions comprised multiple protein bands after separation by polyacrylamide gel electrophoresis. Immunoblotting demonstrated that these proteins included alpha-synuclein, alphaB-crystallin, tubulins, ubiquitin, and prominent, possibly truncated alpha-synuclein species as high-molecular-weight aggregates. Our study provides the first biochemical evidence that oligodendroglial inclusion filaments consist of multiple protein components, suggesting that these inclusions may form as a result of multiprotein interactions with alpha-synuclein.

Alpha-synuclein accumulates in Lewy bodies in Parkinson's disease and dementia with Lewy bodies but not in Alzheimer's disease beta-amyloid plaque cores.

A growing body of evidence suggests that the non-Abeta component of Alzheimer's disease amyloid precursor protein (NACP) or alpha-synuclein contributes to the neurodegenerative processes in Alzheimer's disease (AD), Parkinson's disease (PD) and dementia with Lewy bodies (DLB). In the present study antisera to the N terminus and the NAC domain of the alpha-synuclein protein were employed to elucidate the expression pattern in brains of patients with AD, PD, DLB and control specimen. Alpha-synuclein exhibited an overall punctuate expression profile compatible with a synaptic function. Interestingly, while Lewy bodies were strongly immunoreactive, none of the alpha-synuclein antisera revealed staining in mature beta-amyloid plaques in AD. These observations suggest that alpha-synuclein does not contribute to late neurodegenerative processes in AD brains.

Intracellular accumulation of detergent-soluble amyloidogenic A beta fragment of Alzheimer's disease precursor protein in the hippocampus of aged transgenic mice.

To study amyloid beta-protein (A beta) production and aggregation in vivo, we created two transgenic (Tg) mouse lines expressing the C-terminal 100 amino acids of human amyloid precursor protein (APP): Tg C100.V717F and Tg C100.WT. Western blot analysis showed that human APP-C100 and A beta were produced in brain and some peripheral tissues and A beta was produced in serum. Using antibodies specific for the A beta C terminus we found that Tg C100.V717F produced a 1.6-fold increase in A beta42/A beta40 compared with Tg C100.WT. Approximately 30% of total brain A beta (approximately 122 ng/g of wet tissue) was water-soluble. The remaining 70% of A beta partitioned into the particulate fraction and was completely sodium dodecyl sulfate-soluble. In contrast, human Alzheimer's disease brain has predominantly sodium dodecyl sulfate-insoluble A beta. Immunohistochemistry with an A beta(5-8) antibody showed that A beta or A beta-containing fragments accumulated intracellularly in the hippocampus of aged Tg C100.V717F mice. The soluble A beta levels in Tg brain are similar to those in normal human brain, and this may explain the lack of microscopic amyloid deposits in the Tg mice. However, this mouse model provides a system to study the intracellular processing and accumulation of A beta or A beta-containing fragments and to screen for compounds directed at the gamma-secretase activity.

Subcellular localization of the Alzheimer's disease amyloid precursor protein and derived polypeptides expressed in a recombinant yeast system.

Different isoforms and derived polypeptides of the Alzheimer's disease amyloid protein precursor (A beta PP) have been expressed in the yeast Pichia pastoris. The expression characteristics of the different A beta PP polypeptides were studied by post-embedding immunogold electron microscopy with various A beta PP antibodies. The site of intracellular expression could be readily identified with specific antibodies. Full length A beta PP was expressed in association with the nuclear membrane and the endoplasmic reticulum. Secretory derivatives of A beta PP were localized in membrane-bound secretory vesicles. A construct encoding two copies of beta A4[1-42] linked head-to-tail (beta A4duplex) accumulated as irregular dense cytoplasmic and intranuclear inclusions which reacted with all beta A4 antibodies tested. A beta A4-C-terminal construct accumulated into membranous structures in the cytoplasm and nucleus and reacted with most antibodies to beta A4 and the cytoplasmic domain of A beta PP. The two shorter constructs containing the beta A4 sequence formed similar intranuclear aggregates to those reported for intranuclear inclusions of polyglutamine peptides from huntingtin (in Huntington's disease) and ataxin protein fragments (in spinocerebellar ataxia). This is of interest because intracellular aggregation of the polyglutamine and beta A4 peptides may affect cells by similar toxic mechanisms. These studies demonstrate clear differences in the expression properties of different A beta PP polypeptides.

CD36-dependent adhesion and knob expression of the transmission stages of Plasmodium falciparum is stage specific.

Plasmodium falciparum trophozoites sequester from the peripheral circulation by adherence to host endothelium. Gametocytes, also sequester during maturation. Analysis of the adhesion phenotype of stage I to V gametocytes of several isolates/clones was assessed by binding of infected cells to C32 melanoma cells (C32MC) and the purified adhesion proteins, leucocyte differentiation antigen (CD36) and intercellular adhesion molecule-1 (ICAM-1). These cells and proteins, have previously been shown to be receptors for adherence of trophozoites. Early gametocytes (stages I-IIA) were found to bind to C32MC as well as the purified receptor CD36 but not to ICAM-1. Early gametocytes bound to C32MC via CD36 and the parasite ligand involved in this binding was trypsin sensitive. Stage IIB to V gametocytes did not adhere to C32MC, CD36 nor ICAM-1. Electron-dense protruberances known as knobs and histidine rich protein 1 (HRP 1) expression have been associated with trophozite adhesion to CD36. Knobs were present at the surface of early but not late gametocyte infected cells. Stage-specific patterns of HRP 1 expression, consistent with a role for this molecule in CD36 adhesion of early gametocytes, were also observed. The adhesion phenotype of these young gametocytes was indistinguishable from that of the trophozoites by all criteria examined. These data support the hypothesis that other host receptors mediate the binding of late gametocytes.

Alzheimer's disease-associated presenilin 1 in neuronal cells: evidence for localization to the endoplasmic reticulum-Golgi intermediate compartment.

The recently identified Alzheimer's disease-associated presenilin 1 and 2 (PS1 and PS2) genes encode two homologous multi membrane-spanning proteins. Rabbit antibodies to the N-terminal domain of PS1 detected PS1 in human neuroblastoma SH-SY5Y wild type and PS1 transfectants (SY5Y-PS1) as well as in mouse P19, in CHO-K1 and CHO-APP770 transfected cells, in rat cerebellar granule and hippocampal neurons, and astrocytes. Immunoblotting detected full-length protein of 50 kDa, and a major presumptive cleavage product of 30 kDa. The immunofluorescence pattern resembled labeling of the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) marker protein ERGIC-53. PS1 distribution showed slight condensation after brefeldin A and more marked condensation after incubation of cells at 16 degrees C, characteristic of the ERGIC compartment. Double labeling showed colocalization of ERGIC-53 with PS1 in the SY5Y-PS1 cells. PS1 labeling of SY5Y-PS1 and P19 cells showed overlap of the cis-Golgi marker p210 and colocalization with p210 after brefeldin A which causes redistribution of p210 to the ERGIC. Expression of PS1 did not change in level or cellular distribution during development of neurons in culture. Double labeling for the amyloid precursor protein (APP) and PS1 on SY5Y-PS1 cells and CHO-APP770 cells showed some overlap under control conditions. These results indicate that PS1 is a resident protein of the ERGIC and could be involved in trafficking of proteins, including APP, between the ER and Golgi compartments.

Ligand-regulated transport of the Menkes copper P-type ATPase efflux pump from the Golgi apparatus to the plasma membrane: a novel mechanism of regulated trafficking.

The Menkes P-type ATPase (MNK), encoded by the Menkes gene (MNK; ATP7A), is a transmembrane copper-translocating pump which is defective in the human disorder of copper metabolism, Menkes disease. Recent evidence that the MNK P-type ATPase has a role in copper efflux has come from studies using copper-resistant variants of cultured Chinese hamster ovary (CHO) cells. These variants have MNK gene amplification and consequently overexpress MNK, the extents of which correlate with the degree of elevated copper efflux. Here, we report on the localization of MNK in these copper-resistant CHO cells when cultured in different levels of copper. Immunofluorescence studies demonstrated that MNK is predominantly localized to the Golgi apparatus of cells in basal medium. In elevated copper conditions there was a rapid trafficking of MNK from the Golgi to the plasma membrane. This shift in steady-state distribution of MNK was reversible and not dependent on new protein synthesis. In media containing basal copper, MNK accumulated in cytoplasmic vesicles after treatment of cells with a variety of agents that inhibit endosomal recycling. We suggest that MNK continuously recycles between the Golgi and the plasma membrane and elevated copper shifts the steady-state distribution from the Golgi to the plasma membrane. These data reveal a novel system of regulated protein trafficking which ultimately leads to the efflux of an essential yet potentially toxic ligand, where the ligand itself appears directly and specifically to stimulate the trafficking of its own transporter.

Expression of the amyloid precursor protein of Alzheimer's disease on the surface of transfected HeLa cells.

The principal component of the amyloid which accumulates in Alzheimer's Disease brain is a 4-kDa beta A4 fragment of the amyloid precursor protein (APP). Although APP has the structural features of an integral transmembrane receptor, there has been limited evidence for expression of APP at the plasma membrane. The function of APP and related molecules is unknown. Using rabbit antisera to purified human brain APP, surface labeling of APP is demonstrable in HeLa cells transfected with the APP695 isoform. Indirect immunofluorescence indicates the presence of APP at the surface of unfixed or aldehyde-fixed cells; preembedding immunoelectron microscopy using 5- or 1-nm gold particles and silver enhancement confirms plasma membrane labeling as well as labeling within intracellular membrane vesicles. Immunolabeling of unfixed cells at 4 degrees C followed by incubation at 37 degrees C shows APP within endocytic vesicles. Transfected HeLa cells with prominent surface APP were larger with more extensive microvilli than nonimmunoreactive HeLa cells. This is consistent with the postulated role of APP as a mediator of cell surface adhesion and membrane-matrix stabilization.

Candidate gamma-secretases in the generation of the carboxyl terminus of the Alzheimer's disease beta A4 amyloid: possible involvement of cathepsin D.

beta A4 amyloid peptide, the main constituent of amyloid plaques and cerebrovascular amyloid deposits associated with Alzheimer's disease, derives from a large precursor protein (APP) by the action of beta- and gamma-secretases, the unidentified endoproteases which release the amino and carboxyl termini of beta A4, respectively. Several gamma-secretase cleavage sites exist which yield the more soluble (1-39/40) forms of beta A4 and the longer forms (1-42/43) which have a greater tendency to aggregate into amyloid plaques. gamma-Secretase activity may therefore be critical in amyloid formation. In this study, a synthetic peptide which encompasses the various gamma-secretase cleavage sites was used as a substrate to probe proteases of various classes and specificities. Elastase, collagenase, and cathepsin D cleaved at the amyloidogenic sites (after Ala42 or after Thr43) to release the carboxyl termini of the aggregating forms. In addition, collagenase and pepsin released the carboxyl terminus of the more soluble forms. Human brain fractions enriched in lysosomes contained a proteolytic activity that cleaved the substrate at the amyloidogenic site(s). This activity was more active at acidic pH and was inhibited by pepstatin, two characteristics of the lysosomal aspartyl proteinase cathepsin D. The same lysosomal fractions were found to contain APP carboxyl-terminal fragments which are potentially amyloidogenic. These were degraded, only in acidic conditions, by an endogenous protease activity inhibited by pepstatin. Thus, a cathepsin D-like activity from human brain is a candidate for APP gamma-secretase(s).