Peptide backbone modifications of amyloid ß (1-40) impact fibrillation behavior and neuronal toxicity.

Fibril formation of amyloid ß (Aß) peptides is one of the key molecular events connected to Alzheimer's disease. The pathway of formation and mechanism of action of Aß aggregates in biological systems is still object of very active research. To this end, systematic modifications of the Phe19-Leu34 hydrophobic contact, which has been reported in almost all structural studies of Aß40 fibrils, helps understanding Aß folding pathways and the underlying free energy landscape of the amyloid formation process. In our approach, a series of Aß40 peptide variants with two types of backbone modifications, namely incorporation of (i) a methylene or an ethylene spacer group and (ii) a N-methylation at the amide functional group, of the amino acids at positions 19 or 34 was applied. These mutations are expected to challenge the inter-ß-strand side chain contacts as well as intermolecular backbone ß-sheet hydrogen bridges. Using a multitude of biophysical methods, it is shown that these backbone modifications lead, in most of the cases, to alterations in the fibril formation kinetics, a higher local structural heterogeneity, and a somewhat modified fibril morphology without generally impairing the fibril formation capacity of the peptides. The toxicological profile found for the variants depend on the type and extent of the modification.

Construction of a 3D brain extracellular matrix model to study the interaction between microglia and T cells in co-culture.

Neurodegenerative disorders are characterised by the activation of brain-resident microglia cells and by the infiltration of peripheral T cells. However, their interplay in disease has not been clarified yet. It is difficult to investigate complex cellular dynamics in living animals, and simple two-dimensional (2D) cell culture models do not resemble the soft 3D structure of brain tissue. Therefore, we developed a biomimetic 3D in vitro culture system for co-cultivation of microglia and T cells. As the activation and/or migration of immune cells in the brain might be affected by components of the extracellular matrix, defined 3D fibrillar collagen I-based matrices were constructed and modified with hyaluronan and/or chondroitin sulphate, resembling aspects of brain extracellular matrix. Murine microglia and spleen-derived T cells were cultured alone or in co-culture on the constructed matrices. Microglia exhibited in vivo-like morphology and T cells showed enhanced survival when co-cultured with microglia or to a minor degree in the presence of glia-conditioned medium. The open and porous fibrillar structure of the matrix allowed for cell invasion and direct cell-cell interaction, with stronger invasion of T cells. Both cell types showed no dependence on the matrix modifications. Microglia could be activated on the matrices by lipopolysaccharide resulting in interleukin-6 and tumour necrosis factor-α secretion. The findings herein indicate that biomimetic 3D matrices allow for co-cultivation and activation of primary microglia and T cells and provide useful tools to study their interaction in vitro.

Incorporation of the Nonproteinogenic Amino Acid ß-Methylamino-alanine Affects Amyloid ß Fibril Properties and Toxicity.

The nonproteinogenic amino acid ß-methylamino alarelevant example for environmental hazards are nonnine (BMAA) is a neurotoxin and represents a potential risk factor for neurodegenerative diseases. Despite intense research over the last years, the pathological mechanism of BMAA is still unclear. One of the main open questions is whether BMAA can be misincorporated into proteins, especially as a substitute for serine, and whether this has structural and functional consequences for the afflicted proteins leading to early onset neurodegeneration. In this study, we hypothesize that BMAA was indeed incorporated into Aß40 molecules and study the structural and dynamical consequences of such misincorporation along with the effect such mutated Aß40 peptides have on neuronal cells. We used the synthetic ß-amyloid peptide (Aß40), a known key player in the development of Alzheimer's disease, to incorporate BMAA substitutions at three different positions in the peptide sequence: Ser8BMAA at the peptide's N-terminus, Phe19BMAA in the hydrophobic core region, and S26BMAA in the flexible turn region of Aß40 fibrils. We performed a set of biophysical experiments including fluorescence, circular dichroism, solid-state NMR spectroscopy, transmission electron microscopy, and X-ray diffraction to investigate structural and functional aspects of the mutated peptides compared to wildtype Aß40. All variants showed high structural tolerance to BMAA misincorporation. In contrast, the cellular response and neuronal survival were affected in a mutation site-specific manner. As a consequence, we can state from the physicochemical point of view that, if BMAA was misincorporated into proteins, it could indeed represent a risk factor that could potentially play a role in neurodegeneration. Further research addressing the role of BMAA, especially its protein-associated form, should be performed to obtain a better understanding of neurodegenerative diseases and to develop new therapeutic strategies.

Endogenous mouse huntingtin is highly abundant in cranial nerve nuclei, co-aggregates to Abeta plaques and is induced in reactive astrocytes in a transgenic mouse model of Alzheimer's disease.

Pathogenic variants of the huntingtin (HTT) protein and their aggregation have been investigated in great detail in brains of Huntington's disease patients and HTT-transgenic animals. However, little is known about the physiological brain region- and cell type-specific HTT expression pattern in wild type mice and a potential recruitment of endogenous HTT to other pathogenic protein aggregates such as amyloid plaques in cross seeding events. Employing a monoclonal anti-HTT antibody directed against the HTT mid-region and using brain tissue of three different mouse strains, we detected prominent immunoreactivity in a number of brain areas, particularly in cholinergic cranial nerve nuclei, while ubiquitous neuronal staining appeared faint. The region-specific distribution of endogenous HTT was found to be comparable in wild type rat and hamster brain. In human amyloid precursor protein transgenic Tg2576 mice with amyloid plaque pathology, similar neuronal HTT expression patterns and a distinct association of HTT with Abeta plaques were revealed by immunohistochemical double labelling. Additionally, the localization of HTT in reactive astrocytes was demonstrated for the first time in a transgenic Alzheimer's disease animal model. Both, plaque association of HTT and occurrence in astrocytes appeared to be age-dependent. Astrocytic HTT gene and protein expression was confirmed in primary cultures by RT-qPCR and by immunocytochemistry. We provide the first detailed analysis of physiological HTT expression in rodent brain and, under pathological conditions, demonstrate HTT aggregation in proximity to Abeta plaques and Abeta-induced astrocytic expression of endogenous HTT in Tg2576 mice.

Defined astrocytic expression of human amyloid precursor protein in Tg2576 mouse brain.

Transgenic Tg2576 mice expressing human amyloid precursor protein (hAPP) with the Swedish mutation are among the most frequently used animal models to study the amyloid pathology related to Alzheimer's disease (AD). The transgene expression in this model is considered to be neuron-specific. Using a novel hAPP-specific antibody in combination with cell type-specific markers for double immunofluorescent labelings and laser scanning microscopy, we here report that-in addition to neurons throughout the brain-astrocytes in the corpus callosum and to a lesser extent in neocortex express hAPP. This astrocytic hAPP expression is already detectable in young Tg2576 mice before the onset of amyloid pathology and still present in aged Tg2576 mice with robust amyloid pathology in neocortex, hippocampus, and corpus callosum. Surprisingly, hAPP immunoreactivity in cortex is restricted to resting astrocytes distant from amyloid plaques but absent from reactive astrocytes in close proximity to amyloid plaques. In contrast, neither microglial cells nor oligodendrocytes of young or aged Tg2576 mice display hAPP labeling. The astrocytic expression of hAPP is substantiated by the analyses of hAPP mRNA and protein expression in primary cultures derived from Tg2576 offspring. We conclude that astrocytes, in particular in corpus callosum, may contribute to amyloid pathology in Tg2576 mice and thus mimic this aspect of AD pathology.

Dipeptidyl-Peptidase Activity of Meprin ß Links N-truncation of Aß with Glutaminyl Cyclase-Catalyzed pGlu-Aß Formation.

The formation of amyloid-ß (Aß) peptides is causally involved in the development of Alzheimer's disease (AD). A significant proportion of deposited Aß is N-terminally truncated and modified at the N-terminus by a pGlu-residue (pGlu-Aß). These forms show enhanced neurotoxicity compared to full-length Aß. Although the truncation may occur by aminopeptidases after formation of Aß, recently discovered processing pathways of amyloid-ß protein precursor (AßPP) by proteases such as meprin ß may also be involved. Here, we assessed a role of meprin ß in forming Aß3-40/42, which is the precursor of pGlu-Aß3-40/42 generated by glutaminyl cyclase (QC). Similar to QC, meprin ß mRNA is significantly upregulated in postmortem brain from AD patients. A histochemical analysis supports the presence of meprin ß in neurons and astrocytes in the vicinity of pGlu-Aß containing deposits. Cleavage of AßPP-derived peptides by meprin ß in vitro results in peptides Aß1-x, Aß2-x, and Aß3-x. The formation of N-truncated Aß by meprin ß was also corroborated in cell culture. A subset of the generated peptides was converted into pGlu-Aß3-40 by an addition of glutaminyl cyclase, supporting the preceding formation of Aß3-40. Further analysis of the meprin ß cleavage revealed a yet unknown dipeptidyl-peptidase-like activity specific for the N-terminus of Aß1-x. Thus, our data suggest that meprin ß contributes to the formation of N-truncated Aß by endopeptidase and exopeptidase activity to generate the substrate for QC-catalyzed pGlu-Aß formation.

Pyroglutamate-Modified Amyloid ß (11- 40) Fibrils Are More Toxic than Wildtype Fibrils but Structurally Very Similar.

The morphology, structure, and dynamics of mature amyloid ß (Aß) fibrils formed by the Aß variant, which is truncated at residue 11 and chemically modified by enzymatic pyroglutamate formation (pGlu11 -Aß(11-40)), was studied along with the investigation of the toxicity of these Aß variants to neurons and astrocytes. The fibrils of pGlu11 -Aß (11-40) were more toxic than wildtype Aß (1-40) and the longer pGlu3-Aß (3-40) especially at higher concentration, whereas the overall morphology was quite similar. The secondary structure of pGlu11 -Aß (11-40) fibrils shows the typical two ß-strands connected by a short turn as known for mature fibrils of Aß (1-40) and also pGlu3 -Aß (3-40). Further insights into tertiary contacts exhibit some similarities of pGlu11 -Aß (11-40) fibrils with wildtype Aß (1-40), but also a so far not described contact between Gly25 and Ile31 . This highlights the biological importance of chemical modifications on the molecular structure of Aß.

Differential transgene expression patterns in Alzheimer mouse models revealed by novel human amyloid precursor protein-specific antibodies.

Alzheimer's disease (AD) is histopathologically characterized by neurodegeneration, the formation of intracellular neurofibrillary tangles and extracellular Aß deposits that derive from proteolytic processing of the amyloid precursor protein (APP). As rodents do not normally develop Aß pathology, various transgenic animal models of AD were designed to overexpress human APP with mutations favouring its amyloidogenic processing. However, these mouse models display tremendous differences in the spatial and temporal appearance of Aß deposits, synaptic dysfunction, neurodegeneration and the manifestation of learning deficits which may be caused by age-related and brain region-specific differences in APP transgene levels. Consequentially, a comparative temporal and regional analysis of the pathological effects of Aß in mouse brains is difficult complicating the validation of therapeutic AD treatment strategies in different mouse models. To date, no antibodies are available that properly discriminate endogenous rodent and transgenic human APP in brains of APP-transgenic animals. Here, we developed and characterized rat monoclonal antibodies by immunohistochemistry and Western blot that detect human but not murine APP in brains of three APP-transgenic mouse and one APP-transgenic rat model. We observed remarkable differences in expression levels and brain region-specific expression of human APP among the investigated transgenic mouse lines. This may explain the differences between APP-transgenic models mentioned above. Furthermore, we provide compelling evidence that our new antibodies specifically detect endogenous human APP in immunocytochemistry, FACS and immunoprecipitation. Hence, we propose these antibodies as standard tool for monitoring expression of endogenous or transfected APP in human cells and APP expression in transgenic animals.

Proteolytic degradation of neuropeptide Y (NPY) from head to toe: Identification of novel NPY-cleaving peptidases and potential drug interactions in CNS and Periphery.

The bioactivity of neuropeptide Y (NPY) is either N-terminally modulated with respect to receptor selectivity by dipeptidyl peptidase 4 (DP4)-like enzymes or proteolytic degraded by neprilysin or meprins, thereby abrogating signal transduction. However, neither the subcellular nor the compartmental differentiation of these regulatory mechanisms is fully understood. Using mass spectrometry, selective inhibitors and histochemistry, studies across various cell types, body fluids, and tissues revealed that most frequently DP4-like enzymes, aminopeptidases P, secreted meprin-A (Mep-A), and cathepsin D (CTSD) rapidly hydrolyze NPY, depending on the cell type and tissue under study. Novel degradation of NPY by cathepsins B, D, L, G, S, and tissue kallikrein could also be identified. The expression of DP4, CTSD, and Mep-A at the median eminence indicates that the bioactivity of NPY is regulated by peptidases at the interphase between the periphery and the CNS. Detailed ex vivo studies on human sera and CSF samples recognized CTSD as the major NPY-cleaving enzyme in the CSF, whereas an additional C-terminal truncation by angiotensin-converting enzyme could be detected in serum. The latter finding hints to potential drug interaction between antidiabetic DP4 inhibitors and anti-hypertensive angiotensin-converting enzyme inhibitors, while it ablates suspected hypertensive side effects of only antidiabetic DP4-inhibitors application. The bioactivity of neuropeptide Y (NPY) is either N-terminally modulated with respect to receptor selectivity by dipeptidyl peptidase 4 (DP4)-like enzymes or proteolytic degraded by neprilysin or meprins, thereby abrogating signal transduction. However, neither the subcellular nor the compartmental differentiation of these regulatory mechanisms is fully understood. Using mass spectrometry, selective inhibitors and histochemistry, studies across various cell types, body fluids, and tissues revealed that most frequently DP4-like enzymes, aminopeptidases P, secreted meprin-A (Mep-A), and cathepsin D (CTSD) rapidly hydrolyze NPY, depending on the cell type and tissue under study. Novel degradation of NPY by cathepsins B, D, L, G, S, and tissue kallikrein could also be identified. The expression of DP4, CTSD, and Mep-A at the median eminence indicates that the bioactivity of NPY is regulated by peptidases at the interphase between the periphery and the CNS. Detailed ex vivo studies on human sera and CSF samples recognized CTSD as the major NPY-cleaving enzyme in the CSF, whereas an additional C-terminal truncation by angiotensin-converting enzyme could be detected in serum. The latter finding hints to potential drug interaction between antidiabetic DP4 inhibitors and anti-hypertensive angiotensin-converting enzyme inhibitors, while it ablates suspected hypertensive side effects of only antidiabetic DP4-inhibitors application.

Secretome protein enrichment identifies physiological BACE1 protease substrates in neurons.

Cell surface proteolysis is essential for communication between cells and results in the shedding of membrane-protein ectodomains. However, physiological substrates of the contributing proteases are largely unknown. We developed the secretome protein enrichment with click sugars (SPECS) method, which allows proteome-wide identification of shedding substrates and secreted proteins from primary cells, even in the presence of serum proteins. SPECS combines metabolic glycan labelling and click chemistry-mediated biotinylation and distinguishes between cellular and serum proteins. SPECS identified 34, mostly novel substrates of the Alzheimer protease BACE1 in primary neurons, making BACE1 a major sheddase in the nervous system. Selected BACE1 substrates-seizure-protein 6, L1, CHL1 and contactin-2-were validated in brains of BACE1 inhibitor-treated and BACE1 knock-out mice. For some substrates, BACE1 was the major sheddase, whereas for other substrates additional proteases contributed to total substrate shedding. The new substrates point to a central function of BACE1 in neurite outgrowth and synapse formation. SPECS is also suitable for quantitative secretome analyses of primary cells and may be used for the discovery of biomarkers secreted from tumour or stem cells.

Heteroarylketones inhibit astroglial interleukin-6 expression via a STAT3/NF-κB signaling pathway.

BACKGROUND: Elevated brain levels of the pleiotropic cytokine interleukin-6, which is mainly secreted from activated local astrocytes, contribute to pathological events including neuroinflammation and neurodegeneration. Thus, inhibition of pathological IL-6 expression provides a rationale strategy for targeting the onset or further progression of neurological disorders including Alzheimer's disease, multiple sclerosis, Parkinson's disease and traumatic brain injury. The purpose of this study was to identify and to characterize new potent inhibitors of astrocytic IL-6 expression for further therapeutic development of novel anti-inflammatory and neuroprotective drugs. METHODS: Oncostatin M (OSM)-treated human glioma U343 cells were used as model for induction of astrocytic IL-6 expression. This model was characterized by immunoblotting, siRNA technique, ELISA and qRT-PCR and used to screen low molecular weight compound libraries for IL-6-lowering effects. To validate bioactive compounds identified from library screens, bacterial lipopolysaccharide was used to induce IL-6 expression in cultivated primary astrocytes and in mice in vivo. To dissect underlying molecular mechanisms, protein extracts from OSM-treated U343 cells were analyzed by phospho-specific immunoblotting and immunocytochemistry as well as by co-immunoprecipitation. RESULTS: OSM-treatment (100 ng/ml; 24 h) led to 30-fold increase of IL-6 secretion from U343 cells. The temporal profile of IL-6 mRNA induction displayed a biphasic induction pattern with peak synthesis at 1 h (6.5-fold) and 16 h (5.5-fold) post stimulation. IL-6 protein release did not show that biphasic pattern and was detected as early as 3 h post stimulation reaching a maximum at 24 h. The screen of compound libraries identified a set of heteroarylketones (HAKs) as potent inhibitors of IL-6 secretion. HAK compounds affected the second peak in IL-6 mRNA synthesis, whereas the first peak was insensitive to HAK treatment. HAK compounds also suppressed lipopolysaccharide-induced IL-6 expression in primary murine astrocytes as well as in brain and plasma samples from lipopolysaccharide-treated mice. Finally, HAK compounds were demonstrated to specifically suppress the OSM-induced phosphorylation of STAT3 at serine 727 and the physical interaction of pSTAT3S727 with p65. CONCLUSION: Heteroarylketone compounds are potent inhibitors of IL-6 expression in vitro and in vivo and may represent a new class of potent anti-inflammatory and neuroprotective drugs.

Glutaminyl cyclase contributes to the formation of focal and diffuse pyroglutamate (pGlu)-Aß deposits in hippocampus via distinct cellular mechanisms.

In the hippocampal formation of Alzheimer's disease (AD) patients, both focal and diffuse deposits of Aß peptides appear in a subregion- and layer-specific manner. Recently, pyroglutamate (pGlu or pE)-modified Aß peptides were identified as a highly pathogenic and seeding Aß peptide species. Since the pE modification is catalyzed by glutaminyl cyclase (QC) this enzyme emerged as a novel pharmacological target for AD therapy. Here, we reveal the role of QC in the formation of different types of hippocampal pE-Aß aggregates. First, we demonstrate that both, focal and diffuse pE-Aß deposits are present in defined layers of the AD hippocampus. While the focal type of pE-Aß aggregates was found to be associated with the somata of QC-expressing interneurons, the diffuse type was not. To address this discrepancy, the hippocampus of amyloid precursor protein transgenic mice was analysed. Similar to observations made in AD, focal (i.e. core-containing) pE-Aß deposits originating from QC-positive neurons and diffuse pE-Aß deposits not associated with QC were detected in Tg2576 mouse hippocampus. The hippocampal layers harbouring diffuse pE-Aß deposits receive multiple afferents from QC-rich neuronal populations of the entorhinal cortex and locus coeruleus. This might point towards a mechanism in which pE-Aß and/or QC are being released from projection neurons at hippocampal synapses. Indeed, there are a number of reports demonstrating the reduction of diffuse, but not of focal, Aß deposits in hippocampus after deafferentation experiments. Moreover, we demonstrate in neurons by live cell imaging and by enzymatic activity assays that QC is secreted in a constitutive and regulated manner. Thus, it is concluded that hippocampal pE-Aß plaques may develop through at least two different mechanisms: intracellularly at sites of somatic QC activity as well as extracellularly through seeding at terminal fields of QC expressing projection neurons.

ADAM10 is the physiologically relevant, constitutive alpha-secretase of the amyloid precursor protein in primary neurons.

The amyloid precursor protein (APP) undergoes constitutive shedding by a protease activity called alpha-secretase. This is considered an important mechanism preventing the generation of the Alzheimer's disease amyloid-beta peptide (Abeta). alpha-Secretase appears to be a metalloprotease of the ADAM family, but its identity remains to be established. Using a novel alpha-secretase-cleavage site-specific antibody, we found that RNAi-mediated knockdown of ADAM10, but surprisingly not of ADAM9 or 17, completely suppressed APP alpha-secretase cleavage in different cell lines and in primary murine neurons. Other proteases were not able to compensate for this loss of alpha-cleavage. This finding was further confirmed by mass-spectrometric detection of APP-cleavage fragments. Surprisingly, in different cell lines, the reduction of alpha-secretase cleavage was not paralleled by a corresponding increase in the Abeta-generating beta-secretase cleavage, revealing that both proteases do not always compete for APP as a substrate. Instead, our data suggest a novel pathway for APP processing, in which ADAM10 can partially compete with gamma-secretase for the cleavage of a C-terminal APP fragment generated by beta-secretase. We conclude that ADAM10 is the physiologically relevant, constitutive alpha-secretase of APP.

Bepridil and amiodarone simultaneously target the Alzheimer's disease beta- and gamma-secretase via distinct mechanisms.

The two proteases beta-secretase and gamma-secretase generate the amyloid beta peptide and are drug targets for Alzheimer's disease. Here we tested the possibility of targeting the cellular environment of beta-secretase cleavage instead of the beta-secretase enzyme itself. beta-Secretase has an acidic pH optimum and cleaves the amyloid precursor protein in the acidic endosomes. We identified two drugs, bepridil and amiodarone, that are weak bases and are in clinical use as calcium antagonists. Independently of their calcium-blocking activity, both compounds mildly raised the membrane-proximal, endosomal pH and inhibited beta-secretase cleavage at therapeutically achievable concentrations in cultured cells, in primary neurons, and in vivo in guinea pigs. This shows that an alkalinization of the cellular environment could be a novel therapeutic strategy to inhibit beta-secretase. Surprisingly, bepridil and amiodarone also modulated gamma-secretase cleavage independently of endosomal alkalinization. Thus, both compounds act as dual modulators that simultaneously target beta- and gamma-secretase through distinct molecular mechanisms. In addition to Alzheimer's disease, compounds with dual properties may also be useful for drug development targeting other membrane proteins.

BACE1 is a newly discovered protein secreted by the pancreas which cleaves enteropeptidase in vitro.

CONTEXT: Activity of beta-site APP-cleaving enzyme1 (BACE1) is required for the generation of beta-amyloid peptides, the principal constituents of plaques in the brains of patients with Alzheimer's disease. Strong BACE1 expression has also been described in pancreatic tissue. OBJECTIVE: The aim of the present study was to reveal the cell type-specific expression of BACE1 in the pancreas and to identify a substrate for BACE1 in this organ. METHODS: RT-PCR of microdissected rat pancreatic tissue was carried out in order to analyze BACE1 expression within pancreatic acini. Pancreatic juice was examined by western blot analysis and by an enzymatic activity assay in order to reveal the presence of secreted BACE1. Database analysis suggested enteropeptidase as a putative substrate for BACE1 in pancreatic juice. In vitro digestion of enteropeptidase by BACE1 was performed to demonstrate this cleavage. RESULTS: We demonstrate the expression of BACE1 in the islets of Langerhans and at the apical pole of pancreatic acinar cells. Recombinant BACE1 cleaves enteropeptidase in vitro. Furthermore, some results suggested the presence of BACE1 enzymatic activity in pancreatic juice and pancreatic tissue. DISCUSSION: We hypothesize that enteropeptidase is a BACE1 substrate in vivo. If so, BACE1 could protect the pancreas from premature trypsinogen activation due to the occasionally occurring reflux of enteropeptidase.

Glutaminyl cyclase inhibition attenuates pyroglutamate Abeta and Alzheimer's disease-like pathology.

Because of their abundance, resistance to proteolysis, rapid aggregation and neurotoxicity, N-terminally truncated and, in particular, pyroglutamate (pE)-modified Abeta peptides have been suggested as being important in the initiation of pathological cascades resulting in the development of Alzheimer's disease. We found that the N-terminal pE-formation is catalyzed by glutaminyl cyclase in vivo. Glutaminyl cyclase expression was upregulated in the cortices of individuals with Alzheimer's disease and correlated with the appearance of pE-modified Abeta. Oral application of a glutaminyl cyclase inhibitor resulted in reduced Abeta(3(pE)-42) burden in two different transgenic mouse models of Alzheimer's disease and in a new Drosophila model. Treatment of mice was accompanied by reductions in Abeta(x-40/42), diminished plaque formation and gliosis and improved performance in context memory and spatial learning tests. These observations are consistent with the hypothesis that Abeta(3(pE)-42) acts as a seed for Abeta aggregation by self-aggregation and co-aggregation with Abeta(1-40/42). Therefore, Abeta(3(pE)-40/42) peptides seem to represent Abeta forms with exceptional potency for disturbing neuronal function. The reduction of brain pE-Abeta by inhibition of glutaminyl cyclase offers a new therapeutic option for the treatment of Alzheimer's disease and provides implications for other amyloidoses, such as familial Danish dementia.

The transcription factor Yin Yang 1 is an activator of BACE1 expression.

The beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1) is a prerequisite for the generation of beta-amyloid peptides, the principle constituents of senile plaques in the brains of patients with Alzheimer's disease (AD). BACE1 expression and enzymatic activity are increased in the AD brain, but the regulatory mechanisms of BACE1 expression are largely unknown. Here we show that Yin Yang 1 (YY1), a highly conserved and multifunctional transcription factor, binds to its putative recognition sequence within the BACE1 promoter and stimulates BACE1 promoter activity in rat pheochromocytoma 12 (PC12) cells, rat primary neurones and astrocytes. In rat brain YY1 and BACE1 are widely expressed by neurons, but there was only a minor proportion of neurones that co-expressed YY1 and BACE1, suggesting that YY1 is not required for constitutive neuronal BACE1 expression. Resting astrocytes in the untreated rat brain did not display either YY1 or BACE1 immunoreactivity. When chronically activated, however, astrocytes expressed both YY1 and BACE1 proteins, indicating that YY1 is important for the stimulated BACE1 expression by reactive astrocytes. This is further emphasized by the expression of YY1 and BACE1 by reactive astrocytes in proximity to beta-amyloid plaques in the AD brain. Our observations suggest that interfering with expression, translocation or binding of YY1 to its BACE1 promoter-specific sequence may have therapeutic potential for treating patients with AD.

Brain prolyl endopeptidase expression in aging, APP transgenic mice and Alzheimer's disease.

Prolyl endopeptidase (PEP) is believed to inactivate neuropeptides that are present in the extracellular space. However, the intracellular localization of PEP suggests additional, yet unidentified physiological functions for this enzyme. Here we studied the expression, enzymatic activity and subcellular localization of PEP in adult and aged mouse brain as well as in brains of age-matched APP transgenic Tg2576 mice and in brains of Alzheimer's disease patients. In mouse brain PEP was exclusively expressed by neurons and displayed region- and age-specific differences in expression levels, with the highest PEP activity being present in cerebellum and a significant increase in hippocampal but not cortical or cerebellar PEP activity in aged mouse brain. In brains of young APP transgenic Tg2576 mice, hippocampal PEP activity was increased compared to wild-type littermates in the pre-plaque phase but not in aged mice with beta-amyloid plaque pathology. This "accelerated aging" with regard to hippocampal PEP expression in young APP transgenic mice might be one factor contributing to the observed cognitive deficits in these mice in the pre-plaque phase and could also explain in part the cognition-enhancing effects of PEP inhibitors in several experimental paradigms.

Subcellular localization suggests novel functions for prolyl endopeptidase in protein secretion.

For a long time, prolyl endopeptidase (PEP) was believed to inactivate neuropeptides in the extracellular space. However, reports on the intracellular activity of PEP suggest additional, as yet unidentified, physiological functions for this enzyme. Here, we demonstrate using biochemical methods of subcellular fractionation, immunocytochemical double-labelling procedures and localization of PEP-enhanced green fluorescent protein fusion proteins that PEP is mainly localized to the perinuclear space, and is associated with the microtubulin cytoskeleton in human neuroblastoma and glioma cell lines. Disassembly of the microtubules by nocodazole treatment disrupts both the fibrillar tubulin and PEP labelling. Furthermore, in a two-hybrid screen, PEP was identified as binding partner of tubulin. These findings indicate novel functions for PEP in axonal transport and/or protein secretion. Indeed, a metabolic labelling approach revealed that both PEP inhibition and PEP antisense mRNA expression result in enhanced peptide/protein secretion from human U-343 glioma cells. Because disturbances in intracellular transport and protein secretion mechanisms are associated with a number of ageing-associated neurodegenerative diseases, cell-permeable PEP inhibitors may be useful for the application in a variety of related clinical conditions.

Alzheimer's disease beta-secretase BACE1 is not a neuron-specific enzyme.

The brains of Alzheimer's disease (AD) patients are morphologically characterized by neurofibrillar abnormalities and by parenchymal and cerebrovascular deposits of beta-amyloid peptides. The generation of beta-amyloid peptides by proteolytical processing of the amyloid precursor protein (APP) requires the enzymatic activity of the beta-site APP cleaving enzyme 1 (BACE1). The expression of this enzyme has been localized to the brain, in particular to neurons, indicating that neurons are the major source of beta-amyloid peptides in brain. Astrocytes, on the contrary, are known to be important for beta-amyloid clearance and degradation, for providing trophic support to neurons, and for forming a protective barrier between beta-amyloid deposits and neurons. However, under certain conditions related to chronic stress, the role of astrocytes may not be beneficial. Here we present evidence demonstrating that astrocytes are an alternative source of BACE1 and therefore may contribute to beta-amyloid plaque formation. While resting astroyctes in brain do not express BACE1 at detectable levels, cultured astrocytes display BACE1 promoter activity and express BACE1 mRNA and enzymatically active BACE1 protein. Additionally, in animal models of chronic gliosis and in brains of AD patients, there is BACE1 expression in reactive astrocytes. This would suggest that the mechanism for astrocyte activation plays a role in the development of AD and that therapeutic strategies that target astrocyte activation in brain may be beneficial for the treatment of AD. Also, there are differences in responses to chronic versus acute stress, suggesting that one consequence of chronic stress is an incremental shift to different phenotypic cellular states.