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Short communication
Region-specific attenuation of a trypsin-like protease in substantia
nigra following dopaminergic neurotoxicity by
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
b a a b
Rajamma Usha , Dhanasekharan Muralikrishnan , Bobby Thomas , Sagarmoy Ghosh ,
b a ,
*
Chhabinath Mandal , Kochupurackal P. Mohanakumar
a
Laboratory of Neurochemistry, Division of Pharmacology and Experimental Therapeutics, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Calcutta 700 032, India
b
Protein Design and Engineering Division, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Calcutta 700 032, India
Accepted 2 August 2000
Abstract
We analysed apoptosis, caspase-1 and -3, and trypsin-like protease activity in the nigrostriatal pathway during 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity. MPTP injected (30 mg / kg, i.p., twice, 16 h apart) mice were sacrificed on 1, 2 and 7 days. DNA extracted from nucleus caudatus putamen (NCP) and substantia nigra (SN) was subjected to agarose gel electrophoresis. Typical apoptotic-like DNA cleavage was absent in SN or NCP after this dose of MPTP. A trypsin-like protease activity was significantly decreased in SN and not in NCP. While caspase-3 activity in the whole brain was increased significantly, caspase-1 activity was unaffected. Striatal dopamine content was decreased to 75% by 7 days. The absence of typical DNA ‘ladder’ when there was severe striatal dopamine depletion suggests that in vivo MPTP-mediated dopaminergic neurotoxicity may not involve apoptotic cell death, and explains why in mice MPTP-induced dopamine depletion is transient. The region-specific decrease in trypsin-like protease activity and absence of caspase-3 activation in SN signify the importance of trypsin-like protease in the regulation of apoptosis in MPTP-neurotoxicity in mice. 2000 Elsevier Science B.V. All rights reserved.
Theme: Disorders of the nervous system
Topic: Neurotoxicity
Keywords: Experimental parkinsonism; Apoptosis; Caspase-1; Caspase-3; Dopamine depletion; Trypsin inhibition; Striatum
The potent neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetra- metabolite of MPTP, 1-methyl-4-phenylpyridinium
1
hydropyridine (MPTP), is known to selectively damage (MPP ), is shown to induce apoptosis in vitro in cerebellar nigrostriatal dopaminergic neurons, and it is now exten- granule cells [5,6,13], mesencephalic-striatal co-culture sively used to investigate pathophysiology and pathogene- [16] and in PC12 cells [9]. MPTP administration in mice sis of Parkinson’s disease. The biochemical and molecular too has been shown to cause apoptotic cell death in mechanisms that are activated to result in selective destruc- substantia nigra (SN) [23].
tion of the dopaminergic neurons are mostly unknown. Caspases 1–10, members of interleukin-1b-converting One of the mechanisms that is proposed is apoptotic-like enzyme (ICE)-like proteases are now shown to have an cell death. Though, apoptosis is a process of physiological active role in the apoptotic cell death, including neurons cell death, non-physiological events too can trigger apop- [24]. An involvement of caspase-3-like cysteine protease
1
tosis as in stroke, ischemia, free radical-induced neuro- in MPP -mediated apoptosis in cerebellar granule cells has toxicity, neurological disorders [2,7,12,25], etc. The active been demonstrated recently [6]. Interestingly, inhibitors of trypsin-like serine proteases are shown to protect PC12 cells and sympathetic neurons from apoptotic-like cell
*Corresponding author. Tel.:191-33-473-3491 / 473-6793 (ext. 213);
death [21]. Regulation of ICE proteases activation,
proba-fax:191-33-473-5197 / 473-0284.
E-mail address: [email protected] (K.P. Mohanakumar). bly, is the key step in apoptosis. Interestingly, inhibitors of
several kinds of proteases have been shown to inhibit bated with the substrate (0.1 mM) in a total volume of 100 apoptosis in cells [3,4,8,19,20]. In the present study we ml containing 50 mM HEPES buffer, pH 7.4 for 30 min at investigated changes in dopamine, caspase-1, caspase-3 378C. Similarly, caspase-1 and -3 activities were assayed and a trypsin-like protease activities as well as DNA using, respectively, N-acetyl-Tyr-Val-Ala-Asp-7-amido-4-cleavage in Balb / c mice during the development of methylcoumarin and acetyl-Asp-Glu-Val-Asp-7-amido-4-MPTP-induced neurotoxicity. Our aim was to investigate methylcoumarin (Sigma Chemical Co., St Louis) as sub-the involvement, if any, of a non-caspase protease in sub-the strates [22]. The reaction was carried out in a total volume action of MPTP. of 100ml, which contained 50 mM HEPES buffer, pH 7.4, Adult inbred Balb / c mice from the institute colony were 100 mM NaCl, 2.0 mM DTT, 0.1 mM substrate and 40ml used in the present study. They were housed under tissue homogenate at 378C for 30 min. The reactions were standard conditions of temperature (22618C), humidity terminated by the addition of ethanol and the fluorescent (6065%) and illumination (12 h light / dark cycles) with product was estimated in a Hitachi spectrofluorimeter at free access to food and water. The experimental protocols excitation 380 nm and emission 460 nm. Student’s t-test were approved by the Animal Ethics Committee of the was used for finding significant differences between two Institute. MPTP hydrochloride (Research Biochemicals means. Values of P#0.05 were considered significant. International, Natick) was administered (30 mg / kg, i.p.) Levels of dopamine in NCP did not change on the first two times, 16 h apart to induce dopamine depletion [17]. day, decreased by 65% on the second day and severely Animals were sacrificed by decapitation, brains were depleted (75%) on the seventh day following the adminis-dissected out, frozen quickly in dry ice and 0.5–1.0-mm- tration of MPTP, 30 mg / kg, two times at 16 h intervals thick sections were cut. SN and nucleus caudatus putamen (Fig. 1). We investigated whether the dopaminergic neu-(NCP) were micropunched on a cold plate with the help of rons at the cell body region, SN, or the cells at the nerve fabricated stainless steel punching needles. terminal region, NCP, had undergone apoptosis 1, 2 and 7 DNA was extracted from NCP and SN. The tissues were days after the neurotoxin treatment by studying the DNA homogenized in lysis buffer containing, 10 mM Tris–HCl,
2 mM EDTA, 0.5% sodium dodecyl sulphate and 20 ml pancreatic RNase at pH 8.0. Following a 30 min incuba-tion at 378C, 200 mg / ml proteinase K was added and further incubated for 3 h at 508C. The DNA was extracted [18] twice with phenol equilibrated with equal volume of Tris, 0.5 M, pH 8.0 buffer, chloroform as well as ethanol (double volume) containing ammonium acetate (2 M) and sodium acetate (0.6 M), pH 5.2. The precipitate was washed twice with ethanol (–208C) and vacuum dried. DNA was dissolved in Tris (10 mM)–EDTA (1 mM), pH 8.0 and loaded on 1.5% (w / v) agarose horizontal slab gel containing 10mg / ml ethidium bromide [18]. Electropho-resis was carried out in Tris–borate (450 mM)–EDTA (1 mM) buffer, pH 8.0 for 3 h at 40 V.
NCP was sonicated (50 Hz for 30 s) in 0.1 M HClO4
containing EDTA (0.01%). The samples were centrifuged at 60003g for 5 min and the supernatant was directly
injected into a HPLC equipped with electrochemical detector (Waters Associates) for the analysis of dopamine [17]. The separation was achieved using a C18, ion pairing, analytical column (4.63250 mm; Ultrasphere IP; Beckman, USA), with a particle size of 5 mm. The flow rate was 0.7 ml / min and the electrodetection was per-formed at 0.74 V.
For assaying proteases activities, whole brain, NCP and SN were homogenized in HEPES buffer, 50 mM, pH 7.4, in a glass–Teflon homogenizer. The samples were cen-trifuged at 80003g at 48C for 15 min.
N-t-Boc-Leu-Arg-Fig. 1. Dopamine concentration in the striatum of mice at 1, 2 and 7 days
Arg-7-amido-4-methylcoumarin (Sigma Chemical Co., St
following MPTP administration. Dopamine was estimated in
micro-Louis) was used as the fluorigenic substrate for the assay punched nucleus caudatus putamen employing HPLC coupled with of trypsin-like activity as described earlier [22], with minor electrochemical detection. Saline treated mice served as control. Data
Table 1
fragmentation pattern. A typical representative result, out a
Effects of MPTP on caspase-3 activity in the brain of mice
of four each independent analyses, is shown in Fig. 2 for
Tissue Enzyme activity (pmol AMC liberated / mg tissue)
the second and seventh days samples. As shown in Fig. 2,
no fragmentation of DNA was observed in SN or NCP of Control 1st day 2nd day
Balb / c mice at 1, 2, or 7 days following neurotoxic doses SN 15.8761.27 13.7362.55 17.6060.24 of MPTP. However, DNA degradation was evident (Fig. NCP 29.3363.65 39.2660.20* 34.5660.17
2). Whole brain 6.5361.15 12.0260.41** 13.9060.15**
We checked whether caspase-1 or trypsin-like activities a
Caspase-3 activity in substantia nigra (SN), nucleus caudatus putamen
were altered in these discrete brain regions as well as in (NCP) and in the whole brain of mice sacrificed on days 1 and 2
the whole brain. Caspase-1 activity did not show any following the treatment of MPTP (30 mg / kg, i.p., two times, 16 h apart). The enzyme activity is expressed as pmol amino-methylcoumarin (AMC)
change at 1, 2 or 7 days after MPTP administration (data
liberated / mg tissue.
not shown). Caspase-3 activity was unaffected in SN. The
Data are mean6S.E.M., n53, *P#0.05; **P#0.01.
enzyme activity was significantly elevated in NCP on the first day, but this increase disappeared by the second day.
toxin, MPTP. MPTP-induced decrease in the activity of However a two-fold increase in caspase-3 activity was
this enzyme was specifically restricted to SN indicating a observed in whole brain homogenates 24 and 48 h
neurotoxin-specific effect, since MPTP is known to destroy following MPTP (Table 1). Trypsin-like activity, as
mea-dopamine-containing neurons of this region. Conversely, sured by amino-methylcoumarin liberation from
N-t-Boc-while caspase-3 activity was increased in the whole brain, Leu-Arg-Arg-7-amido-4-methylcoumarin was
compara-in SN the enzyme activity was not affected. This study also tively higher in normal SN and NCP than in the whole
demonstrates the absence of apoptosis after 60 mg / kg brain. The enzyme activity was significantly inhibited in
cumulative doses of MPTP that cause more than 70% SN on first (34%), second (30%) and seventh (42%) days
dopamine depletion in the striatum by 7 days. after MPTP treatment. The enzyme activity showed an
Balb / c mice are known to be susceptible to MPTP-inhibition (26%) only on the seventh day in NCP (Table
neurotoxicity [14,17]. In the present study we have used 2). The inhibition of the enzyme activity was not evident
neurotoxic doses of MPTP that can result in more than in the whole brain homogenate.
70% decrease in dopamine. The severe decrease in dopa-The present study demonstrates for the first time a
mine at the nerve terminal region, NCP, suggests neuronal region-specific inhibition of a trypsin-like protease activity
loss at SN. It is also possible that the decrease in dopamine following the administration of the dopaminergic
neuro-as observed might be due to biochemical lesions, such neuro-as decrease in tyrosine hydroxylase. Although, the biochemi-cal and molecular mechanisms by which MPTP or its
1
biologically active metabolite, MPP , cause dopaminergic neuronal lesion is unclear, there are several hypotheses that have been proposed, including mitochondrial damage, free radical generation, excitotoxicity, etc. [17]. One or more of these mechanisms, if active, can result in DNA strand break and neuronal death. In vitro studies have demon-strated apoptosis in neuronal and PC12 cultures
1
[5,6,9,13,16] due to the presence of MPP in the medium. However, the present study failed to detect any typical DNA fragmentation following 60 mg / kg cumulative doses
Table 2
a
Effects of MPTP on trypsin-like activity in the brain of mice
Tissue Enzyme activity (nmol AMC liberated / mg tissue)
Control 1st day 2nd day 7th day
SN 1.9660.16 1.3060.14** 1.3960.13** 1.1360.05** NCP 1.8260.17 1.7560.17 2.0260.28 1.5360.05* Whole brain 0.9960.02 0.9560.01 0.9460.04 0.9960.03 Fig. 2. Agarose gel electrophoresis of DNA extracted from the
micro-a
punched substantia nigra (SN) and nucleus caudatus putamen (NCP) Trypsin-like activity in substantia nigra (SN), nucleus caudatus putamen from mice treated with saline or MPTP. Lane 1, control SN; Lanes 2 and (NCP) and in the whole brain of mice sacrificed on days 1, 2 and 7 3, SN from animals treated with MPTP on 2nd and 7th day, respectively; following the treatment of MPTP (30 mg / kg, i.p., two times, 16 h apart) Lane 4, control NCP; Lanes 5 and 6, NCP from animals treated with were analysed employing a fluorimetric procedure [22]. The enzyme MPTP, on 2nd and 7th day, respectively. No typical ladder-type DNA activity is expressed as nmol amino-methylcoumarin (AMC) liberated / cleavage was detected for the dose of MPTP administered in the present mg tissue.
of MPTP. This finding is confirmatory to the earlier report the triggering of apoptosis following MPTP neurotoxicity. which failed to show apoptosis in mice after 80 mg / kg Further studies on the trypsin-like activity following MPTP MPTP using in situ end-labelling technique [11]. Interest- are important in the light of the report that apoptosis takes ingly, 30 mg / kg dose of MPTP administered systemically place in dopaminergic neurons of SN during normal aging everyday for 2 days has resulted in apoptosis as detected and in idiopathic Parkinson’s disease [1]. Our future by terminal deoxynucleotidyl transferase labelling tech- studies are aimed at understanding the regulation by nique [23]. The discrepancy between these studies may be trypsin-like protease, of caspase-3-like protease activity in due to the differences in the techniques used. It is also MPTP-neurotoxicity.
possible that we failed to detect apoptotic-like pattern due to the fact that in an in vivo system it is impractical to
select the time when apoptosis occurs. However, the DNA Acknowledgements breakdown products in our gels suggest nuclear
degenera-tion, may be due to necrosis as seen in cerebellar granule Financed by CSIR and DST. UR is a Senior Research cells exposed to this neurotoxin [6]. Associate (Scientists’ Pool) of CSIR, Government of India. Alternately, there may be some endogenous regulatory BT, DM and SG are recipients of CSIR Senior Research mechanisms operating in animals that prevent apoptosis to Fellowships.
occur. Many studies have confirmed that caspases are essential mediators of apoptotic response in cells [11]. We have observed an activation of caspase-3 in the whole
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