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Cathepsin D plays a crucial role in the trimethyltin-induced hippocampal neurodegeneration process
Neuroscience 174 (2011) 160 –170
CATHEPSIN D PLAYS A CRUCIAL ROLE IN THE TRIMETHYLTININDUCED HIPPOCAMPAL NEURODEGENERATION PROCESS S. CECCARIGLIA,a A. D’ALTOCOLLE,a A. DEL FA’,a F. PIZZOLANTE,a E. CACCIA,b F. MICHETTIa* AND C. GANGITANOa
the hippocampus being a key target of its action. The neurotoxicant induces in the rat hippocampus a selective loss of pyramidal neurons, especially in the CA1/CA3 subfields, together with reactive astrogliosis (Dyer et al., 1982; McCann et al., 1996; Bruccoleri et al., 1998; Koczyk and Oderfeld-Nowak, 2000; Tsutsumi et al., 2002). The pyramidal cells of the CA3 region are particularly sensitive to acute toxicity with single doses of TMT (Ishida et al., 1997; Imai et al., 2001). The action of TMT has been shown to be mediated by disregulation of intracellular Ca2⫹ homeostasis, leading to a marked increase in [Ca2⫹]i in cultured rat hippocampal neurons (Piacentini et al., 2008), in HeLa and neuroblastoma cells (Florea et al., 2005a,b) and also in spiral ganglion cells (Fechter and Liu, 1995). Interestingly, rat hippocampal neurons expressing the Ca2⫹-binding proteins calretinin and parvalbumin are selectively spared by the neuroxicant (Geloso et al., 1996, 1997, 1998; Businaro et al., 2002; Gangitano et al., 2006), possibly through a calcium-buffering effect that counteracts the TMT-induced calcium overload. The molecular mechanisms that mediate TMT-induced toxicity and cellular death are not completely understood. However, numerous studies using different experimental models have demonstrated that TMT induces apoptosis via a series of mediators, such as protein kinase C activation, oxidative stress due to the generation of reactive oxygen species, over-expression of stannin, cyclooxygenase metabolism and caspase activation (Toggas et al., 1992; Kane et al., 1998; Fiedorowicz et al., 2001; Gunasekar et al., 2001a,b; Geloso et al., 2002; Jenkins and Barone, 2004; Buck-Koehntop et al., 2005; Mundy and Freudenrich, 2006; Nagashima et al., 2008). A substantial body of research indicates that the lysosomal system, especially lysosomal proteases such as the cathepsins, seems to be involved in the early stages of several neurodegenerative processes that occur in different conditions, such as transient forebrain ischaemia, kainate-evoked brain damage, age-related neurodegeneration and Alzheimer’s disease (Cataldo and Nixon, 1990; Diedrich et al., 1991; Nakanishi et al., 1993, 1995; Cataldo et al., 1995; Hetman et al., 1995; Nixon and Cataldo, 1995; Sato et al., 2006). Cat D is one of the main lysosomal aspartic proteases widely distributed throughout the brain (Whitaker et al., 1981; Banay-Schwartz et al., 1992). Although the major function of Cat D is related to intracellular catabolism in lysosomal compartments (Dean, 1975) this protease seems also to play an important role in the processes that regulate apoptosis (Deiss et al., 1996; Shibata et al., 1998; Bidère et al., 2003; Fusek and Vetvicka, 2005; Liaudet-
a Institute of Anatomy and Cell Biology, Catholic University, L. go F. Vito 1, 00168 Rome, Italy b Department of Environmental Sciences, Tuscia University, L. go Dell= Università, 01100 Viterbo, Italy
Abstract—Trimethyltin chloride (TMT) is known to produce neuronal damage in the rat hippocampus, especially in the CA1/CA3 subfields, together with reactive astrogliosis. Previous studies indicate that in cultured rat hippocampal neurons the Ca2ⴙ cytosolic increase induced by TMT is correlated with apoptotic cell death, although some molecular aspects of the hippocampal neurodegeneration induced by this neurotoxicant still remain to be clarified. Cathepsin D (Cat D) is a lysosomal aspartic protease involved in some neurodegenerative processes and also seems to play an important role in the processes that regulate apoptosis. We investigated the specific activity and cellular expression of Cat D in the rat hippocampus in vivo and in cultured organotypic rat hippocampal slices. The role of Cat D in cell death processes and the mechanisms controlling Cat D were also investigated. Cat D activity was assayed in hippocampus homogenates of control and TMT-treated rats. In order to visualize the distribution of Cat D immunoreactivity in the hippocampus, double-label immunofluorescence for Cat D and Neu N, GFAP, OX42 was performed. In addition, in order to clarify the possible relationship between Cat D activity, neuronal calcium overload and neuronal death processes, organotypic hippocampal cultures were also treated with a Cat D inhibitor (Pepstatin A) or Calpain inhibitor (Calpeptin) or an intracellular Ca2ⴙ chelator (BAPTA-AM) in the presence of TMT. TMT treatment in rat hippocampus induced high levels of Cat D activity both in vivo and in vitro, in glial cells and in CA3 neurons, where a marked TMT-induced neuronal loss also occurred. Cat D is actively involved in CA3 neuronal death and the protease increase is a calcium-Calpain dependent phenomenon. © 2011 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: trimethyltin, Cathepsin D, neurodegeneration, hippocampus, organotypic culture.
Trimethyltin (TMT) is known to produce significant and selective neuronal degeneration in the limbic system, with *Corresponding author. Tel: ⫹39-06-30155848; fax: ⫹39-06-30155753. E-mail address: [email protected] (F. Michetti). Abbreviations: BAPTA-AM, (2-aminophenoxi)ethane-N-N-N’,N’-tetraacetic acid tetrakis(acetoxymethyl) ester; Cat D, Cathepsin D; GFAP, glial acidic fibrillary protein; HBSS, Hank’s balanced salt solution; HEPES, 4-2-hydroxyethyl-1-piperazineethanesulfonic acid; i.p., intraperitoneal; PB, phosphate buffer; PBS, phosphate-buffered saline; PFA, paraformaldehyde; RT, room temperature; TMT, trimethyltin.
0306-4522/11 $ - see front matter © 2011 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2010.11.024
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Coopman et al., 2006; Minarowska et al., 2007; ZuzarteLuis et al., 2007a,b). To date, the role of cathepsins in TMT-neurotoxicity has not been studied, while the initiating events in the neurodegeneration induced by the neurotoxicant still remain to be clarified. In this study, we investigated the specific activity, distribution and cellular localization of Cat D in the rat hippocampus in vivo after TMT intoxication. We also used organotypic hippocampal cultures in order to clarify the role and possible relationship between Cat D, neuronal calcium overload and neuronal death processes.
EXPERIMENTAL PROCEDURES Animals and treatment Experiments were performed using adult female Wistar rats (200 – 250 g body weight, age 2 months). Animals were housed in an air-conditioned environment with constant temperature and a standardized light/dark schedule, food and water being supplied ad libitum. The rats were treated with a single intraperitoneal (i.p.) injection of TMT (Sigma, St Louis, MO, USA) dissolved in saline at a dose of 8 mg/kg body weight in a volume of 1 ml/kg body weight. Control rats received the same volume of saline. The animals were anaesthetized with 2 mg/100g i.p. diazepam (Biologici Italia Laboratories, Milano) followed by i.m. injection of 4 mg/100g ketamine (Intervet International, GmbH, Germany). The rats were sacrificed at 3, 5, 7, 14 and 21 days after treatment. All the animal protocols were approved by the Animal Experimentation Committee of Catholic University, Rome. In particular, animal experiments were performed in accordance with the European Community Council Directive of 24 November 1986 (86/609/EEC). All efforts were made to minimize the number of animals used and their suffering.
Cathepsin D enzymatic activity Total Cat D enzymatic activity was assayed at 3, 5, 7, 14, 21 days post-TMT intoxication in the rat hippocampi (10 rats/time point). Enzymatic activity of Cat D was also measured in hippocampi of control rats (n⫽10). Animals were killed by decapitation, the brains were removed and the entire hippocampi dissected. The tissues were homogenized in five volumes of 0.05 M citrate buffer, pH 3.2, containing 0.5% Triton X-100 with a glass homogenizer (Bracco et al., 1982); the samples were centrifuged at 25,000 g for 30 min at 4 °C. The supernatant was removed and aliquots were used for enzymatic assay. We also performed parallel experiments (9 rats/time point) to assay specific activity of Cat D in lysosomal and cytosolic fractions obtained according to the modified method of Banay-Schwartz et al. (1987). Briefly, the hippocampi of control and TMT-treated rats were homogenized in 0.01 M phosphate buffer pH 7.4 containing 0.3 M sucrose and centrifuged at 1000 g for 10 min at 4 °C. After discarding the pellet, the supernatant was centrifuged at 25,000 g for 45 min at 4 °C. The supernatant was then collected as cytosolic fraction and the pellet (crude lysosomal fraction) was re-suspended in the same buffer previously used with 0.5% Triton X-100, re-centrifuged at 25,000 g for 45 min at 4 °C, and the resulting supernatant was collected as enriched lysosomal fraction. Both lysosomal and cytosolic fractions were used to measure the Cat D specific activities in the corresponding cellular compartments. Cat D enzymatic activities were assayed using the method described by Barrett (1977). Briefly, lysates from hippocampus tissue were incubated with 0.5 M citrate buffer, pH 3.2 and 3% specific haemoglobin substrate (Sigma) for 1 h at 45 °C. Using a modified alkaline copper reagent, the haemoglobin breakdown
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was measured as tyrosine equivalent formed, read at 700 nm using the Beckman DU®-70 spectrophotometer. Enzyme activity was expressed as nmol/mg protein/h of tyrosine equivalent formed. In all cases about 90% of Cat D activity was inhibited by 12 g/ml Pepstatin A (Sigma). Protein quantification of the tissue homogenates was performed according to the method of Lowry et al. (1951), using bovine serum albumin as standard.
Organotypic slice cultures Hippocampal organotypic cultures from neonatal rats were prepared following the method of Stoppini et al. (1991). Briefly, female Wistar rats (20 –21 days old) were anaesthetized with 100 g/g ketamine and decapitated. The brains were removed and placed in an ice-cold dissection medium consisting of Hank’s balanced salt solution (HBSS, Gibco, BRL Life Technologies, Scotland) with 25 mM 4-2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES, Gibco) and 6% glucose, pH 7.2. The brains were then hemisected along the longitudinal fissure. From each hemisection, 400 m thick serial slices were cut transversely using a vibratome (Leica VT 1000S). Once cut, individual slices were immediately removed from the vibratome bath with a fire-polished Pasteur pipette and four to five slices were transferred onto each Millicell-CM (Millipore, Bedford, MA, USA) membrane insert which was placed in a six-well plate (Costar, Corning, NY, USA) containing 1 ml culture medium/well. The tissue surrounding the hippocampus was removed under a dissection microscope (Leitz Wetzlar Germany). The culture medium consisted of 50% Minimum Essential Medium with Earle’s salts (Gibco, BRL Life Technologies, Scotland), 25% heat-inactived horse serum (Gibco, Invitrogen Corporation, Carlsbad, CA, USA), 25% HBSS, supplemented with 1 mM L-glutamine (Gibco, Invitrogen Corporation), 20 mM HEPES buffer, 5 mg/ml glucose and 0.1 mg/ml gentamicin (Gibco, BRL Life Technologies, Scotland). The slice cultures were incubated at 37 °C, 5% CO2 for 7– 8 days before treatments were performed. The culture medium was changed three times a week. Seven/eight-day-old interface slices were incubated with TMT dissolved in the culture medium to the concentration of 10 M (Noraberg et al., 1998) for 24 h, 48 h and 48 h followed by 24 h in normal medium without the neurotoxicant (48 h⫹24 h). In order to evaluate the relationship between Cat D and neuronal degeneration, we preincubated (24 h) the cultures with 10 M Cat D inhibitor (Pepstatin A). Pepstatin A was again added (10 M) to the culture medium at the same time as TMT and remained in the culture until fixation. Parallel cultures were treated either with TMT or with Pepstatin A alone. To investigate the “Calpain¡Cathepsin D” hypothesis, the cultures were exposed to 100 M Calpain inhibitor or Calpeptin (Merk-Calbiochem, Darmstadt, Germany) dissolved in dimethyl sulfoxide (Sigma), before (24 h) and during toxic treatment. Parallel cultures were treated either with TMT or with Calpeptin alone. Finally, to investigate whether Cat D overexpression is regulated by intracellular calcium overload, the slices were preincubated for 45 min with the 50 M Ca2⫹ chelator 1,2– bis (2-aminophenoxi)ethane-N-N-N’,N’-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM, Sigma) and then treated with 10 M TMT at different times of incubation, in the presence of BAPTA-AM. Parallel cultures were treated either with TMT or with BAPTA-AM alone.
Immunohistochemical localization of Cathepsin D in vivo and in vitro Under deep anaesthesia, the animals were perfused by a peristaltic pump through the aorta with 250 ml of saline solution, followed by 250 ml of 4% paraformaldehyde (PFA) at a rate of 4 –5 ml/min. About 30 min after perfusion the brains were removed from the skull, post-fixed in 4% PFA overnight at 4 °C, transferred
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in phosphate buffer (PB) 0.1 M pH 7.4. Serial 40 m-thick coronal sections were cut on a vibratome and collected in a free-floating manner. Tissue sections of control and 3, 5, 7, 14, 21-days TMTtreated animals (5 animals/group) were processed for doubleimmunofluorescence labelling. Non-specific binding sites were blocked with 5% normal donkey serum in phosphate buffered saline (PBS) 0.01 M pH 7.4 for 1 h at room temperature (RT). The sections were incubated overnight at ⫹4 °C with goat anti-Cat D (1:250, Santa Cruz Biotecnology, Inc., CA, USA) in combination with different cellular markers: (1) mouse anti-Neu N antibody for neurons (1:500, Chemicon, Temecula, CA, USA), or (2) mouse anti-OX-42 antibody for resting/activated microglia and macrophages (1:1000, Serotec, Kidlington, UK) or (3) rabbit anti-glial acidic fibrillary protein (GFAP) antibody for astrocytes (1:1000, Dako Cytomatetion, Glostrup, Denmark). After incubation in primary antisera, sections were rinsed three times with PBS and then exposed to the appropriate secondary antibodies: Cy3 anti-goat (1:400, Jackson Immunorsearch Laboratories, West Grove, PA, USA), Cy2 antimouse (1:200, Jackson Immunorsearch Laboratories) and Cy3 anti-rabbit (1:400, Jackson Immunorsearch Laboratories) for 1 h at RT. After further washing, sections were mounted on glass slides, coverslipped with gel-mount anti-fading mounting medium (Sigma-Aldrich, St Louis, MO, USA), examined and photographed under a Zeiss LSM 510 confocal laser scanning microscopy system. The specificity of the labelling was confirmed by incubation of sections with the secondary antibodies, omitting the primary antisera. No immunoreactivity was observed under these conditions. Five non consecutive sections were immunostained from each animal/time point. The expression of Cat D was assessed also in hippocampal organotypic cultures after TMT exposure in the presence or absence of Pepstatin A, Calpeptin or BAPTA-AM. After toxic incubation the slices were fixed in 4% PFA overnight and then transferred to 30% sucrose in PB for 72 h at 4 °C. Subsequently, serial 40 m-thick coronal sections were cut by cryostat (Leica CM 1850). For Neu N/Cat D double-immunostaining, the sections were treated as previously described in vivo. A total of 25 non-adjacent sections were stained from five separate cultures. Background staining was not observed in negative control sections.
Cell counts in TMT treated rat hippocampus in vivo and in vitro In order to evaluate further the loss of neuronal cells in vivo, after TMT treatment, we have counted the number of Neu N immunoreactive cells in the CA3 area of the hippocampus. Since reduction of Neu N immunolabelling is regarded as a good biomarker for predicting delayed neuronal degeneration (Collombet et al., 2006), Neu N expression in the CA3 area was evaluated by immunofluorescence analysis. Progressive neurodegeneration was measured by cell counts according to Shirakawa et al. (2007). In particular, to measure cell survival, five regularly spaced (every 100 m) brain sagittal sections of control and TMT treated rats (3, 5, 7, 14 and 21 days, 5 animals/group) were analysed. The number of Neu N-positive hippocampal cells was counted in a randomly chosen microscopic field (0.0063 mm2) in each CA3 region and examined under a fluorescence microscope (Axiophot Zeiss) equipped with AxioVision Rel 4.5 software (Zeiss), with a 20⫻ objective. Only neurons with the nucleus in the focal plane and with confirmation of Neu N antibody immunostaining were considered. In organotypic cultures, cell counts were performed to evaluate the survival of neurons in control and in TMT treated slices as
previously described. In particular, Neu N positive cells were counted within two randomly chosen microscopic fields (each of 0.003 mm2) in each CA3 hippocampal region and were expressed as cells number/mm2 from 25 non-adjacent sections derived from five separate cultures.
Statistical analysis For all results, data are expressed as mean⫾SEM. Statistical analysis for enzymatic activity, Neu N staining in vivo and in vitro was performed with Student’s t-test, assuming the levels of probability: * P⬍0.05, ** P⬍0.001 and ## P⬍0.001 as significant.
RESULTS Neuronal cell death in the CA3 hippocampal region in vivo and in organotypic cultures Neuronal cell loss was examined by Neu N immunostaining of the CA3 region of rat hippocampus and of hippocampal organotypic slices after TMT treatment. In vivo, we observed that the neuronal loss became statistically significant starting from 5 days after treatment and gradually increased until 21 days after administration of the toxicant (Fig. 1). In organotypic cultures (Fig. 2) marked neuronal loss occurred after 48 h and became more pronounced at 48 h⫹24 h of TMT incubation. To evaluate whether Cat D overexpression plays a role in neurodegenerative processes, we exposed hippocampal slices to a Cat D inhibitor (Pepstatin A) before and during the toxic treatment. Pretreatment and treatment with Pepstatin A attenuated cellular damage and markedly reduced neuronal death in the CA3 region of 48 h TMT and 48 h⫹24 h TMT incubated slices, reaching the same vitality rate that we observed in the control cultures. Further, the treatments with Calpain inhibitor (Calpeptin) or calcium chelator BAPTA-AM significantly reduced neuronal death in the CA3 region at 48 h TMT and 48 h⫹24 h TMT of intoxication as evidenced by cell counts.
Fig. 1. Neuronal viability in the hippocampal CA3 area of control and TMT treated rats. The numbers of Neu N-positive cells gradually declined starting from days 5 until days 21 after toxic administration. Each column indicates the mean⫾SEM of 25 sections obtained from five animals, ** P⬍0.001, Student’s t-test. All statistical data are compared with control values.
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Fig. 2. Neuronal viability in the CA3 area of organotypic hippocampal cultures after TMT treatment. Histogram shows the number of surviving neurons (Neu N-positives) in untreated (control) and hippocampal slices treated with 10 M TMT for 24 h, 48 h and 48 h (TMT)⫹24 h (in absence of TMT). The 48 h and 48 h⫹24 h TMT-treated cultures were also incubated with 10 M Pepstatin A (Cat D inhibitor) or 100 M Calpeptin (Calpain inhibitor) or 50 M Ca2⫹ chelator (BAPTA-AM). In TMT-treated cultures neuronal cell loss occurred at 48 h and became more pronounced at 48 h⫹24 h of TMT treatment. In all TMT-treated cultures in the presence of inhibitors or BAPTA-AM cellular viability is similar to that observed in control experiments. Values represent the mean⫾SEM of 25 sections of five separate cultures, ** P⬍0.001, Student’s t-test.
TMT treatment increases in vivo Cathepsin D enzymatic activity Total Cat D enzymatic activity was assayed in hippocampus homogenates of control and TMT-treated rats (Fig. 3). Activity levels of the protease increased significantly and progressively between the 5th and 14th day after TMT treatment. The moderate increase at 5 days was followed by a more pronounced one at 7 days, but the peak value was observed on the 14th day, reaching a value of 312 nmol eq/mg protein/h compared with the control value (201 nmol eq/mg protein/h). The data obtained at 21 days
showed a subsequent decrease in the total activity level (278 nmol eq/mg protein/h), but the values of TMT-treated samples remained significantly higher compared with controls. Lysosomal and cytosolic Cat D enzymatic activities were also assayed in lysosomal and cytosolic fractions of hippocampus homogenates of control and TMTtreated rats (Fig. 3). The values of the lysosomal and cytosolic control activities were respectively 401 and 65 nmol eq/mg protein/h. Both lysosomal and cytosolic activities of the protease increased progressively and sig-
Fig. 3. Cathepsin D enzymatic activity in hippocampus homogenates of control and TMT-treated rats. Total activity of the protease increased significantly between 5 and 14 days of toxic administration; on day 21 after treatment the activity value decreased, although still significantly higher than the control. Both lysosomal and cytosolic activities of the protease increased progressively and significantly between the 5 and 14 days after TMT treatment. The values are the mean⫾SEM and are indicated for each group: the control rats (n⫽18) and 3, 5, 7, 14, 21 days TMT-treated rats (n⫽18/group), * P⬍0.05, ## P⬍0.001, Student’s t-test. Statistical data are compared with control values.
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nificantly between the 5 and 14 days after TMT treatment. In order to confirm that the enzymatic activity was due only to Cat D, we added the specific Cat D inhibitor Pepstatin A to the samples of tissue homogenates. The presence of Pepstatin A inhibited about 90% of the enzymatic activity. In particular, in controls and in rats up to 14 days after TMT treatment total Cat D activity in the presence of Pepstatin A was reduced respectively to 20 and 31 nmol eq/mg protein/h. Cat D immunoreactivity is enhanced by TMT in neurons and in glial cells in vivo In order to visualize the distribution and cellular localization of the Cat D-immunoreactivity in the hippocampus of normal and TMT-treated rats, double-labelling immunofluorescence studies using an antibody to Cat D in combination with a variety of cell-specific markers, Neu N/GFAP/ OX42, were performed.
We observed Cat D co-localization with Neu N in few and scattered CA3 neurons starting from 3 days after TMT intoxication (Fig. 4D–F). At 5 days after TMT treatment, immunofluorescence analysis showed that most hippocampal neurons of the CA3 region were markedly Cat D-reactive (Fig. 4G–I), while only a few labelled neuronal cells were observed in the CA1/CA4 subfields and in the dentate gyrus (data not shown). Parallel double-labelling experiments, Cat D/GFAP and Cat D/OX42, at the same time-point after treatment (5 days), indicated the presence of few labelled astroglial cells (data not shown). Starting from the 7th day of treatment, when neuron loss was increasing and Cat D overexpression became less evident in neurons (Fig. 5A–C), Cat D immunolabelling in astrocytes exhibited a marked and progressive increase, which extended to all hippocampal regions, in particular in CA3 area (Fig. 6B), but not in microglial cells (Fig. 6F). At 14 days the Cat D-immunoreactivity was present only in scattered neurons (Fig. 5D–F) but intensively and predominantly associated with reactive astrocytes (GFAP-positive cells) (Fig. 6C) and, more rarely, with OX-42-positive micro-
Fig. 4. Progressive expression of Cat D in rat hippocampal neurons after TMT treatment (control, 3, 5 days). Sections of the CA3 fields at different TMT treatment time-points are double-labelled for Neu N (green), Cat D (red) and Neu N/Cat D (Merge). Control sections (A–C). After 3 days of TMT administration Cat D immunoreactivity was observed in few and scattered Neu N-positive cells (D–F). After 5 days of treatment a marked and notable increase in the number of Cat D-positive neurons was observed (G–I). The inserts show details. Scale bar: 50 m. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.
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glial cells (Fig. 6G). Finally, we found that the Cat D-staining largely co-localized with activated microglia at 21 days after TMT treatment (Fig. 6H) it was persistent in astrocytes (Fig. 6D) but essentially absent from residual neurons (Fig. 5G–I). These Cat D immunolabelling data are consistent with the specific activity of the enzyme that we assayed at different time points of TMT treatment. All the experiments performed on the control animals evidenced only few and weakly Cat D-immunoreactive neurons (Fig. 4A–C) and glial/microglial cells (Fig. 6A, E) in CA3 area analysed. Increased neuronal Cat D in hippocampal slices cultured in vitro after TMT treatment To confirm further the data regarding neuronal Cat D obtained in vivo, we repeated double-immunolabelling experiments, Cat D/Neu N, on rat hippocampal slices cultured in vitro.
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According to our in vivo findings there was marked and progressive neuronal Cat D-immunoreactivity localized only in the CA3 area, starting from 24 h after TMT treatment (Fig. 7B). Cultures exposed to TMT for 48 h showed that the labelling for neuronal Cat D in the CA3 subfield was the most pronounced and extensive (Fig. 7C) and it began to decrease after 48 h⫹24 h of TMT incubation because of the neuronal loss (Fig. 7D). No significant Cat D labelling was observed in control culture slices (Fig. 7A). Pretreatment with Pepstatin A inhibitor in 48 h TMTincubated hippocampal slices, in which the maximal overexpression of Cat D was observed, markedly decreased neuronal Cat D immunoreactivity (Fig. 7E). Since a calcium-induced Calpain-Cathepsin cascade in brain neuronal death was reported (Yamashima et al., 1998; Yamashima, 2000, 2004), we examined the effects of Calpain inhibitor on Cat D expression in CA3 hippocam-
Fig. 5. Progressive expression of Cat D in rat hippocampal neurons after TMT treatment (7, 14, 21 days). Sections of the CA3 fields at different TMT-treatment time-points are double-labelled for Neu N (green), Cat D (red) and Neu N/Cat D (Merge). After 7 days of TMT administration Cat D immunoreactivity associated with Neu N-positive cells was reduced (A–C) compared with 5 days-treated rats (Fig. 4G–I). After 14 days of intoxication Cat D labelling was only occasional in neurons and the number of Neu N-positive cells was considerably reduced (D–F). After 21 days of TMT treatment Cat D staining was essentially absent from residual neurons (G–I). Note that the number of Neu N positive cells is reduced progressively in TMT-treated rats (A–D–G) compared with control rats (Fig. 4A). The inserts show details. Scale bar: 50 m. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.
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Fig. 6. Merged images of sections double-labelled with GFAP (green)/Cat D (red) and OX-42 (green)/Cat D (red) in CA3 rat hippocampal area after TMT treatment. Control sections (A, E). After 7 days of TMT treatment only some astrocytes (GFAP positives, B) and occasional microglial cells (OX-42 positives, F) were Cat D-immunoreactive. After 14 days of intoxication Cat D staining was associated mainly with reactive astrocytes (C) and, to a lesser extent, with microglia (G). After 21 days of TMT treatment Cat D labelling was persistent in astrocytes (D) but was also largely extended with activated microglia (H). The inserts show details. Scale bar: 50 m. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.
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Fig. 7. Merged images of sections double-labelled with Neu N (green)/Cat D (red) in the CA3 area of rat hippocampal organotypic cultures. Control section (A). After 24 h of TMT incubation Cat D immunoreactivity was expressed in a few scattered Neu N-positive cells (B). After 48 h TMT Cat D staining was extensively and markedly colocalized with neuronal cells (C). After 48 h⫹24 h TMT Cat D immunoreactivity associated with Neu N staining was reduced and the number of Neu N-positive cells was considerably reduced (D). The Cat D immunolabelling pattern observed in 48 h TMT⫹Pepstatin A (E), in 48 h TMT⫹Calpeptin (F) and in 48 h TMT⫹BAPTA-AM (G) sections was markedly reduced. The inserts show details. Scale bar: 50 m. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.
pal neurons of organotypic slices in order to investigate this molecular mechanism in our experimental model. We administered Calpain inhibitor, Calpeptin, to 48 h TMTtreated slices. The Calpeptin treatment markedly and significantly reduced the reactivity of neuronal Cat D, as shown by Cat D/Neu N double immunolabelling images (Fig. 7F). Immunofluorescence analysis showed that these inhibitors are not toxic to the cultures, as shown in the control slices treated with Pepstatin A and Calpeptin alone (data not shown). Finally, since Calpain is known to exert its action in a calcium-dependent manner, in order to determine whether the increase in Cat D observed following treatment with TMT was also calcium-dependent, we incubated hippocampal slices (48 h TMT treated) with the membrane-permeable form of the intracellular Ca2⫹ chelator BAPTA-AM. In the presence of BAPTA-AM, the immunoreactivity for neuronal Cat D in the CA3 area was significantly reduced (Fig. 7F). No histological changes were detected in hippocampi treated with BAPTA-AM alone (data not shown).
DISCUSSION The present results show that Cat D plays a crucial role in neurodegenerative processes triggered by TMT. Both enzymatic activity, which appears, as expected, to be elevated especially in the lysosomal fraction, and cellular expression of the protein are significantly increased after neurotoxic treatment in the rat hippocampus. Interestingly, we demonstrate that Cat D inhibition, obtained either directly through a specific Cat D inhibitor or indirectly through
inhibition of molecules that control the enzyme, significantly reduces neuronal TMT-induced death. It is noteworthy that also CatD expression appears to be reduced under the influence of different molecules controlling the enzyme. In this respect, it is noteworthy that Pepstatin A has already been reported to decrease Cat D expression (Fan et al., 2010). In addition, since TMT activity has been shown to be dependent on intracellular calcium overload (Piacentini et al., 2008), the reduced Cat D immunoreactivity after calcium chelator treatment, or treatment with the inhibitor of calcium-dependent calpain, might be related to the reduced cellular reaction to the neurotoxicant, which is reasonably inactivated concomintantly with the impairment of calcium overload or of calpain inactivation. Cat D upregulation has already been shown in other experimental models of neurodegeneration (Hetman et al., 1997; Moechars et al., 1999; German et al., 2002; Gowran and Campbell, 2008; Wirths et al., 2010). Although apoptotic hallmarks have been shown for cell death mechanisms in TMTinduced degeneration, Cat D upregulation is not surprising. In the last decade, in fact, lysosomal permeabilization, with subsequent release of proteolytic enzymes into the cytosol and their active contribution to the apoptotic signalling pathways, has been described in several models of apoptosis (rev Benes et al., 2008), so that Cat D can effectively be regarded as playing an active role in apoptosis. Notwithstanding this, conclusive data are lacking to clarify whether in the present experimental conditions Cat D participates in an apoptotic pathway or is mainly involved in necrotic and autophagic cell death. In any case, in TMT-
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induced neurodegeneration, Cat D is controlled by Calpain activity and, consequently, by intracellular calcium concentration. This latter aspect is consistent with previous data indicating that TMT-induced cell death is both accompanied by and dependent on intracellular calcium overload. As revealed by double immunolabelling analysis in vivo, Cat D is markedly expressed 5 days after TMT treatment in neuronal populations of the CA3 area, which is a key site of TMT-induced neurodegeneration. Then, when neuron loss is increasing and Cat D overexpression becomes less evident in neurons, starting from the 7th day of treatment, the protease is more diffusely overexpressed in reactive astrocytes throughout the hippocampus, where a marked and progressive increase in Cat D occurs. Cat D expression is associated with the glial cells in accordance with the fact that TMT neurotoxicity in the rat hippocampus is accompanied by characteristic features of reactive gliosis, including increased expression of astrocytes and activation of microglia. Finally, 20 –21 days after treatment, when the neurodegenerative process is almost complete, staining for Cat D is also significantly extended to activated microglia and is still evident in astrocytes. This dynamic cell distribution reasonably reflects cell interactions underlying the progression of TMT-induced neurodegeneration. The possible relationships between neuron and glial Cat D expression, as already noted for other experimental models (Hetman et al., 1995, 1997), as well as the molecular messengers involved in this process constitute a promising field for future research. As far as microglial upregulation is concerned, it should be noted that in vitro Cathepsins B and D released by microglia have been reported to be involved in extracellular proteolysis, thereby participating in neuronal death processes (Kingham and Pocock, 2001; Kim et al., 2007). A similar process might by hypothesized for astroglial-Cathepsin D upregulation. As part of the neuron-glial crosstalk already observed in degenerative neuronal processes, the strict correspondence of neurodegeneration and reactive gliosis suggests that both astroglia and microglia are not only sensitive indicators of injury, but seem also to play an important role in tissue reaction (Gehrmann et al., 1992; Bechmann and Nitsch, 1997; Koczyk and Oderfeld-Nowak, 2000; McTigue et al., 2000). Furthermore, the data presented show that Cat D upregulation in neurons of the CA3 region is also markedly evident at different times of incubation in organotypic cultures exposed to the neurotoxicant, in agreement with data obtained from in vivo experiments. Since the cultures are derived from 3-week-old rats, an age at which maturation of hippocampal architecture and synaptic connectivity/activity reaches adult levels (Pokorný and Yamamoto, 1981), the hippocampal slices represent a convenient model for the study of neurodegeneration and of the molecular processes involved in it.
CONCLUSIONS The present results indicate that Cat D is upregulated after TMT treatment, exhibiting different cell localizations during the degenerative process, and even more importantly, that
Cat D inhibition, obtained directly or indirectly through Calpain inhibition or intracellular calcium chelation, preserves neurons from death. This study provides new information to clarify neurodegenerative processes induced by TMT, significantly implicating Cat D and the molecular mechanisms related to this protease, even offering clues regarding the prevention of neuronal death in this experimental model of neurodegeneration. The data on organotypic hippocampal cultures delineate a scenario leading to cell death accompanied by intracellular calcium overload via Calpain activation which, in turn, induces Cat D upregulation. Our results also offer possibly fertile insight into the specific action of Cat D, especially regarding the interaction among different cell types (neuronal-glial) during the neurodegenerative processes. Acknowledgments—This work was supported by funds from Catholic University, Rome to C.G. and F.M. We would also like to thank Enrico Guadagni and Roberto Passalacqua for their skilful technical support.
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(Accepted 11 November 2010) (Available online 25 November 2010)
CATHEPSIN D PLAYS A CRUCIAL ROLE IN THE TRIMETHYLTININDUCED HIPPOCAMPAL NEURODEGENERATION PROCESS S. CECCARIGLIA,a A. D’ALTOCOLLE,a A. DEL FA’,a F. PIZZOLANTE,a E. CACCIA,b F. MICHETTIa* AND C. GANGITANOa
the hippocampus being a key target of its action. The neurotoxicant induces in the rat hippocampus a selective loss of pyramidal neurons, especially in the CA1/CA3 subfields, together with reactive astrogliosis (Dyer et al., 1982; McCann et al., 1996; Bruccoleri et al., 1998; Koczyk and Oderfeld-Nowak, 2000; Tsutsumi et al., 2002). The pyramidal cells of the CA3 region are particularly sensitive to acute toxicity with single doses of TMT (Ishida et al., 1997; Imai et al., 2001). The action of TMT has been shown to be mediated by disregulation of intracellular Ca2⫹ homeostasis, leading to a marked increase in [Ca2⫹]i in cultured rat hippocampal neurons (Piacentini et al., 2008), in HeLa and neuroblastoma cells (Florea et al., 2005a,b) and also in spiral ganglion cells (Fechter and Liu, 1995). Interestingly, rat hippocampal neurons expressing the Ca2⫹-binding proteins calretinin and parvalbumin are selectively spared by the neuroxicant (Geloso et al., 1996, 1997, 1998; Businaro et al., 2002; Gangitano et al., 2006), possibly through a calcium-buffering effect that counteracts the TMT-induced calcium overload. The molecular mechanisms that mediate TMT-induced toxicity and cellular death are not completely understood. However, numerous studies using different experimental models have demonstrated that TMT induces apoptosis via a series of mediators, such as protein kinase C activation, oxidative stress due to the generation of reactive oxygen species, over-expression of stannin, cyclooxygenase metabolism and caspase activation (Toggas et al., 1992; Kane et al., 1998; Fiedorowicz et al., 2001; Gunasekar et al., 2001a,b; Geloso et al., 2002; Jenkins and Barone, 2004; Buck-Koehntop et al., 2005; Mundy and Freudenrich, 2006; Nagashima et al., 2008). A substantial body of research indicates that the lysosomal system, especially lysosomal proteases such as the cathepsins, seems to be involved in the early stages of several neurodegenerative processes that occur in different conditions, such as transient forebrain ischaemia, kainate-evoked brain damage, age-related neurodegeneration and Alzheimer’s disease (Cataldo and Nixon, 1990; Diedrich et al., 1991; Nakanishi et al., 1993, 1995; Cataldo et al., 1995; Hetman et al., 1995; Nixon and Cataldo, 1995; Sato et al., 2006). Cat D is one of the main lysosomal aspartic proteases widely distributed throughout the brain (Whitaker et al., 1981; Banay-Schwartz et al., 1992). Although the major function of Cat D is related to intracellular catabolism in lysosomal compartments (Dean, 1975) this protease seems also to play an important role in the processes that regulate apoptosis (Deiss et al., 1996; Shibata et al., 1998; Bidère et al., 2003; Fusek and Vetvicka, 2005; Liaudet-
a Institute of Anatomy and Cell Biology, Catholic University, L. go F. Vito 1, 00168 Rome, Italy b Department of Environmental Sciences, Tuscia University, L. go Dell= Università, 01100 Viterbo, Italy
Abstract—Trimethyltin chloride (TMT) is known to produce neuronal damage in the rat hippocampus, especially in the CA1/CA3 subfields, together with reactive astrogliosis. Previous studies indicate that in cultured rat hippocampal neurons the Ca2ⴙ cytosolic increase induced by TMT is correlated with apoptotic cell death, although some molecular aspects of the hippocampal neurodegeneration induced by this neurotoxicant still remain to be clarified. Cathepsin D (Cat D) is a lysosomal aspartic protease involved in some neurodegenerative processes and also seems to play an important role in the processes that regulate apoptosis. We investigated the specific activity and cellular expression of Cat D in the rat hippocampus in vivo and in cultured organotypic rat hippocampal slices. The role of Cat D in cell death processes and the mechanisms controlling Cat D were also investigated. Cat D activity was assayed in hippocampus homogenates of control and TMT-treated rats. In order to visualize the distribution of Cat D immunoreactivity in the hippocampus, double-label immunofluorescence for Cat D and Neu N, GFAP, OX42 was performed. In addition, in order to clarify the possible relationship between Cat D activity, neuronal calcium overload and neuronal death processes, organotypic hippocampal cultures were also treated with a Cat D inhibitor (Pepstatin A) or Calpain inhibitor (Calpeptin) or an intracellular Ca2ⴙ chelator (BAPTA-AM) in the presence of TMT. TMT treatment in rat hippocampus induced high levels of Cat D activity both in vivo and in vitro, in glial cells and in CA3 neurons, where a marked TMT-induced neuronal loss also occurred. Cat D is actively involved in CA3 neuronal death and the protease increase is a calcium-Calpain dependent phenomenon. © 2011 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: trimethyltin, Cathepsin D, neurodegeneration, hippocampus, organotypic culture.
Trimethyltin (TMT) is known to produce significant and selective neuronal degeneration in the limbic system, with *Corresponding author. Tel: ⫹39-06-30155848; fax: ⫹39-06-30155753. E-mail address: [email protected] (F. Michetti). Abbreviations: BAPTA-AM, (2-aminophenoxi)ethane-N-N-N’,N’-tetraacetic acid tetrakis(acetoxymethyl) ester; Cat D, Cathepsin D; GFAP, glial acidic fibrillary protein; HBSS, Hank’s balanced salt solution; HEPES, 4-2-hydroxyethyl-1-piperazineethanesulfonic acid; i.p., intraperitoneal; PB, phosphate buffer; PBS, phosphate-buffered saline; PFA, paraformaldehyde; RT, room temperature; TMT, trimethyltin.
0306-4522/11 $ - see front matter © 2011 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2010.11.024
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Coopman et al., 2006; Minarowska et al., 2007; ZuzarteLuis et al., 2007a,b). To date, the role of cathepsins in TMT-neurotoxicity has not been studied, while the initiating events in the neurodegeneration induced by the neurotoxicant still remain to be clarified. In this study, we investigated the specific activity, distribution and cellular localization of Cat D in the rat hippocampus in vivo after TMT intoxication. We also used organotypic hippocampal cultures in order to clarify the role and possible relationship between Cat D, neuronal calcium overload and neuronal death processes.
EXPERIMENTAL PROCEDURES Animals and treatment Experiments were performed using adult female Wistar rats (200 – 250 g body weight, age 2 months). Animals were housed in an air-conditioned environment with constant temperature and a standardized light/dark schedule, food and water being supplied ad libitum. The rats were treated with a single intraperitoneal (i.p.) injection of TMT (Sigma, St Louis, MO, USA) dissolved in saline at a dose of 8 mg/kg body weight in a volume of 1 ml/kg body weight. Control rats received the same volume of saline. The animals were anaesthetized with 2 mg/100g i.p. diazepam (Biologici Italia Laboratories, Milano) followed by i.m. injection of 4 mg/100g ketamine (Intervet International, GmbH, Germany). The rats were sacrificed at 3, 5, 7, 14 and 21 days after treatment. All the animal protocols were approved by the Animal Experimentation Committee of Catholic University, Rome. In particular, animal experiments were performed in accordance with the European Community Council Directive of 24 November 1986 (86/609/EEC). All efforts were made to minimize the number of animals used and their suffering.
Cathepsin D enzymatic activity Total Cat D enzymatic activity was assayed at 3, 5, 7, 14, 21 days post-TMT intoxication in the rat hippocampi (10 rats/time point). Enzymatic activity of Cat D was also measured in hippocampi of control rats (n⫽10). Animals were killed by decapitation, the brains were removed and the entire hippocampi dissected. The tissues were homogenized in five volumes of 0.05 M citrate buffer, pH 3.2, containing 0.5% Triton X-100 with a glass homogenizer (Bracco et al., 1982); the samples were centrifuged at 25,000 g for 30 min at 4 °C. The supernatant was removed and aliquots were used for enzymatic assay. We also performed parallel experiments (9 rats/time point) to assay specific activity of Cat D in lysosomal and cytosolic fractions obtained according to the modified method of Banay-Schwartz et al. (1987). Briefly, the hippocampi of control and TMT-treated rats were homogenized in 0.01 M phosphate buffer pH 7.4 containing 0.3 M sucrose and centrifuged at 1000 g for 10 min at 4 °C. After discarding the pellet, the supernatant was centrifuged at 25,000 g for 45 min at 4 °C. The supernatant was then collected as cytosolic fraction and the pellet (crude lysosomal fraction) was re-suspended in the same buffer previously used with 0.5% Triton X-100, re-centrifuged at 25,000 g for 45 min at 4 °C, and the resulting supernatant was collected as enriched lysosomal fraction. Both lysosomal and cytosolic fractions were used to measure the Cat D specific activities in the corresponding cellular compartments. Cat D enzymatic activities were assayed using the method described by Barrett (1977). Briefly, lysates from hippocampus tissue were incubated with 0.5 M citrate buffer, pH 3.2 and 3% specific haemoglobin substrate (Sigma) for 1 h at 45 °C. Using a modified alkaline copper reagent, the haemoglobin breakdown
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was measured as tyrosine equivalent formed, read at 700 nm using the Beckman DU®-70 spectrophotometer. Enzyme activity was expressed as nmol/mg protein/h of tyrosine equivalent formed. In all cases about 90% of Cat D activity was inhibited by 12 g/ml Pepstatin A (Sigma). Protein quantification of the tissue homogenates was performed according to the method of Lowry et al. (1951), using bovine serum albumin as standard.
Organotypic slice cultures Hippocampal organotypic cultures from neonatal rats were prepared following the method of Stoppini et al. (1991). Briefly, female Wistar rats (20 –21 days old) were anaesthetized with 100 g/g ketamine and decapitated. The brains were removed and placed in an ice-cold dissection medium consisting of Hank’s balanced salt solution (HBSS, Gibco, BRL Life Technologies, Scotland) with 25 mM 4-2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES, Gibco) and 6% glucose, pH 7.2. The brains were then hemisected along the longitudinal fissure. From each hemisection, 400 m thick serial slices were cut transversely using a vibratome (Leica VT 1000S). Once cut, individual slices were immediately removed from the vibratome bath with a fire-polished Pasteur pipette and four to five slices were transferred onto each Millicell-CM (Millipore, Bedford, MA, USA) membrane insert which was placed in a six-well plate (Costar, Corning, NY, USA) containing 1 ml culture medium/well. The tissue surrounding the hippocampus was removed under a dissection microscope (Leitz Wetzlar Germany). The culture medium consisted of 50% Minimum Essential Medium with Earle’s salts (Gibco, BRL Life Technologies, Scotland), 25% heat-inactived horse serum (Gibco, Invitrogen Corporation, Carlsbad, CA, USA), 25% HBSS, supplemented with 1 mM L-glutamine (Gibco, Invitrogen Corporation), 20 mM HEPES buffer, 5 mg/ml glucose and 0.1 mg/ml gentamicin (Gibco, BRL Life Technologies, Scotland). The slice cultures were incubated at 37 °C, 5% CO2 for 7– 8 days before treatments were performed. The culture medium was changed three times a week. Seven/eight-day-old interface slices were incubated with TMT dissolved in the culture medium to the concentration of 10 M (Noraberg et al., 1998) for 24 h, 48 h and 48 h followed by 24 h in normal medium without the neurotoxicant (48 h⫹24 h). In order to evaluate the relationship between Cat D and neuronal degeneration, we preincubated (24 h) the cultures with 10 M Cat D inhibitor (Pepstatin A). Pepstatin A was again added (10 M) to the culture medium at the same time as TMT and remained in the culture until fixation. Parallel cultures were treated either with TMT or with Pepstatin A alone. To investigate the “Calpain¡Cathepsin D” hypothesis, the cultures were exposed to 100 M Calpain inhibitor or Calpeptin (Merk-Calbiochem, Darmstadt, Germany) dissolved in dimethyl sulfoxide (Sigma), before (24 h) and during toxic treatment. Parallel cultures were treated either with TMT or with Calpeptin alone. Finally, to investigate whether Cat D overexpression is regulated by intracellular calcium overload, the slices were preincubated for 45 min with the 50 M Ca2⫹ chelator 1,2– bis (2-aminophenoxi)ethane-N-N-N’,N’-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM, Sigma) and then treated with 10 M TMT at different times of incubation, in the presence of BAPTA-AM. Parallel cultures were treated either with TMT or with BAPTA-AM alone.
Immunohistochemical localization of Cathepsin D in vivo and in vitro Under deep anaesthesia, the animals were perfused by a peristaltic pump through the aorta with 250 ml of saline solution, followed by 250 ml of 4% paraformaldehyde (PFA) at a rate of 4 –5 ml/min. About 30 min after perfusion the brains were removed from the skull, post-fixed in 4% PFA overnight at 4 °C, transferred
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in phosphate buffer (PB) 0.1 M pH 7.4. Serial 40 m-thick coronal sections were cut on a vibratome and collected in a free-floating manner. Tissue sections of control and 3, 5, 7, 14, 21-days TMTtreated animals (5 animals/group) were processed for doubleimmunofluorescence labelling. Non-specific binding sites were blocked with 5% normal donkey serum in phosphate buffered saline (PBS) 0.01 M pH 7.4 for 1 h at room temperature (RT). The sections were incubated overnight at ⫹4 °C with goat anti-Cat D (1:250, Santa Cruz Biotecnology, Inc., CA, USA) in combination with different cellular markers: (1) mouse anti-Neu N antibody for neurons (1:500, Chemicon, Temecula, CA, USA), or (2) mouse anti-OX-42 antibody for resting/activated microglia and macrophages (1:1000, Serotec, Kidlington, UK) or (3) rabbit anti-glial acidic fibrillary protein (GFAP) antibody for astrocytes (1:1000, Dako Cytomatetion, Glostrup, Denmark). After incubation in primary antisera, sections were rinsed three times with PBS and then exposed to the appropriate secondary antibodies: Cy3 anti-goat (1:400, Jackson Immunorsearch Laboratories, West Grove, PA, USA), Cy2 antimouse (1:200, Jackson Immunorsearch Laboratories) and Cy3 anti-rabbit (1:400, Jackson Immunorsearch Laboratories) for 1 h at RT. After further washing, sections were mounted on glass slides, coverslipped with gel-mount anti-fading mounting medium (Sigma-Aldrich, St Louis, MO, USA), examined and photographed under a Zeiss LSM 510 confocal laser scanning microscopy system. The specificity of the labelling was confirmed by incubation of sections with the secondary antibodies, omitting the primary antisera. No immunoreactivity was observed under these conditions. Five non consecutive sections were immunostained from each animal/time point. The expression of Cat D was assessed also in hippocampal organotypic cultures after TMT exposure in the presence or absence of Pepstatin A, Calpeptin or BAPTA-AM. After toxic incubation the slices were fixed in 4% PFA overnight and then transferred to 30% sucrose in PB for 72 h at 4 °C. Subsequently, serial 40 m-thick coronal sections were cut by cryostat (Leica CM 1850). For Neu N/Cat D double-immunostaining, the sections were treated as previously described in vivo. A total of 25 non-adjacent sections were stained from five separate cultures. Background staining was not observed in negative control sections.
Cell counts in TMT treated rat hippocampus in vivo and in vitro In order to evaluate further the loss of neuronal cells in vivo, after TMT treatment, we have counted the number of Neu N immunoreactive cells in the CA3 area of the hippocampus. Since reduction of Neu N immunolabelling is regarded as a good biomarker for predicting delayed neuronal degeneration (Collombet et al., 2006), Neu N expression in the CA3 area was evaluated by immunofluorescence analysis. Progressive neurodegeneration was measured by cell counts according to Shirakawa et al. (2007). In particular, to measure cell survival, five regularly spaced (every 100 m) brain sagittal sections of control and TMT treated rats (3, 5, 7, 14 and 21 days, 5 animals/group) were analysed. The number of Neu N-positive hippocampal cells was counted in a randomly chosen microscopic field (0.0063 mm2) in each CA3 region and examined under a fluorescence microscope (Axiophot Zeiss) equipped with AxioVision Rel 4.5 software (Zeiss), with a 20⫻ objective. Only neurons with the nucleus in the focal plane and with confirmation of Neu N antibody immunostaining were considered. In organotypic cultures, cell counts were performed to evaluate the survival of neurons in control and in TMT treated slices as
previously described. In particular, Neu N positive cells were counted within two randomly chosen microscopic fields (each of 0.003 mm2) in each CA3 hippocampal region and were expressed as cells number/mm2 from 25 non-adjacent sections derived from five separate cultures.
Statistical analysis For all results, data are expressed as mean⫾SEM. Statistical analysis for enzymatic activity, Neu N staining in vivo and in vitro was performed with Student’s t-test, assuming the levels of probability: * P⬍0.05, ** P⬍0.001 and ## P⬍0.001 as significant.
RESULTS Neuronal cell death in the CA3 hippocampal region in vivo and in organotypic cultures Neuronal cell loss was examined by Neu N immunostaining of the CA3 region of rat hippocampus and of hippocampal organotypic slices after TMT treatment. In vivo, we observed that the neuronal loss became statistically significant starting from 5 days after treatment and gradually increased until 21 days after administration of the toxicant (Fig. 1). In organotypic cultures (Fig. 2) marked neuronal loss occurred after 48 h and became more pronounced at 48 h⫹24 h of TMT incubation. To evaluate whether Cat D overexpression plays a role in neurodegenerative processes, we exposed hippocampal slices to a Cat D inhibitor (Pepstatin A) before and during the toxic treatment. Pretreatment and treatment with Pepstatin A attenuated cellular damage and markedly reduced neuronal death in the CA3 region of 48 h TMT and 48 h⫹24 h TMT incubated slices, reaching the same vitality rate that we observed in the control cultures. Further, the treatments with Calpain inhibitor (Calpeptin) or calcium chelator BAPTA-AM significantly reduced neuronal death in the CA3 region at 48 h TMT and 48 h⫹24 h TMT of intoxication as evidenced by cell counts.
Fig. 1. Neuronal viability in the hippocampal CA3 area of control and TMT treated rats. The numbers of Neu N-positive cells gradually declined starting from days 5 until days 21 after toxic administration. Each column indicates the mean⫾SEM of 25 sections obtained from five animals, ** P⬍0.001, Student’s t-test. All statistical data are compared with control values.
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Fig. 2. Neuronal viability in the CA3 area of organotypic hippocampal cultures after TMT treatment. Histogram shows the number of surviving neurons (Neu N-positives) in untreated (control) and hippocampal slices treated with 10 M TMT for 24 h, 48 h and 48 h (TMT)⫹24 h (in absence of TMT). The 48 h and 48 h⫹24 h TMT-treated cultures were also incubated with 10 M Pepstatin A (Cat D inhibitor) or 100 M Calpeptin (Calpain inhibitor) or 50 M Ca2⫹ chelator (BAPTA-AM). In TMT-treated cultures neuronal cell loss occurred at 48 h and became more pronounced at 48 h⫹24 h of TMT treatment. In all TMT-treated cultures in the presence of inhibitors or BAPTA-AM cellular viability is similar to that observed in control experiments. Values represent the mean⫾SEM of 25 sections of five separate cultures, ** P⬍0.001, Student’s t-test.
TMT treatment increases in vivo Cathepsin D enzymatic activity Total Cat D enzymatic activity was assayed in hippocampus homogenates of control and TMT-treated rats (Fig. 3). Activity levels of the protease increased significantly and progressively between the 5th and 14th day after TMT treatment. The moderate increase at 5 days was followed by a more pronounced one at 7 days, but the peak value was observed on the 14th day, reaching a value of 312 nmol eq/mg protein/h compared with the control value (201 nmol eq/mg protein/h). The data obtained at 21 days
showed a subsequent decrease in the total activity level (278 nmol eq/mg protein/h), but the values of TMT-treated samples remained significantly higher compared with controls. Lysosomal and cytosolic Cat D enzymatic activities were also assayed in lysosomal and cytosolic fractions of hippocampus homogenates of control and TMTtreated rats (Fig. 3). The values of the lysosomal and cytosolic control activities were respectively 401 and 65 nmol eq/mg protein/h. Both lysosomal and cytosolic activities of the protease increased progressively and sig-
Fig. 3. Cathepsin D enzymatic activity in hippocampus homogenates of control and TMT-treated rats. Total activity of the protease increased significantly between 5 and 14 days of toxic administration; on day 21 after treatment the activity value decreased, although still significantly higher than the control. Both lysosomal and cytosolic activities of the protease increased progressively and significantly between the 5 and 14 days after TMT treatment. The values are the mean⫾SEM and are indicated for each group: the control rats (n⫽18) and 3, 5, 7, 14, 21 days TMT-treated rats (n⫽18/group), * P⬍0.05, ## P⬍0.001, Student’s t-test. Statistical data are compared with control values.
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nificantly between the 5 and 14 days after TMT treatment. In order to confirm that the enzymatic activity was due only to Cat D, we added the specific Cat D inhibitor Pepstatin A to the samples of tissue homogenates. The presence of Pepstatin A inhibited about 90% of the enzymatic activity. In particular, in controls and in rats up to 14 days after TMT treatment total Cat D activity in the presence of Pepstatin A was reduced respectively to 20 and 31 nmol eq/mg protein/h. Cat D immunoreactivity is enhanced by TMT in neurons and in glial cells in vivo In order to visualize the distribution and cellular localization of the Cat D-immunoreactivity in the hippocampus of normal and TMT-treated rats, double-labelling immunofluorescence studies using an antibody to Cat D in combination with a variety of cell-specific markers, Neu N/GFAP/ OX42, were performed.
We observed Cat D co-localization with Neu N in few and scattered CA3 neurons starting from 3 days after TMT intoxication (Fig. 4D–F). At 5 days after TMT treatment, immunofluorescence analysis showed that most hippocampal neurons of the CA3 region were markedly Cat D-reactive (Fig. 4G–I), while only a few labelled neuronal cells were observed in the CA1/CA4 subfields and in the dentate gyrus (data not shown). Parallel double-labelling experiments, Cat D/GFAP and Cat D/OX42, at the same time-point after treatment (5 days), indicated the presence of few labelled astroglial cells (data not shown). Starting from the 7th day of treatment, when neuron loss was increasing and Cat D overexpression became less evident in neurons (Fig. 5A–C), Cat D immunolabelling in astrocytes exhibited a marked and progressive increase, which extended to all hippocampal regions, in particular in CA3 area (Fig. 6B), but not in microglial cells (Fig. 6F). At 14 days the Cat D-immunoreactivity was present only in scattered neurons (Fig. 5D–F) but intensively and predominantly associated with reactive astrocytes (GFAP-positive cells) (Fig. 6C) and, more rarely, with OX-42-positive micro-
Fig. 4. Progressive expression of Cat D in rat hippocampal neurons after TMT treatment (control, 3, 5 days). Sections of the CA3 fields at different TMT treatment time-points are double-labelled for Neu N (green), Cat D (red) and Neu N/Cat D (Merge). Control sections (A–C). After 3 days of TMT administration Cat D immunoreactivity was observed in few and scattered Neu N-positive cells (D–F). After 5 days of treatment a marked and notable increase in the number of Cat D-positive neurons was observed (G–I). The inserts show details. Scale bar: 50 m. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.
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glial cells (Fig. 6G). Finally, we found that the Cat D-staining largely co-localized with activated microglia at 21 days after TMT treatment (Fig. 6H) it was persistent in astrocytes (Fig. 6D) but essentially absent from residual neurons (Fig. 5G–I). These Cat D immunolabelling data are consistent with the specific activity of the enzyme that we assayed at different time points of TMT treatment. All the experiments performed on the control animals evidenced only few and weakly Cat D-immunoreactive neurons (Fig. 4A–C) and glial/microglial cells (Fig. 6A, E) in CA3 area analysed. Increased neuronal Cat D in hippocampal slices cultured in vitro after TMT treatment To confirm further the data regarding neuronal Cat D obtained in vivo, we repeated double-immunolabelling experiments, Cat D/Neu N, on rat hippocampal slices cultured in vitro.
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According to our in vivo findings there was marked and progressive neuronal Cat D-immunoreactivity localized only in the CA3 area, starting from 24 h after TMT treatment (Fig. 7B). Cultures exposed to TMT for 48 h showed that the labelling for neuronal Cat D in the CA3 subfield was the most pronounced and extensive (Fig. 7C) and it began to decrease after 48 h⫹24 h of TMT incubation because of the neuronal loss (Fig. 7D). No significant Cat D labelling was observed in control culture slices (Fig. 7A). Pretreatment with Pepstatin A inhibitor in 48 h TMTincubated hippocampal slices, in which the maximal overexpression of Cat D was observed, markedly decreased neuronal Cat D immunoreactivity (Fig. 7E). Since a calcium-induced Calpain-Cathepsin cascade in brain neuronal death was reported (Yamashima et al., 1998; Yamashima, 2000, 2004), we examined the effects of Calpain inhibitor on Cat D expression in CA3 hippocam-
Fig. 5. Progressive expression of Cat D in rat hippocampal neurons after TMT treatment (7, 14, 21 days). Sections of the CA3 fields at different TMT-treatment time-points are double-labelled for Neu N (green), Cat D (red) and Neu N/Cat D (Merge). After 7 days of TMT administration Cat D immunoreactivity associated with Neu N-positive cells was reduced (A–C) compared with 5 days-treated rats (Fig. 4G–I). After 14 days of intoxication Cat D labelling was only occasional in neurons and the number of Neu N-positive cells was considerably reduced (D–F). After 21 days of TMT treatment Cat D staining was essentially absent from residual neurons (G–I). Note that the number of Neu N positive cells is reduced progressively in TMT-treated rats (A–D–G) compared with control rats (Fig. 4A). The inserts show details. Scale bar: 50 m. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.
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Fig. 6. Merged images of sections double-labelled with GFAP (green)/Cat D (red) and OX-42 (green)/Cat D (red) in CA3 rat hippocampal area after TMT treatment. Control sections (A, E). After 7 days of TMT treatment only some astrocytes (GFAP positives, B) and occasional microglial cells (OX-42 positives, F) were Cat D-immunoreactive. After 14 days of intoxication Cat D staining was associated mainly with reactive astrocytes (C) and, to a lesser extent, with microglia (G). After 21 days of TMT treatment Cat D labelling was persistent in astrocytes (D) but was also largely extended with activated microglia (H). The inserts show details. Scale bar: 50 m. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.
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Fig. 7. Merged images of sections double-labelled with Neu N (green)/Cat D (red) in the CA3 area of rat hippocampal organotypic cultures. Control section (A). After 24 h of TMT incubation Cat D immunoreactivity was expressed in a few scattered Neu N-positive cells (B). After 48 h TMT Cat D staining was extensively and markedly colocalized with neuronal cells (C). After 48 h⫹24 h TMT Cat D immunoreactivity associated with Neu N staining was reduced and the number of Neu N-positive cells was considerably reduced (D). The Cat D immunolabelling pattern observed in 48 h TMT⫹Pepstatin A (E), in 48 h TMT⫹Calpeptin (F) and in 48 h TMT⫹BAPTA-AM (G) sections was markedly reduced. The inserts show details. Scale bar: 50 m. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.
pal neurons of organotypic slices in order to investigate this molecular mechanism in our experimental model. We administered Calpain inhibitor, Calpeptin, to 48 h TMTtreated slices. The Calpeptin treatment markedly and significantly reduced the reactivity of neuronal Cat D, as shown by Cat D/Neu N double immunolabelling images (Fig. 7F). Immunofluorescence analysis showed that these inhibitors are not toxic to the cultures, as shown in the control slices treated with Pepstatin A and Calpeptin alone (data not shown). Finally, since Calpain is known to exert its action in a calcium-dependent manner, in order to determine whether the increase in Cat D observed following treatment with TMT was also calcium-dependent, we incubated hippocampal slices (48 h TMT treated) with the membrane-permeable form of the intracellular Ca2⫹ chelator BAPTA-AM. In the presence of BAPTA-AM, the immunoreactivity for neuronal Cat D in the CA3 area was significantly reduced (Fig. 7F). No histological changes were detected in hippocampi treated with BAPTA-AM alone (data not shown).
DISCUSSION The present results show that Cat D plays a crucial role in neurodegenerative processes triggered by TMT. Both enzymatic activity, which appears, as expected, to be elevated especially in the lysosomal fraction, and cellular expression of the protein are significantly increased after neurotoxic treatment in the rat hippocampus. Interestingly, we demonstrate that Cat D inhibition, obtained either directly through a specific Cat D inhibitor or indirectly through
inhibition of molecules that control the enzyme, significantly reduces neuronal TMT-induced death. It is noteworthy that also CatD expression appears to be reduced under the influence of different molecules controlling the enzyme. In this respect, it is noteworthy that Pepstatin A has already been reported to decrease Cat D expression (Fan et al., 2010). In addition, since TMT activity has been shown to be dependent on intracellular calcium overload (Piacentini et al., 2008), the reduced Cat D immunoreactivity after calcium chelator treatment, or treatment with the inhibitor of calcium-dependent calpain, might be related to the reduced cellular reaction to the neurotoxicant, which is reasonably inactivated concomintantly with the impairment of calcium overload or of calpain inactivation. Cat D upregulation has already been shown in other experimental models of neurodegeneration (Hetman et al., 1997; Moechars et al., 1999; German et al., 2002; Gowran and Campbell, 2008; Wirths et al., 2010). Although apoptotic hallmarks have been shown for cell death mechanisms in TMTinduced degeneration, Cat D upregulation is not surprising. In the last decade, in fact, lysosomal permeabilization, with subsequent release of proteolytic enzymes into the cytosol and their active contribution to the apoptotic signalling pathways, has been described in several models of apoptosis (rev Benes et al., 2008), so that Cat D can effectively be regarded as playing an active role in apoptosis. Notwithstanding this, conclusive data are lacking to clarify whether in the present experimental conditions Cat D participates in an apoptotic pathway or is mainly involved in necrotic and autophagic cell death. In any case, in TMT-
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induced neurodegeneration, Cat D is controlled by Calpain activity and, consequently, by intracellular calcium concentration. This latter aspect is consistent with previous data indicating that TMT-induced cell death is both accompanied by and dependent on intracellular calcium overload. As revealed by double immunolabelling analysis in vivo, Cat D is markedly expressed 5 days after TMT treatment in neuronal populations of the CA3 area, which is a key site of TMT-induced neurodegeneration. Then, when neuron loss is increasing and Cat D overexpression becomes less evident in neurons, starting from the 7th day of treatment, the protease is more diffusely overexpressed in reactive astrocytes throughout the hippocampus, where a marked and progressive increase in Cat D occurs. Cat D expression is associated with the glial cells in accordance with the fact that TMT neurotoxicity in the rat hippocampus is accompanied by characteristic features of reactive gliosis, including increased expression of astrocytes and activation of microglia. Finally, 20 –21 days after treatment, when the neurodegenerative process is almost complete, staining for Cat D is also significantly extended to activated microglia and is still evident in astrocytes. This dynamic cell distribution reasonably reflects cell interactions underlying the progression of TMT-induced neurodegeneration. The possible relationships between neuron and glial Cat D expression, as already noted for other experimental models (Hetman et al., 1995, 1997), as well as the molecular messengers involved in this process constitute a promising field for future research. As far as microglial upregulation is concerned, it should be noted that in vitro Cathepsins B and D released by microglia have been reported to be involved in extracellular proteolysis, thereby participating in neuronal death processes (Kingham and Pocock, 2001; Kim et al., 2007). A similar process might by hypothesized for astroglial-Cathepsin D upregulation. As part of the neuron-glial crosstalk already observed in degenerative neuronal processes, the strict correspondence of neurodegeneration and reactive gliosis suggests that both astroglia and microglia are not only sensitive indicators of injury, but seem also to play an important role in tissue reaction (Gehrmann et al., 1992; Bechmann and Nitsch, 1997; Koczyk and Oderfeld-Nowak, 2000; McTigue et al., 2000). Furthermore, the data presented show that Cat D upregulation in neurons of the CA3 region is also markedly evident at different times of incubation in organotypic cultures exposed to the neurotoxicant, in agreement with data obtained from in vivo experiments. Since the cultures are derived from 3-week-old rats, an age at which maturation of hippocampal architecture and synaptic connectivity/activity reaches adult levels (Pokorný and Yamamoto, 1981), the hippocampal slices represent a convenient model for the study of neurodegeneration and of the molecular processes involved in it.
CONCLUSIONS The present results indicate that Cat D is upregulated after TMT treatment, exhibiting different cell localizations during the degenerative process, and even more importantly, that
Cat D inhibition, obtained directly or indirectly through Calpain inhibition or intracellular calcium chelation, preserves neurons from death. This study provides new information to clarify neurodegenerative processes induced by TMT, significantly implicating Cat D and the molecular mechanisms related to this protease, even offering clues regarding the prevention of neuronal death in this experimental model of neurodegeneration. The data on organotypic hippocampal cultures delineate a scenario leading to cell death accompanied by intracellular calcium overload via Calpain activation which, in turn, induces Cat D upregulation. Our results also offer possibly fertile insight into the specific action of Cat D, especially regarding the interaction among different cell types (neuronal-glial) during the neurodegenerative processes. Acknowledgments—This work was supported by funds from Catholic University, Rome to C.G. and F.M. We would also like to thank Enrico Guadagni and Roberto Passalacqua for their skilful technical support.
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(Accepted 11 November 2010) (Available online 25 November 2010)