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,1
,1
* CAIR Institute, The Mina and Everard Goodman Faculty of Life Sciences,
Leslie Susan Gonda (Goldschmied) Multidisciplinary Brain Research Center;
Department of Chemistry,
¶ Interdisciplinary Department, Bar-Ilan University, Ramat-Gan, Israel.
Laboratory of Neurosciences and
# Laboratory of Immunology, National Institute on Aging Intramural Research Program, Baltimore, Maryland, USA; and
|| Department of Biochemistry, Weill Medical College of Cornell University, New York, New York, USA
2Correspondence: CAIR Institute, The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Keren Hayessod St., Ramat Gan, Israel. E-mail: srednib{at}mail.biu.ac.il
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: neuroprotection • apoptosis • inflammation • cytokines • neurotrophic
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Although rare cases of early-onset PD result from gene mutations, the cause(s) of the common late-onset sporadic form of PD is unknown (2
, 3)
. Environmental toxins such as rotenone, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and related chemicals that inhibit mitochondrial complex I have been implicated in PD because they can induce PD-like neuropathology and motor dysfunction in rodents, monkeys, and humans (4)
. Studies of the pathogenic mechanisms of mutations in PD-linked genes (alpha-synuclein, Parkin and DJ-1) (5)
and neurotoxin-induced PD (6)
suggest that increased oxidative stress, mitochondrial dysfunction, and impaired clearance of damaged proteins are major factors leading to the dysfunction and death of dopaminergic neurons. The damaged neurons may die by apoptosis executed by the cysteine proteases caspases (7)
.
Ammonium trichloro(dioxoethylene-O,O’)tellurate (AS101), an organotelluriumIV compound currently in Phase II clinical trials in cancer patients (8
, 9)
, is a potent immunomodulator (both in vitro and in vivo) with a variety of potential therapeutic applications (10
, 11
, 12)
. AS101 has been shown to have beneficial effects in diverse preclinical and clinical studies. Most of its activities have been primarily attributed to the direct inhibition of the anti-inflammatory cytokine interleukin-10 (IL-10) (13)
. These immunomodulatory properties were found to be crucial for the clinical activities of AS101, demonstrating the protective effects of AS101 in parasite- and virus-infected mice models (14)
, in autoimmune diseases (15)
, in septic mice (16)
, and in kidney diseases (17)
. Furthermore, inhibition of IL-10 by AS101 sensitizes tumors to chemotherapy, resulting in increased survival of tumor-bearing mice (18)
. Moreover, AS101 was recently found to up-regulate GDNF production (19
, 20)
.
Accumulated evidence suggests that much of the biological activity of organotellurium compounds is directly related to their specific chemical interactions with endogenous thiols. Such tellurium-thiol compounds may be important for the manifestation of the biological function itself or for transportation of the tellurium species to its target location. In a previous study, we clarified several mechanistic aspects of this chemistry and discussed its relation to the biological activity of AS101 (21)
. If the reacting thiol is a cysteine residue of an enzyme or another protein, the reaction product may alter the biological activity of that protein. The TellurIV-thiol chemical bond may lead to conformational change, naturally promoted by an endogenous effector, which initiates the biological activity. Alternatively, it may lead to complete loss of the biological activity, if the thiol residue is essential for that activity, as is the case in cysteine proteases. Indeed, we demonstrated that AS101 and other TeIV compounds specifically inactivate cysteine proteases, while exhibiting no effect on the other families of serine-, aspartic- and metalloproteases, in good agreement with the unique TeIV-thiol chemistry. Furthermore, the proteolytic activity of the inactivated cysteine proteases could be recovered by reducing agents such as NaBH4, further supporting the suggestion that the inactivation process involves oxidation of the catalytic thiol to a disulfide (21)
Because of the Te(IV) valence of AS101, it can serve as a reducing or oxidizing agent, depending on the environmental oxidation milieu (22)
.
In recent years, the involvement of neuroinflammatory processes in nigral degeneration has gained increasing attention. The degeneration of dopaminergic neurons in PD is associated with massive microglial activity (23)
, which may be a general consequence of neuronal death or may reflect the active participation of microglia in the neurodegenerative process. Whether microglial activation protects or exacerbates neuronal loss is presently debated, although most evidence gained from both in vitro and in vivo experiments suggests that activated microglia exert a toxic effect on neurons. Microglial activation can be triggered by a variety of elements. When activated, they release proinflammatory molecules, the overproduction of which can be neurotoxic.
Several therapeutic approaches for PD are at various stages of preclinical and clinical investigation. By increasing dopamine production, levodopa temporarily improves symptoms, but does not prevent the death of the dopaminergic neurons (24)
. Antioxidants have demonstrated efficacy in animal models of PD but have either been ineffective or have not yet been tested in PD patients (25
, 26)
. Another approach is to treat patients with glial cell line-derived neurotrophic factor (GDNF), which is known to prevent neuronal death and stimulate dopamine production in animal models of PD (27
, 28)
. However, GDNF must be infused directly into the brain to access the dopaminergic neurons. Because multiple mechanisms are involved in the dysfunction and death of neurons in PD, effective therapies may require administration of several drugs, each targeting a specific neurodegenerative pathway.
In parallel to AS101’s immunomodulatory activities, and in light of the unique Te(IV)-thiol chemistry of AS101, as mentioned above, AS101 is now shown to directly inhibit caspase 3 and 8 activities in vitro, and caspase 1 and 3 in vivo, potentially enabling the compound to exert both anti-inflammatory and anti-apoptotic properties. Coupled with its ability to induce GDNF production in vitro and in vivo, the present study identifies a nontoxic single drug, currently in advanced clinical trials, that, because of its unique mechanisms of action, exerts multifunctional activities that are highly efficacious in protecting dopaminergic neurons and enhancing their function in animal models of PD.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Rat 6-OHDA and mouse MPTP models
Methods for infusion of 6-OHDA into the SN and for evaluation of the resulting motor deficit are detailed in the Supplementary Methods (online). Three to four weeks after 6-OHDA administration, AS101 or saline was infused through a canula placed stereotaxically closely to the SN (0.2 mg/ml in 75 µl saline at 1 µl/h for 72 h; anterior (–4.8 mm) ventral (–8 mm), and lateral (+1.6 mm) of the 6-OHDA-lesioned rats. The methods for MPTP administration, evaluation of motor function, and quantification of tyrosine hydroxylase levels are detailed in the Online supplement. AS101 was administered to mice in two intraperitoneally (i.p.) injections (10 µg of AS101 in a 0.2 ml vol), the first, 30 min prior to MPTP administration and the second 30 min following MPTP administration. Saline was injected into control animals.
Cell cultures and experimental treatments
PC12 cells (wild-type); PC12N17, which express a dominant negative p21ras; and PC12C118S, which express a mutated p21ras in which cysteine 118 was replaced with serine. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 8% heat-inactivated horse serum, 8% heat-inactivated FBS, glutamine (5 mM), and gentamicin (50 µg/ml) at 37°C in a 5% CO2 humidified atmosphere. Human SVG-glial cells were maintained in Eagle’s medium supplemented with fetal calf serum (10%), gentamicin (50 µg/ml), and glutamine (5 mM) at 37°C in a 5% CO2 humidified atmosphere.
Astrocyte isolation and culture
Rat cortices were trypsinized with 0.25% trypsin followed by trituration and plated to culture flasks precoated with 0.1 mg/ml poly-D-lysine. Cells were cultured in DMEM with high glucose, 1.2 mM glutamine, 0.1 mM nonessential amino acid mixture, 0.1% gentamicin, and 10% FCS. On the 10th day of culture, cells were rinsed with complete medium and shaken by an orbital shaker (250 rpm) for 24 h at 37°C, to discard oligodendrocytes by selective detachment. Cells were then trypsinized and cultured again for 1 wk. They were then rinsed and shaken again to discard remaining nonastrocytic cells.
Intracellular GSH and GSSG assays
Isolated astrocytes were plated in 6-well culture plates with or without 1 µg/ml lipopolysaccharide (LPS) or various concentrations of AS101. After 20 h, cells were removed by scraping and washed 3 times. They were immediately lysed with 100 µl of lysis buffer (0.1% Triton-X, 1 M sodium phosphate buffer, 5 mM EDTA, pH 7.5). Thereafter, 15 µl of 0.1 N HCL and 15 µl of 50% sulfosalicylic acid were added. After centrifugation at 14,000 g for 5 min, supernatants were collected for GSH and GSSG assays. The total cellular glutathione concentration was assayed using the GSH-reductase-DTNB recycling procedure. GSSG concentration was assayed according to the method of Sacchetta et al. (29)
.
Cell cycle and apoptosis determinations
The methods for flow cytometry-based analysis of cell cycle and apoptosis are detailed in the Online Supplement.
Caspase activities
Fifty micrograms of total cell lysate were used to evaluate caspase-1 and -3 activities using the colorimetric caspase-1 and -3 assay kits (Biomol Research Laboratories; Plymouth Meeting, PA). For measurement of direct inhibition of caspases, recombinant caspases-3 and -8, following gel permeation chromatography, were used. Caspase-1, -3, and -8 activities were determined by measuring the proteolytic cleavage of the caspase-1, -3, and -8 substrates (YVAD-pNA), Ac-DEVD-pNA and IETD-pNA, respectively. Cleavage of the p-nitroanilide (pNA) from the colorimetric substrates increases absorption at 405 nm. Each assay point was run in duplicate
Gel permeation chromatography
Gel permeation chromatography was used to eliminate DTT from enzyme solution, for the direct inhibition of rCaspase-3 and -8 activity. The chromatography was carried out at 4°C. A 50 µl solution of each rCaspase was loaded on a 1 x 8 cm Sephadex G-15 column, pre-equilibrated with 0.1% CHAPS buffer pH 7.4 containing 0.1 mM EDTA. The enzyme was eluted with the same buffer (degassed) at 0.5 ml/min and 0.5 ml fractions were collected. The A280 of each fraction was measured. Subsequently, 300-µl aliquots were removed from all of the fractions that did not exhibit the 280-nm absorption (not containing protein), mixed with DTNB (20 µl of 10 mM aqueous solution), and A412 was measured.
Transfection
All cDNA expression plasmids were kindly obtained from H. M. Lander, Cornell University, New York. Transfection of wild-type p21ras, p21ras, C118S, p21N17ras, and control pcDNA3 expression plasmids was performed on 100-mm dishes plated at 1 x 106 cells per dish, using the calcium phosphate procedure. The DNA (20 µg) to be transfected was mixed with 25 µl of 10 mM Tris, pH 7.4, and 50 µl of 1.25 M CaCl2, supplemented with H2O to a volume of 250 µl. This DNA mixture was added to an identical vol of 2' HEBS (50 mM HEPES, 280 mM NaCl, 1.5 mM Na2PO4, pH 7.1) over a vortex mixer. A precipitate was allowed to form for 10 min and was then added to dishes. Cells were incubated overnight with this mixture and washed with PBS the next day. The cells were maintained in DMEM supplemented with 10% FCS and G418 (400 µg/ml) for several weeks.
GTPase assay of recombinant p21ras and electrospray ionization-mass spectrometry
GDP-preloaded p21ras was analyzed for guanine nucleotide exchange activity as described previously (30)
. Basal rates of hydrolysis of [
-32P]GTP were 23.5 ± 5 fmol of PO4. released/min/mg. Electrospray ionization-mass spectrometry (ESI-MS) was performed as described previously (30)
.
Immunoblot analysis of samples from 6-OHDA-lesioned rats
Thirty micrograms of cell lysate samples, prepared from punches taken from lesioned and intact parts of the substantia nigra, were boiled for 4 min and separated by SDS-polyacrylamide 12% gel electrophoresis. They were then transferred onto a nitrocellulose membrane and blocked with 5% skim milk in PBST. Blots were developed using horseradish peroxidase-conjugated mouse and goat (secondary antibodies (Jackson Laboratories, Bar Harbor, ME, USA) and the ECL detection system (interleukin; Pierce, Rockford, IL, USA). Each blot was stripped and reblotted with a different antibody. Membranes were immunblotted with the following antibodies: pERK1/ERK2 (Sigma Aldrich; Rehovot, Israel); active IL-1ß, pAkt, BclII, ß-actin (Santa Cruz; Santa Cruz, CA, USA).
Cytokine and GDNF expression
Protein quantification using ELISA kits and quantification of mRNA levels, as detailed in the Online Supplement.
Measurement of Dopa, dopamine and DOPAC levels
Quantitation of the DA, DOPAC, and 5-hydroxytryptamine content of the tissue punch extracts was performed, as described in the Online Supplement.
Statistics
Data are presented as mean ± SE. Comparisons between treatment groups were made using either paired t tests or ANOVA and either Bonferroni or Scheffé post hoc tests. P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A). At 4 mo, postlesioning rats treated with PBS exhibited a partial loss of tyrosine hydroxylase (TH) (the rate-limiting enzyme in dopamine synthesis) immunoreactive neurons in the SN-striatum and a corresponding loss of SN neurons capable of retrograde transport of intrastriatal Fluorogold (Fig. 1B-D
). AS101 increased levels of tyrosine hydroxylase (TH) in the striatal axons, as well as in the SN cell bodies of surviving dopaminergic neurons (17±1% of intact side in control compared to 32±7% in AS101-treated brains; P<0.05). Similarly, AS101 treatment resulted in an increase in Fluorogold (FG)-positive cells in the SN (38±7% compared to 15±4% in the SN of control rats; P<0.01). Colocalization of TH and FG occurred in 80% of the cells demonstrating preservation of functional integrity of the SN-striatal dopaminergic pathway.
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To further evaluate the influence of AS101 on dopaminergic neurons, rats were administered 6-OHDA and AS101 using the same protocol as in the first experiment, and were euthanized 4 wk later. Levels of dopamine (DA) and the dopamine metabolite DOPAC (3,4 dihydroxy-phenylacetic acid) were measured in tissue punches taken from both the left and right SN and striatum. In control rats treated with PBS, 6-OHDA caused marked reductions in levels of dopamine and DOPAC in the SN and striatum (Fig. 1E
). In AS101-treated rats, the concentrations of dopamine and DOPAC in the lesioned SN and striatum were maintained at levels 3- to 4-fold greater than in the control rats. Treatment of the lesioned rats with AS101 did not cause significant changes in the levels of serotonin or in its metabolite (data not shown). The ability of AS101 to sustain cell viability and dopamine production in SN neurons is likely responsible for its ability to ameliorate 6-OHDA-induced motor dysfunction, because even partial recovery of dopaminergic function can dramatically improve symptoms in PD patients and animal models of PD (31)
.
Protection of dopaminergic neurons by AS101 in MPTP-induced PD animal model
The neuroprotective capabilities of AS101 were validated in an additional model of PD. Exposure to the toxin MPTP can cause a PD-like syndrome in humans, monkeys, and mice; after entering the brain, MPTP is converted to MPP+, which is then selectively transported into axon terminals of dopaminergic neurons, where it induces oxidative stress and mitochondrial dysfunction (32
, 33)
. We determined whether peripheral administration of AS101 might modify functional outcome and degeneration of dopaminergic neurons in MPTP-treated mice. Mice were given two i.p. injections of AS101, and 7 days later, motor function was tested using a rotarod apparatus. The dose of AS101(10 µg/injection), was previously demonstrated to exert optimal responses in various in vivo mouse models. The motor dysfunction normally caused by MPTP disappeared completely in the AS101-treated mice (Fig. 2
A, B). Immunoblot analysis of tyrosine hydroxylase levels in the striatum of these mice revealed a marked attenuation of MPTP-induced loss of this dopaminergic marker in AS101-treated mice (Fig. 2C
). Counts of TH immunoreactive cell bodies in the SN revealed a highly significant preservation of dopaminergic neurons in the AS101-treated mice (Fig. 2D
). These results show that peripheral administration of AS101 reduces MPTP-induced damage to dopaminergic neurons and, therefore, may account for the improved behavioral outcome.
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Binding of AS101 to Cys-118 of p21ras is essential for AS101-induced neuronal differentiation
We then wished to address various mechanistic aspects of AS101’s biological activities, related to the Te-thiol moieties of the compound, in order to explain its unique neuroprotective and neurorestorative properties. We have previously reported that AS101 induces the differentiation of the neuronal PC12 cells (34)
. We therefore determined whether the affinity of AS101 to thiols accounts for this property, by testing whether AS101 can directly bind to p21ras and activate it. This monomeric G protein family member, plays a critical role in converting extracellular signals into intracellular biochemical events and plays an important signaling role in neuronal differentiation.
We found that AS101 triggered nucleotide exchange on recombinant Ras in vitro. Using the GTPase assay as a redout of nucleotide exchange, we found that treatment of Ras (1 µM) with 10 ng/ml AS101 for 10 min led to a significant increase in GTPase activity (P<0.005) (Fig. 3
A). The increased exchange activity correlated with our finding of the direct binding of AS101 to Ras in vitro using electospray-ionization mass spectrometry. Using recombinant Ras (1–166), we found that in vitro incubation with AS101 increased the mass of Ras by 301 ± 3 Da, indicating that one AS101 molecule bound to each Ras molecule. Figure 3B
shows that AS101, caused morphological changes in PC12 cells, including enlargement of cell bodies and formation of stable neurites similar to those observed in nerve growth factor-treated cells. These effects of AS101 were concentration dependent with the greatest effect occurring with 0.5 µg/ml of AS101 (data not shown). The rate of dopamine release from PC12 cells was significantly increased in AS101-treated cells (Fig. 3C
), further suggesting a trophic action of AS101. We found that AS101 did not induce differentiation of PC12 cells that express a dominant-negative form (N17) of ras, thus identifying ras as a crucial signaling molecule for AS101-induced differentiation (Fig. 3B
). Because Cys-118 resides on a loop that has contact with the guanine nucleotide, it has been suggested that modification of this cysteine may directly affect the bound GTP/GDP ratio of p21ras, thus changing its activity (32)
. Exposure of PC12 cells that express a point mutation in Cys of p21ras to AS101 did not result in their differentiation, while exposure to NGF did (Fig. 3B
), demonstrating a requirement for Cys118 in p21ras in AS101-induced differentiation. The downstream mediator of P21ras, ERK, as will be shown later, was activated by AS101 in the 6-OHDA PD rat model in vivo. To determine whether ras activation in vitro mediated activation of ERKs in cells treated with AS101, we treated NIH3T3 cells with AS101 for 10 min, in the absence or presence of a farnesyl transferase inhibitor, and then measured ERK activity using a myelin basic protein as an ERK substrate. AS101 increased ERK activity in a concentration-dependent manner, an effect that was completely blocked by the farnesyl transferase inhibitor (Fig. 3D
). These findings suggest that AS101 can directly activate ras and its downstream effector ERK by a mechanism involving cysteine residue 118 of ras.
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AS101 directly inhibits caspase activities
On the basis of the novel TeIV-thiol chemistry of AS101 and the previously demonstrated ability of this compound to specifically inhibit cysteine proteases, while exhibiting no effect on the other families of serine-, aspartic- and metalloproteases, we also tested the interaction of AS101 with the apoptotic caspases—a family of cysteine proteases. Using recombinant caspases, AS101 directly inhibited the activity of the distal effector apoptotic caspase-3 in a dose-dependant manner (Fig. 4
A). Similarly, AS101 directly inhibited the activity of recombinant caspase-8, a proximal effector protein of the cell death pathway, the higher concentration of AS101 yielding 84% inhibition of the enzyme activity.
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Caspase inhibition by AS101 suggested that it may prevent apoptotic cell death of dopaminergic neurons in vitro. Exposure of PC12 cells to 6-OHDA resulted in a concentration-dependent increase in the percentage of cells in the sub-G1 fraction(Fig. 4B
). This was prevented by AS101 treatment (Fig. 4B
), suggesting an antiapoptotic activity. Indeed, AS101 protected PC12 cells, in a dose-dependent manner, against apoptosis induced by 6-OHDA, as quantified by DNA fragmentation using ELISA analysis (Fig. 4C
). AS101 could exert this protective activity whether it was supplemented 1 h before or until 3 h following 6-OHDA addition (Fig. 4D
). Supplementation of AS101 to control cultures did not affect cell survival (not shown). AS101’s ability to prevent neurotoxin-induced dopaminergic neuronal cell death in vitro was associated with decreased caspase-3 activity in AS101-treated cells (Fig. 4E
).
These results prompted us to investigate whether AS101 can inhibit caspases activity in vivo. We found that restoration of dopaminergic neuronal mass and function by AS101 in 6-OHDA-lesioned rats was associated with a substantial decrease in the activity of both inflammatory and apoptotic caspases (Fig. 5
). AS101 treatment prevented the increase of caspase-3 activity induced by 6-OHDA in SN dopaminergic neurons in vivo (Fig. 5A
). Caspase-1 activity was increased by 
10-fold in the SN and 4-fold in the striatum in response to infusion of 6-OHDA into the SN (Fig. 5B, C
). Treatment of rats with AS101 following infusion of 6-OHDA completely prevented the increase of caspase-1 activity. The decrease in both caspase-1 and -3 activities was significant.
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AS101 inhibits IL-10 and up-regulates GDNF
We first tested whether AS101, previously reported to inhibit IL-10 by various cell types in vitro and in vivo (9
, 13
14
15
16
17
18)
, decreases IL-10 produced by astrocytes in vitro and whether this anti-inflammatory cytokine is also inhibited in the SN of 6-OHDA-lesioned rats treated with AS101. Figure 6
A shows that AS101 significantly inhibited LPS-induced IL-10 secretion by primary rat astrocytes in vitro. This activity was also observed in human fetal astrocyte cells (SVG cell line) cultured in the presence of AS101 (Supplementary Table 1). Interestingly, the production of GDNF increased in both types of cells following addition of AS101 (Fig. 6B
; Supplementary Table 1), and levels of IL-6 were also elevated. We show that GDNF levels are regulated by IL-10: inhibition of IL-10 by AS101, in primary astrocytes, or its neutralization by IL-10 neutralizing antibodies resulted in the up-regulation of GDNF. Conversely, the addition of rIL-10 inhibited GDNF production (Fig. 6B
). The dose of the anti IL-10 neutralizing antibody was found to be efficient, as evidenced in Fig. 6A
.
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In 6-OHDA lesioned rats, the levels of IL-6 and GDNF mRNAs species were greater in the SN of AS101 treated rats compared to PBS-treated control rats, whereas levels of IL-10 mRNA and protein were significantly lower in the AS101-treated rats (Fig. 6C, D
). Although IL-10 protein levels in the left lesioned SN were decreased in AS101-treated rats, the protein level of the proinflammatory cytokine IL-1ß was also diminished (Fig. 6C
). The reduced levels of this proinflammatory neurotoxic cytokine in the SN of AS101-treated rats correlated with the inhibitory effect of AS101 on the activity of the inflammatory caspase-1 in that brain area as presented in Fig. 5B
.
Recently, a number of studies have demonstrated that the polarization of Th1/Th2 balance is dependent on the intracellular redox status of cells (35
, 36)
. Cells with high levels of reduced-glutathione (GSHv) secrete low levels of IL-10, while those with low levels of GSH produce much higher levels of this anti-inflammatory cytokine. The Te(IV)-thiol chemistry of AS101 led us to evaluate whether this compound may affect the thiol redox state of stimulated primary astocytes, in which the level of GSHv is low. Figure 6E
shows that LPS stimulation of primary astrocytes significantly decreased the ratio of GSH/GSSG. Treatment of stimulated cells with AS101 significantly increased this ratio to levels similar to those of unstimulated cells. Furthermore, the increase in GSH/GSSG ratio by AS101 was associated with inhibition of IL-10 (Fig. 6A
). These results collectively suggest that inhibition of IL-10 by AS101 may have favorable effects in the PD setting, up-regulating GDNF.
AS101 can stimulate neurotrophic signaling pathways in dopaminergic neurons in vivo
The increase in the expression of the neurotrophic factors GDNF and IL-6 led us to evaluate their signaling pathways. Levels of phosphorylated ERKs and Akt were increased in the SN and striatum of 6-OHDA-lesioned AS101-treated rats compared to PBS-treated 6-OHDA-lesioned rats (Fig. 7
A, B). ERKs and Akt are kinases known to mediate the effects of neurotrophic factors on neuronal survival and differentiation (37
, 38)
. One mechanism by which ERK and Akt signaling pathways promote neuron survival is by up-regulating the expression of the antiapoptotic protein Bcl-2 (38
, 39)
. We found that levels of Bcl-2 were increased in the lesioned SN and striatum of AS101-treated rats compared to PBS-treated controls (Fig. 7A, B
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The main biological activities exerted by AS101, which are closely associated with neuronal survival and disease outcome are neurotrophic, antiapoptotic, and anti-inflammatory capabilities of the compound. The Te-thiol chemistry of AS101 probably accounts for most of its neuroprotective and neurorestorative activities. In vitro, we show that AS101’s induced differentiation of cultured dopamine-producing neural cells is dependent on thiol modification of p21ras: AS101 binds directly to ras and activates it. Moreover, ras is identified as a crucial signaling molecule for AS101-induced differentiation because cells expressing its dominant-negative form did not differentiate in the presence of AS101. More importantly, AS101, as opposed to NGF, failed to induce differentiation of neuronal PC12 cells that express a point mutation in Cys118 of p21ras, demonstrating a requirement for Cys118 in p21ras in AS101-induced differentiation. Tellurium(IV)-Thiol formation on a critical cysteine is apparently responsible for p21ras activation. In fact, others have found that redox agents modulate binding of GTP to p21ras immunoprecipitates (30)
. Because Cys-118 resides on a loop that has contact with the guanine nucleotide, it has been suggested that modification of this cysteine may directly affect the bound GTP/GDP ratio of p21ras, thus changing its activity, as occurring following S-nitrosylation by nitric oxide (30)
.
The Te (IV)-thiol chemistry of AS101 may also account for its direct inhibition of both apoptotic and inflammatory cysteine-containing caspases. These proteases are normally present in cells in a proenzyme form that requires limited proteolysis for enzymatic activity. The processing of caspases needs the reduction of cysteine residues around the catalytic site for enzymatic activity and dimerization via sulfhydryl groups. Therefore, both processing and activity can be inhibited in the presence of thiol-oxidizing agents. AS101 could directly inhibit both the initiator caspase 8 and the downstream executor caspase-3. The activities of caspases-1 and -3 were inhibited in the SN and striatum of 6-OHDA lesioned, AS101-treated rats. The degeneration of dopaminergic neurons of the SN is responsible for primary motor symptoms observed in PD. Although the etiology of nigral neurons degeneration remains unknown, postmortem studies show that dying cells bear the signs of apoptosis, in particular, chromatin condensation, DNA fragmentation, oxidative damage, mitochondrial dysfunction, and caspase activation (40)
. Apoptosis is also observed in in vivo and in vitro models of PD based on the toxicity of MPP+ and 6-OHDA for DA neurons. Caspases-1 and -3 have each been implicated in the deaths of dopaminergic neurons in PD (24
, 41)
. Furthermore, it has been shown that a significantly higher percentage of dopaminergic SN neurons displayed caspase-8 activation in PD patients compared with controls (42)
. The direct inhibition of both the initiator and the executor caspases-8 and -3 is of major importance since it is imperative to rescue neurons before irreversible damage has occurred.
Although caspases-1, -3, and -8 are inhibited by AS101, the possibility still remains that other caspases are affected by AS101. Nevertheless, a differential reactivity of various cysteines toward AS101 may exist, possibly reflecting their variable access to AS101, as well as nucleophilicity, which is determined by hydrogen bonds with the thiol. In fact, Stamler et al. proposed that residues adjacent to cysteines in proteins increase the nucleophilic properties of cysteine, with putative consensus sequences adjacent to the cysteine, providing enhanced susceptibility to S-nitrosylation (43)
. Because apoptotic cell death prevented by AS101 in vitro shows a critical therapeutic window as presented in Fig. 4D
, caspases inhibition in vivo by AS101 probably plays only a partial role in AS101’s beneficial effects in the PD models.
In recent years, the involvement of nonneuronal cells in the pathology of PD has gained increasing attention. The degeneration of dopaminergic neurons in PD is associated with massive microglial activity, which may contribute to the propagation of the disease process by exerting a toxic effect on neurons (23)
. Therefore, the inhibition of caspase-1 activity in the SN/striatum by AS101 treatment followed by the decreased expression of IL-1ß, known to be implicated in the pathogenesis of PD (44)
, may contribute to the anti-inflammatory properties of the compound. Although caspase-1 has been classically implicated in the maturation of the inflammatory IL-1ß and IL-18 cytokines, this protease has also been implicated in cell death. In fact, caspase-1 appears to be involved in some forms of neuronal cell death. Mutant caspase-1 that lacks an active site inhibits trophic factor withdrawal-induced apoptosis in dorsal root cells (47)
. Furthermore, brain injury induced by middle cerebral artery occlusion, a mouse model of stroke, is significantly reduced in caspase-1-deficient mice (45)
. Thus, its inhibition by AS101, along with contributing to AS101’s anti-inflammatory properties, may potentiate its antiapoptotic capabilities in PD.
Our findings suggest roles of the pleiotrophic cytokine IL-10 in PD. It’s beneficial anti-inflammatory properties were previously reported. IL-10 protected against inflammation-mediated degeneration of dopaminergic neurons in the SN (46)
. IL-10 has been shown to improve neurological outcome after central nervous system (CNS) injury and to render neurons in culture less vulnerable to ischemic damage (47)
. IL-10 is notoriously known as an inhibitor of the synthesis of inflammatory cytokines, including tumor necrosis factor-
and IL-1ß (48)
. Some of these cytokines can exacerbate neuronal damage after CNS trauma (50)
; therefore, it has been suggested that the IL-10 ability to improve neurological outcome after CNS injury relies on its anti-inflammatory effects. Furthermore, IL-10 has been reported to prevent cerebellar granule cell death by blocking caspase-3 like activity (52)
. Therefore, one would expect that treatment of PD with compounds that inhibit IL-10 would result in deleterious effects on the disease course. However, our data suggest that, aside to its favorable properties in neurodegenerative diseases, IL-10 exerts detrimental capabilities by inhibiting GDNF production. We show that IL-10 regulates GDNF production in primary astrocytes in vitro: inhibition of IL-10 elevates GDNF. Indeed, this phenomenon could be also seen in vivo where inhibition of IL-10 in the SN by AS101 resulted in elevated expression of GDNF. A similar regulatory pathway has been previously reported by us in mesangial kidney cells (20)
. Because the net effect of IL-10 on neuronal survival is favorable, one might conclude that the inflammatory processes in the brain have more harmful implications than the relative lack of GDNF. However, this study shows that the use of a molecule with specific chemistry that directly inhibits the production of IL-10 in vitro and decreases its secretion in vivo (9
, 13
14
15
, 19
, 20)
, results in favorable effects in animal PD models.
Besides GDNF, AS101 induced the secretion and expression of IL-6 both in vitro and in the animal Parkinson’s model. IL-10 is a potent inhibitor of IL-6 expression (51)
. Because both GDNF (27
, 52)
and IL-6 (53)
can protect neurons against insults relevant to PD, our findings suggest roles for up-regulation of GDNF and IL-6 in the neuroprotective actions of AS101 in the rat and mouse models of PD. As further evidence that AS101 can stimulate neurotrophic signaling pathways in dopaminergic neurons, we found that levels of phosphorylated ERKs and Akt were increased in the SN and striatum of 6-OHDA-lesioned AS101-treated rats compared to PBS-treated 6-OHDA-treated rats. ERKs and Akt are kinases that are known to mediate the effects of neurotrophic factors on neuronal survival and differentiation (37
, 54)
One mechanism by which ERK and Akt signaling pathways promote neuron survival is by up-regulating the expression of the antiapoptotic protein Bcl-2 (38
, 39)
. We found that levels of Bcl-2 were increased in the lesioned SN and striatum of AS101-treated rats compared to PBS-treated control rats. The Bcl-2 family is a group of proteins that regulate apoptotic and nonapoptotic forms of neuronal cell death in both normal cellular development and in acute and chronic pathological insults (55)
. Changes in expression of Bcl-2 family mRNAs and proteins in the processes of neuroprotection in neurodegenerative diseases are well documented (54)
. Moreover, the Bcl-2 protein has been also shown to induce neuronal differentiation (56)
and to promote the regeneration of several axons in mammalian neurons (57)
. Thus, Bcl-2 protein plays a role in both antiapoptosis and neuronal regeneration.
The present study clearly demonstrates neuroprotective properties of AS101 in two animal models of PD. AS101 was effective in protecting SN/striatum dopaminergic neurons against PD neurotoxins and improving motor function when administered either directly into the brain or i.p. Although AS101 significantly elevated dopamine levels in both the SN and the striatum, In the 6-OHDA-induced rat model, these amounted to < 10% of control rats. Nevertheless, the motor functions of the compound were very striking. This can be explained by evidence provided by previous studies that a large depletion of dopamine levels can be tolerated without a major adverse effect on motor function (58)
. Moreover, therapeutic interventions that result in only a small increase in dopamine levels can result in clinically relevant improvement in motor function (59)
. In the MPTP-induced model, the neurotoxin dose used caused a dramatic decrease in the level of TH protein in the substantia nigra, but only a 50% loss of TH-positive neurons, suggesting that TH levels were decreased in many of the surviving neurons. The motor dysfunction caused by MPTP was therefore likely due to a combination of death of some dopaminergic neurons and dysfunction of many of the remaining neurons. AS101 treatment resulted in preservation of TH levels and increased survival of neurons in the substantia nigra and prevented motor dysfunction. It is therefore likely that the improved motor function in AS101-treated MPTP-lesioned mice resulted from a combination of reduced neuronal death and preservation of dopamine levels.
In a clinical setting, each route of AS101 administration used has advantages and disadvantages. For example, intraparenchymal administration ensures direct access of the agent to the dopaminergic neurons and greatly reduces potential adverse effects of the agent on peripheral tissues. On the other hand, systemic administration is more practical for the long-term treatment of large numbers of patients. Preclinical and clinical trials of GDNF have focused on direct infusion into the brain (28
, 45)
or gene therapy approaches (52)
. Here, we show that AS101 preserves neuronal and motor function months after initiation of the disease, at the time when the animals exhibited a severe impairment of motor function. The lack of toxicity of AS101, as determined both in animals and in phase I/II clinical studies suggests that this organothelurium compound probably reacts with specific targeted proteins, thus controlling specific metabolic pathways. Besides AS101, the investigation of therapeutic activities of other tellurium(IV) compounds is scarce in the literature, although tellurium is the fourth most abundant trace element in the human body (after Fe, Zn, and Rb) (60)
. Thus we may envisage, in view of our results, that Te(IV) compounds may have important roles in thiol redox biological activity in the human body, which may involve signal transduction mechanisms as described in Fig. 8
.
|
The use of a nontoxic tellurium compound previously demonstrated an excellent safety profile in clinical trials, which because of its Te(IV)-thiol chemistry, exerts multifunctional activities and suggests that it may be a promising agent for the treatment of PD.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication November 2, 2006. Accepted for publication December 25, 2006.
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REFERENCES |
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levels. J. Immunol. 159,2658-2667[Abstract]
