НЕЙРОХИМИЯ, 2013, том 30, № 2, с. 117-127
GLUTAMATE TREATMENT AND PRECONDITIONING DIFFERENTLY AFFECT CATHEPSIN B RELEASE AND INTRACELLULAR PROTEASES IN PRIMARY CULTURES OF CEREBELLAR GRANULAR CELLS1 © 2013 A. A. Yakovlev", A. A. Kvichansky", *, A. A. Lyzhin4, L. G. Khaspekov4, and N. V. Gulyaeva"
aInstitute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences bResearch Center of Neurology Russian Academy of Medical Sciences
Abstract—Effects of serum deprivation, preconditioning to serum deprivation, and of glutamate toxicity on cathepsin B (CB) release and intracellular activities of CB, caspase-3 and calpain were studied in primary cerebellar granular cell cultures. The cells release CB when they are deprived of trophic factors or treated with glutamate, and this secretion is not accompanied by cell death. Similar CB secretion can be detected in organotypic cultures of hippocampal slices. Preconditioning reduces CB release in cerebellar cultures and modulates effects of glutamate on intracellular proteolytic activities. Glutamate augments the ratio of CB activity in the cultural medium and within cells, while preconditioning results in a decrease of this ratio. We suggest that extracellular CB is a potential player in mechanisms of neuronal cell preconditioning.
Keywords:preconditioning, cathepsin B, caspase-3, calpain, cerebellar cultures, serum deprivation
LIST OF ABBREVATIONS
BSS—Hank's balanced salt solution; CA074—L-3-trans-(propylcarbamyl)oxirane-2-carbonyl)-L-isoleucyl-L-proline methyl ester; CB—cathepsin B; DTT—dithiothreitol; EDTA—ethylenediaminetetraacetic acid; PAAG—polyacrilamide gel; LDH—lactate dehydrogenase; PMSF—phenylmethanesulfonylfluoride; Suc-LLVY-AMC—Suc-Leu-Leu-Val-Tyr-7-ami-do-4-methylcoumarin hydrochloride;
Nervous cells, like most other cells of the living organism, are constantly synthesizing and degrading lots
1 The article was submitted by the authors in English.
* Corresponding author; address: 5a Butlerov Street, Moscow 117486 Russia, e-mail: firstname.lastname@example.org.
of proteins, including cytoskeletal, signal transduction and extracellular matrix proteins [1—3]. Processing of synthesized proteins, regulation of their activities/functions, as well as their degradation require a number of proteolytic activities. During the last two decades there was a burst of studies focused on pro-teolytic enzymes in the central nervous system. The majority of these studies deals with caspase-dependent programmed neuronal cell death in both normal  and pathological conditions [5, 6], and as a rule, is related to caspase-3, an essential effector caspase in mammals . Autophagy, representing another major pathway of programmed cell death, is mediated by lyso-somal proteases, first of all by the acidic proteases cathepsins . Calpains may be regarded functionally as links between apoptotic-like and autophagic cell death since they are involved in both. Indeed, they may participate in caspasedependent neuronal cell death [9, 10], but also may regulate autophagy [11, 12].
Along with the intracellular brain proteases, there is a growing interest in the secreted brain proteases, such as metalloproteases and tissue plasminogen activator . These enzymes, secreted mostly by the glial cells, are involved in the nervous tissue reorganization and brain plasticity in normal and pathological conditions [14, 15]. Cathepsin B (CB), a cysteine protease, can be also secreted by brain cells. Its release from glial cells is well documented [16, 17]. Unlike other acidic lysosomal cysteine proteases, this enzyme retains its activity at almost neutral pH values . CB is believed to be involved in extracellular matrix remodel-
ing , angiogenesis , tau protein  and amyloid precursor protein proteolysis . Recently, we have shown that, similar to glial cells, the cerebellar granular neurons in primary cultures can secrete CB
, however, putative functional role of the neuronal CB secretion still remains obscure.
Preconditioning is a natural adaptive phenomenon promoting the development of a tolerance to factors inducing potentially lethal damage in organs and tissues by exposure to sublethal factors; the level of the tolerance and the time period of its maintenance depend on the nature of the stimulus and its intensity
. Since trophic factors deprivation can be a preconditioning stimulus and can induce CB secretion in neuronal cultures as well , we have hypothesized that extracellular CB together with the intracellular proteases can be involved in the neuronal cell preconditioning. This study aimed to explore this possibility.
MATERIALS AND METHODS
CB inhibitor CA074 (L-3-trans-(propylcarbam-yl)oxirane-2-carbonyl)-L-isoleucylL-proline methyl ester), trypsin inhibitor aprotinin, general cysteine protease inhibitor glutathione disulfide, general serine protease inhibitor phenylmethanesulfonylfluoride (PMSF), CB and trypsin substrate Z-Arg-Arg-7-ami-do-4-methylcoumarin hydrochloride (Z-RR-AMC), cathepsin substrate Z-Phe-Arg—7-amido-4-methylcou-marin hydrochloride (Z-FR-AMC), caspase-3 substrate Ac-Asn-Glu-Val-Asn-7-amido-4methylcoumarin hydrochloride (Ac-DEVD-AMC), calpain substrate Suc-Leu-Tyr-7amido-4-methylcoumarin hydrochlo-ride (Suc-LY-AMC), chymotrypsin substrate Su-cLeu-Leu-Vil-Tyr-7-amido-4-methylcoumarin hydrochloride (Suc- LLVY-AMC), Amicon Ultracell 3 kDa filters, and Coomassie G-250 were from Sigma. Other chemicals were from Sigma, unless otherwise indicated. Cathepsin antibody FL-339 was from Santa Cruz Biotechnology, Inc. Supplements of culture media and BSS were from Gibco unless otherwise indicated.
Cell Cultures and Their Treatments
Cell cultures. Dissociated primary rat cerebellar granular cell cultures of 7 days old rat pups were cultivated as described by Andreeva et al.  in a CO2-in-cubator (5% C02, 95% air, 35.5°C) for 7 or 10 days. All experimental procedures were approved by the Institutional Committee for Animal Care and Use. The cell suspension was added to 24 well plates covered with polyethyleneimine (150 ^L in each well); material from 3 wells was pooled for each experimental sample. No less than 3 parallel samples from the sister cultures were analyzed. Hippocampal slices from 7 days old rat pups were cultivated on the membranes (4 explants per a membrane) according to Stoppini et al. . The cul-
tural medium of both culture types contained B27 and Glutamax.
Preconditioning. The cultures were placed into a balanced salt solutions (BSS) containing 10 mM glucose, 143.4 mM NaCl, 25 mM KCl, 2 mM CaCl2, 1.2 mM NaH2PO4, 5 mM HEPES, pH 7.4 on the seventh day of cultivation, kept for an hour at 35.5°C and returned to the usual medium.
Treatment with glutamate. To induce acute glutamate toxicity the cerebellar cultures were placed into BSS containing glutamate (100 or 200 ^M) for 15 or 30 min a day after preconditioning, then to BSS for 5 h. Ten days old organotypic cultures were placed into BSS containing 0.5 mM glutamate for 30 min and then transferred to BSS for 5 h.
To induce chronic glutamate toxicity the ten days old cerebellar cultures were placed into cultural medium containing 250 ^M glutamate, and the samples were taken 24 h later. Samples of cultural medium, BSS or cell lysates were taken for analysis.
Cell lysis. After the withdrawal of BSS or cultural medium, the cells were lysed in a buffer containing 20 mM HEPES, 0.5 mM EDTA, 10 mM KCl, 1.5 mM MgCl2, 0.01% NP-40, pH 7.5.
Measurement of Proteolytic Activities
Proteolytic activities were assessed at pH 7.5 (trypsin, caspase-3, calpain, chymotrypsin) or pH 6.0 (CB and caspase-3-like activity) with respective fluorescent substrates and inhibitors using plate reader Perki-nElmer Victor 3 (excitation/emission 370 nm/430 nm). The concentration of Z-FR-AMC was 25 ^M, of other substrates 100 ^M. AMC (10 ^M) was used as a standard. DTT (20 mM) and EDTA (1 mM) were added in buffers for cathepsin B, caspase, and caspase-like activity measurement, while CaCl2 was added in buffers for trypsin, chymotrypsin (4 mM) and calpain (2 mM) activity measurement. The activities were measured using 384-well microplates for fluoromety (Grenier Bio One) in a volume 50 ^L/well. Proteolytic activities were calculated as pmol substrate/min/mL of medium/BSS or pmol substrate/min/mg protein for cell lysates.
Concentration of Proteins and PAAG Electrophoresis
The cultural medium was concentrated about 100-fold using Amicon Ultracell 3 kDa filters (Milli-pore). The concentrated samples were analysed using gel electrophoresis according to Laemmli in 15% PAAG on Mini Protean II (Bio-Rad) using the method described earlier .
Immunoblotting of Proteinases
Cerebellar granular cells were lysed in a buffer containing 12.5 mM Tris, pH 6.8, 10% glycerol, 2% SDS, 25 mM Tris(2-carboxyethyl)phosphine hydrochloride, 0.001% bromophenol blue, and heated at 95°C for 5 min. The samples were subjected to electrophoresis in 7.5% PAAG and a subsequent transfer to a nitrocellulose membrane. The blots were developed using SIGMAFAST™ 3,3'-Diaminobenzidine tablets (Sigma), primary antibodies Rabbit Cleaved Caspase-3 (Asp175) Antibody (Cell Signal Technology), secondary antibodies Biotin-SP-AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson Immunoresearch), and streptavidine-peroxidase complex (IMTEK, Russia). To stain products of calpain-and caspase-3-mediated cleavage of spectrin the spectrin a II Antibody (H-105, Santa Cruz Biotech) was used.
Analysis of Cell Death (Lactate Dehydrogenase Release)
Lactate dehydrogenase (LDH) release in cultural medium
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