Steffen Roßner

ADAM10 is the physiologically relevant, constitutive α‐secretase of the amyloid precursor protein in primary neurons

The amyloid precursor protein (APP) undergoes constitutive shedding by a protease activity called α‐secretase. This is considered an important mechanism preventing the generation of the Alzheimers disease amyloid‐β peptide (Aβ). α‐Secretase appears to be a metalloprotease of the ADAM family, but its identity remains to be established. Using a novel α‐secretase‐cleavage site‐specific antibody, we found that RNAi‐mediated knockdown of ADAM10, but surprisingly not of ADAM9 or 17, completely suppressed APP α‐secretase cleavage in different cell lines and in primary murine neurons. Other proteases were not able to compensate for this loss of α‐cleavage. This finding was further confirmed by mass‐spectrometric detection of APP‐cleavage fragments. Surprisingly, in different cell lines, the reduction of α‐secretase cleavage was not paralleled by a corresponding increase in the Aβ‐generating β‐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 γ‐secretase for the cleavage of a C‐terminal APP fragment generated by β‐secretase. We conclude that ADAM10 is the physiologically relevant, constitutive α‐secretase of APP.

The regulation of amyloid precursor protein metabolism by cholinergic mechanisms and neurotrophin receptor signaling

The increased expression and/or abnormal processing of the amyloid precursor protein (APP) is associated with the formation of amyloid plaques and cerebrovascular amyloid deposits, which are one of the major morphological hallmarks of Alzheimers disease (AD). Among the processes regulating APP metabolism, the proteolytic cleavage of APP into amyloidogenic or nonamyloidogenic fragments is of special interest. The cleavage of the APP by the alpha-secretase within the beta-amyloid sequence generates nonamyloidogenic C-terminal APP fragments and soluble APPs alpha, which has neurotrophic and neuroprotective activities. Proteolytic processing of APP by beta-secretase, on the other hand, exposes the N-terminus of beta-amyloid, which is liberated after gamma-secretase cleavage at the variable amyloid C-terminus. The resulting 39-43 amino acid beta-amyloid may be neurotoxic and disrupt neuronal connectivity after its accumulation in senile plaques. In this review, we discuss evidence derived from in vitro experiments, suggesting that the stimulation of protein kinase C (PKC)-coupled M1/M3 muscarinic acetylcholine receptors increases the nonamyloidogenic, secretory pathway of APP processing. It has also been shown in animal models that under conditions of reduced M1/M3 muscarinic acetylcholine receptor stimulation the secretory pathway of APP processing is inhibited and that constitutive upregulation of M1/M3-associated PKC increases APP secretion. Thus, the cortical cholinergic hypoactivity characteristic of AD may inhibit the nonamyloidogenic APP processing pathway and lead to increased beta-amyloid generation. It has been shown in vitro that nerve growth factor (NGF)-associated signaling also influences the expression and catabolism of APP. Recent experiments with NGF-responsive cells revealed a specific role for the high-affinity NGF receptor, TrkA, in the increases in secretory APP processing and a role for the low-affinity neurotrophin receptor, p75NTR, in the transcriptional regulation of APP. Therefore, treatments with NGF could ameliorate cortical cholinergic dysfunction in AD. These findings may influence the design of therapeutic strategies aimed at stimulating cholinergic function and at increasing nonamyloidogenic APP processing without elevating APP expression.

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.

Secretome Protein Enrichment with Click Sugars Identifies Physiological Substrates of the Alzheimer Protease BACE1 in Primary 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.

Differential regulation of BACE1 promoter activity by nuclear factor-κB in neurons and glia upon exposure to β-amyloid peptides

The brains of Alzheimers disease (AD) patients display cerebrovascular and parenchymal deposits of β‐amyloid (Aβ) peptides, which are derived by proteolytic processing by the β‐site APP‐cleaving enzyme 1 (BACE1) of the amyloid precursor protein (APP). The rat BACE1 promoter has a nuclear factor‐κB (NF‐κB) binding site. Deletion studies with a BACE1 promoter/luciferase reporter suggest that the NF‐κB binding DNA consensus sequence plays a suppressor role, when occupied by NF‐κB, in the regulation of neuronal brain BACE1 expression. Here we characterize a signal transduction pathway that may be responsible for the increases in Aβ associated with AD. We propose that the transcription factor NF‐κB acts as a repressor in neurons but as an activator of BACE1 transcription in activated astrocytes present in the CNS under chronic stress, a feature present in the AD brain. The activated astrocytic stimulation of BACE1 may in part account for increased BACE1 transcription and subsequent processing of Aβ in a cell‐specific manner in the aged and AD brain. As measured by reporter gene promoter constructs and endogenous BACE1 protein expression, a functional NF‐κB site was stimulatory in activated astrocytes and Aβ‐exposed neuronal cells and repressive in neuronal and nonactivated astrocytic cells. Given the evidence for increased levels of activated astrocytes in the aged brain, the age‐ and AD‐associated increases in NF‐κB in brain may be significant contributors to increases in Aβ, acting as a positive feedback loop of chronic inflammation, astrocyte activation, increased p65/p50 activation of BACE1 transcription, and further inflammation.

Astrocytic expression of the Alzheimer's disease β-secretase (BACE1) is stimulus-dependent

The beta‐site APP‐cleaving enzyme (BACE1) is a prerequisite for the generation of β‐amyloid peptides, which give rise to cerebrovascular and parenchymal β‐amyloid deposits in the brain of Alzheimers disease patients. BACE1 is neuronally expressed in the brains of humans and experimental animals such as mice and rats. In addition, we have recently shown that BACE1 protein is expressed by reactive astrocytes in close proximity to β‐amyloid plaques in the brains of aged transgenic Tg2576 mice that overexpress human amyloid precursor protein carrying the double mutation K670N‐M671L. To address the question whether astrocytic BACE1 expression is an event specifically triggered by β‐amyloid plaques or whether glial cell activation by other mechanisms also induces BACE1 expression, we used six different experimental strategies to activate brain glial cells acutely or chronically. Brain sections were processed for the expression of BACE1 and glial markers by double immunofluorescence labeling and evaluated by confocal laser scanning microscopy. There was no detectable expression of BACE1 protein by activated microglial cells of the ameboid or ramified phenotype in any of the lesion paradigms studied. In contrast, BACE1 expression by reactive astrocytes was evident in chronic but not in acute models of gliosis. Additionally, we observed BACE1‐immunoreactive astrocytes in proximity to β‐amyloid plaques in the brains of aged Tg2576 mice and Alzheimers disease patients. GLIA 41:169–179, 2003.

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

The brains of Alzheimers disease (AD) patients are morphologically characterized by neurofibrillar abnormalities and by parenchymal and cerebrovascular deposits of β‐amyloid peptides. The generation of β‐amyloid peptides by proteolytical processing of the amyloid precursor protein (APP) requires the enzymatic activity of the β‐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 β‐amyloid peptides in brain. Astrocytes, on the contrary, are known to be important for β‐amyloid clearance and degradation, for providing trophic support to neurons, and for forming a protective barrier between β‐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 β‐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.

Neuronal and glial β-secretase (BACE) protein expression in transgenic Tg2576 mice with amyloid plaque pathology

We measured tissue distribution and expression pattern of the beta‐site amyloid precursor protein (APP)‐cleaving enzyme (BACE) in the brains of transgenic Tg2576 mice that show amyloid pathology. BACE protein was expressed at high levels in brain; at lower levels in heart and liver; and at very low levels in pancreas, kidney, and thymus and was almost absent in spleen and lung when assayed by Western blot analysis. We observed strictly neuronal expression of BACE protein in the brains of nontransgenic control mice, with the most robust immunocytochemical labeling present in the cerebral cortex, hippocampal formation, thalamus, and cholinergic basal forebrain nuclei. BACE protein levels did not differ significantly between control and transgenic mice or as a result of aging. However, in the aged, 17‐month‐old Tg2576 mice there was robust amyloid plaque formation, and BACE protein was also present in reactive astrocytes present near amyloid plaques, as shown by double immunofluorescent labeling and confocal laser scanning microscopy. The lack of astrocytic BACE immunoreactivity in young transgenic Tg2576 mice suggests that it is not the APP overexpression but rather the amyloid plaque formation that stimulates astrocytic BACE expression in Tg2576 mice. Our data also suggest that the neuronal overexpression of APP does not induce the overexpression of its metabolizing enzyme in neurons. Alternatively, the age‐dependent accumulation of amyloid plaques in the Tg2576 mice does not require increased neuronal expression of BACE. Our data support the hypothesis that neurons are the primary source of β‐amyloid peptides in brain and that astrocytic β‐amyloid generation may contribute to amyloid plaque formation at later stages or under conditions when astrocytes are activated. J. Neurosci. Res. 64:437–446, 2001.

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.

Reduced infarct volume and differential effects on glial cell activation after hyperbaric oxygen treatment in rat permanent focal cerebral ischaemia

Permanent middle cerebral artery occlusion (MCAO) causes neurodegeneration and a robust activation of glial cells primarily in sensorimotor brain regions of rats. It has been shown that hyperbaric oxygen (HBO) increases oxygen supply to ischaemic areas and reduces neuronal cell loss. The effects of HBO treatment on microgliosis and astrogliosis in permanent cerebral ischaemia have not been addressed so far, but might be critical for neurodegeneration and neuroprotection, respectively. Therefore, we used spontaneously hypertensive rats with permanent MCAO to investigate the time window to start HBO and to compare the effects of different HBO treatment frequencies on infarct volume and on differences with regard to microgliosis and astrogliosis. Seven days after MCAO the infarct volume was calculated from Nissl‐stained brain sections by image analysis. HBO significantly decreased the infarct volume when used as early as 15, 90 or 180 min post‐MCAO by 24%, 16% and 13%, respectively, in the single‐treatment group. Repetitive HBO treatment (first HBO session 90 min after MCAO) was not effective. Microglial cells and astrocytes were detected by cytochemical fluorescent labelling and confocal laser scanning microscopy. In the single‐treatment group we observed significantly higher astrocyte immunoreactivity but decreased microglial density in the peri‐infarct region. These effects of HBO treatment on glial cells were not present in rats where HBO did not reduce the infarct volume (360 min after MCAO). Our data indicate that HBO‐induced suppression of microgliosis and aggravated response of astrocytes might contribute to the reported beneficial effects of early HBO treatment in cerebral ischaemia.