Search

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.

The pathogenesis of some neurodegenerative disorders has been linked to excitotoxicity, excess generation of nitric oxide (NO) and apoptosis. Here, we used a model of NO-triggered neuronal apoptosis that was strictly dependent on autocrine NMDA receptor (NMDA-R) activation and intracellular Ca2 increase. We investigated the efficiency and potentially beneficial effects of calpain inhibition. Three calpain inhibitors that prevented intracellular fodrin proteolysis also blocked apoptotic features such as decrease in mitochondrial membrane potential, chromatin breakdown, and subsequent death of cerebellar granule neurons exposed to NO donors (S-nitroso- L-glutathione, S-nitroso-N-acetyl D,L-penicillamine, and diethylamino- diazenolate-2-oxide). Since inhibitors did not interfere with NMDA-R activation, we suggest that block of calpains blunts NO-triggered neuronal apoptosis by stopping the cascade downstream of primary autocrine excitotoxic events.

In cerebellar granule cells, microtubule disassembling by colchicine induced degeneration of the axodendritic network followed by apoptotic demise of neuronal somata. Colchicine-induced apoptosis was accompanied by mitochondrial cytochrome c release, activation of execution caspase-3, advanced chromatin condensation, oligonucleosomal DNA fragmentation, and phosphatidylserine translocation to the outer surface of the plasma membrane. In the presence of peptide caspase inhibitors, apoptotic changes were prevented, although cytochrome c was released from mitochondria and the axodendritic network was still destroyed. Depletion of intracellular ATP blocked caspase activation, nuclear apoptotic features, and phosphatidylserine exposure, while the primary effect of microtubule disruption and neurite loss still occurred. Conversely, repletion of intracellular ATP restored all apoptotic features. Thus, sufficient ATP is required for the execution of neuronal apoptosis and energy deficiency may lead to the persistence of degenerating and dysfunctional neurons. Neither caspase inhibition by ATP depletion or pharmacological agents nor antiapoptotic Bcl-2 prevented microtubule breakdown or neurite loss. But neuronal somata were protected from apoptosis under these conditions. However, after a lag period caspase-inhibited as well as Bcl-2 overexpressing neurons underwent delayed cell death. This implies that death programs for neurite degeneration and apoptosis can occur independently from each other and that block of caspases may not rescue neurons from demise under degenerative conditions. The delayed caspase-independent death was characterized by partial chromatin condensation, large scale DNA fragmentation and phosphatidylserine exposure. Inhibitors of the proteasome reduced caspase-independent apoptosis, implying that noncaspase proteases can take over execution of apoptosis in neurons.

Plasma membrane Ca²+ ATPase (PMCA) pump isoforms 2, 3, and 1CII are expressed in large amounts in the cerebellum of adult rats but only minimally in neonatal cerebellum. These isoforms were almost undetectable in rat neonatal cerebellar granule cells 1 3 days after plating, but they became highly expressed after 7 9 days of culturing under membrane depolarizing conditions (25 mM KCl). The behavior of isoform 4 was different: it was clearly detectable in adult cerebellum but was down-regulated by the depolarizing conditions in cultured cells. 25 mM KCl-activated L-type Ca²+ channels, significantly increasing cytosolic Ca²+. Changes in the concentration of Ca²+ in the culturing medium affected the expression of the pumps. L-type Ca²+ channel blockers abolished both the up-regulation of the PMCA1CII, 2, and 3 isoforms and the down-regulation of PMCA4 isoform. When granule cells were cultured in high concentrations of N-methyl-D-aspartic acid, a condition that increased cytosolic Ca²+ through the activation of glutamate-operated Ca²+ channels, up-regulation of PMCA1CII, 2, and 3 and down-regulation of PMCA4 was also observed. The activity of the isoforms was estimated by measuring the phosphoenzyme intermediate of their reaction cycle: the up-regulated isoforms, the activity of which was barely detectable at plating time, accounted for a large portion of the total PMCA activity of the cells. No up-regulation of the sarcoplasmic/ endoplasmic reticulum calcium pump was induced by the depolarizing conditions.

Background: Early loss of neurites followed by delayed damage of neuronal somata is a feature of several neurodegenerative diseases. Death by apoptosis would ensure the rapid removal of injured neurons, whereas conditions that prevent apoptosis may facilitate the persistence of damaged cells and favor inflammation and disease progression. Materials and Methods: Cultures of cerebellar granule cells (CGC) were treated with microtubule disrupting agents. These compounds induced an early degeneration of neurites followed by apoptotic destruction of neuronal somata. The fate of injured neurons was followed after co-exposure to caspase inhibitors or agents that decrease intracellular ATP (deoxyglucose, S-nitrosoglutathione, l-methyl 4-phenylpyridinium). We examined the implications of energy loss for caspase activation, exposure of phagocytosis markers, and long-term persistence of damaged cells. Results: In CGC exposed to colchicine or nocodazole, axodendritic degeneration preceded caspase activation and apoptosis. ATP-depleting agents or protein synthesis inhibition prevented caspase activation, translocation of the phagocytosis marker, phosphatidylserine, and apoptotic death. However, they did not affect the primary neurite loss. Repletion of ATP by enhanced glycolysis restored all apoptotic features. Peptide inhibitors of caspases also prevented the apoptotic changes in the cell bodies, although the axodendritic net was lost. Under this condition cell demise still occurred 48 hr later in a caspase-independent manner and involved plasma membrane lysis at the latest stage. Conclusions: Inhibition of the apoptotic machinery by drugs, energy deprivation, or endogenous mediators may result in the persistence and subsequent lysis of injured neurons. In vivo, this may favor the onset of inflammatory processes and perpetuate neurodegeneration.