QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: mol.106.028415v1 71/1/30    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Ahtoniemi, T. Articles by Koistinaho, J. Search for Related Content PubMed PubMed Citation Articles by Ahtoniemi, T. Articles by Koistinaho, J.

Pyrrolidine Dithiocarbamate Inhibits Induction of Immunoproteasome and Decreases Survival in a Rat Model of Amyotrophic Lateral Sclerosis

Department of Neurobiology, A. I. Virtanen Institute for Molecular Sciences (T.A., G.G., V.K.-G., T.M., K.K., J.K.), Department of Neuroscience and Neurology (A.S.), University of Kuopio, and Department of Oncology (J.K.), Kuopio University Hospital, Kuopio, Finland

Received June 29, 2006; accepted September 27, 2006

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Pyrrolidine dithiocarbamate (PDTC), an inhibitor of nuclear transcription factor -B (NF-B) and an antioxidant, has beneficial effects in animal models of various diseases, including arthritis, brain ischemia, spinal cord injury, Alzheimer's disease, and Duchenne muscular dystrophy. Because inflammation and oxidative damage are also hallmarks of amyotrophic lateral sclerosis (ALS), we studied the effect of oral PDTC treatment on G93A-superoxide dismutase 1 (SOD1) transgenic (TG) rat model of human ALS and observed that PDTC treatment significantly decreases the survival. PDTC treatment evoked the end stage of the disease at 121 ± 21 days, whereas untreated TG animals reached the end stage at 141 ± 13 days (p < 0.01). The DNA binding activity of NF-B was not altered in G93A-SOD1 TG rats by PDTC treatment. The copper concentration in the spinal cord was increased after PDTC treatment both in G93A-SOD1 TG and wild-type rats, suggesting that increased copper may enhance the neurotoxicity of mutant SOD1. The amount of ubiquitinated proteins were significantly higher and proteasomal activity was decreased in the spinal cords of PDTC-treated TG rats compared with other groups, suggesting that PDTC treatment decreases proteasome function. Immunoblotting and immunocytochemistry showed that the level of immunoproteasome but not constitutive proteasome was increased in glia of G93A-SOD1 TG rats along with disease development. PDTC treatment completely blocked the induction of immunoproteasome expression without affecting constitutive proteasome. These results suggest that PDTC acts as an immunoproteasome inhibitor in mutant SOD1 rats and that immunoproteasome may help the nervous system to cope with deleterious effects of SOD1-G93A mutation.

Amyotrophic lateral sclerosis (ALS) is a late-onset motor neuron degenerative disease of the spinal cord, brainstem, and cortex occurring both sporadically and as a familial disorder, the latter accounting for approximately 10% of the cases. The patients with ALS typically become progressively paralyzed, and respiratory failure eventually leads to death within 3 to 5 years (Mulder et al., 1986). The exact mechanism responsible for the motor degeneration is largely unknown, and there is no known effective treatment for this fatal disease. However, mutations in the ubiquitously expressed protein Cu,Zn-superoxide dismutase (SOD1) are associated with approximately 20% of familial ALS cases (Rosen et al., 1993). Because the pathology and clinical symptoms of familial and sporadic ALS cannot be distinguished, TG animal models overexpressing mutant SOD1 offer a valuable tool for understanding the pathogenic mechanisms shared by both sporadic and familial forms of ALS. It is noteworthy that several pathogenic SOD1 mutations do not affect SOD1 activity significantly, and "a toxic gain of function" of the mutated protein rather than a lack of its antioxidant function has been postulated. The nature of this gained toxic function is not known, even though several putative mechanisms have been proposed, including the formation of protein aggregates, mislocalization and aggregation of neurofilaments, increased free radical generation, mitochondrial dysfunction, and proapoptotic alterations (Bruijn et al., 2004). In addition, inflammation is strongly implicated in the pathogenesis of ALS, because microgliosis and astrogliosis with activation of stress-induced kinases and transcription factor NF-B accompany motor neuron degeneration either in ALS or SOD1 mutant mice (Migheli et al., 1997; Tortarolo et al., 2003). Moreover, expression of several proinflammatory mediators is increased both in ALS and SOD1 mutant mice (Alexianu et al., 2001; Elliott, 2001; Nguyen et al., 2001), and numerous anti-inflammatory compounds prolong survival of TG SOD1 mutant mice (Drachman et al., 2002; Kriz et al., 2002; Van Den Bosch et al., 2002; Zhu et al., 2002; Kiaei et al., 2006). In addition, animal models that express mutant SOD1 exclusively either in motor neurons (Pramatarova et al., 2001; Lino et al., 2002) or in astrocytes (Gong et al., 2000) do not develop the disease or pathologic phenotype, suggesting that interplay between glia and motor neurons is required for the ALS pathogenesis.

Because oxidative damage, inflammation, and even apoptosis play major roles in the pathology of ALS, we decided to test the effect of PDTC treatment on G93A-SOD1 TG rats, a disease model for human amyotrophic lateral sclerosis (Howland et al., 2002). We show that daily oral treatment with PDTC significantly decreases the survival of G93A-SOD1 TG rats. Our data indicate that this unexpected effect may be mediated by the prevention of induction of immunoproteasome, suggesting that immunoproteasome is a beneficial response in ALS to cope with accumulating protein aggregates. Because we found that the immunoproteasome was induced exclusively in astrocytes and microglia, our results support the idea of the important role of the non-neuronal cells adjacent of dying motor neurons.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
PDTC Treatment of ALS Rats. All animal studies were carried out under permission of The Institutional Animal Care and Use Committee of the University of Kuopio and the Provincial Government according to the National Institutes of Health guidelines for the care and use of laboratory animals. Transgenic rats [Tac:N:(SD)-TgN(SOD1G93A)L26H, Emerging Models Program sponsored by Amyotrophic Lateral Sclerosis Association, Taconic] expressing human mutant G93A-SOD1 were treated with PDTC on a dose of 50 mg/kg/day starting at 70 days of age (n = 19). Fresh PDTC (Sigma, St. Louis, MO) was administered into drinking water every other day. The control transgenic rats (n = 12) received plain water. In addition, wild-type (WT) litter mates of corresponding age received either PDTC (n = 6) or plain water (n = 7). Neurological signs of disease, such as overall locomotor activity, ataxia, extension reflex of hind limps, limp gait, paralysis of hind limp, or paralysis of fore limp and weight gain of the animals were followed daily, and appearance of limp gait was determined as the onset of the disease. New dose of PDTC was calculated weekly according to animal weight and water consumption, and the treatment was continued till the end stage of the disease when the animals were killed. The end stage of the disease was determined by righting reflex test where animal was placed on its side, and if it was not able to right itself in 30 s, it was scored as death. Sacrificed animals were perfused transcardially with saline, and extracted tissues were either frozen in liquid nitrogen and stored at -80°C or postfixed with 4% paraformaldehyde for 24 h and cryoprotected with 30% sucrose for 3 days and then frozen in liquid nitrogen and stored at -80°C.

Electrophoretic Mobility Shift Assay. Electrophoretic mobility shift assays (EMSA) for NF-B-DNA binding were carried out as described earlier in detail (Helenius et al., 1996) with 5 µg of nuclear protein of the spinal cord tissues at the presymptomatic age (100 d) and endstage. Double-stranded oligonucleotides for NF-B binding sites were from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA). The probe was labeled with T4 polynucleotide kinase (Promega, Madison, WI). Nonspecific binding was blocked with 2 µg of poly(dI-dC)/poly(dI-dC) (Roche Applied Science, Basel, Switzerland) in a 20-µl assay volume. Bound and free probes were separated in a native 4% polyacrylamide gel. Radioactive bands were visualized with a Storm 860 Imager (GE Healthcare, Little Chalfont, Buckinghamshire, UK) and pixel volumes of specific bands were calculated with ImageQuant software (GE Healthcare).

Atomic Absorption Spectrophotometry. Copper concentrations in the spinal cord, cortex, and liver tissues from each treatment group were measured by atomic absorption spectrophotometry at the City of Kuopio Environmental Health Laboratory by Hitachi Z-8100 Polarized Zeeman (Hitachi, Tokyo, Japan) graphite furnace atomic absorption spectrophotometry from pyrolyzed samples. Copper concentrations shown as microgram of copper per gram of tissue wet weight.

Proteasomal Activity. Proteasomal activity was measured from cytosolic fractions of the spinal cord samples as chymotrypsin-like activity by cleavage of N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Sigma). The activity was measured as increasing fluorescence of cleaved 7-amino-4-methylcoumarinpeptides. Tissues were homogenized in ice-cold buffer (50 mM Tris-HCl pH 7.5, 1 mM dithiothreitol, 0.25 M sucrose, 5 mM MgCl₂, 0.5 mM EDTA, and 2 mM ATP) and centrifuged 3500 rpm at 4°C for 7 min. Protein concentration of the supernatant was determined using Protein Assay reagent (Bio-Rad Laboratories, Hercules, CA). Proteasomal activity was measured in aliquots of 10 µg of protein in 50-µl volume of assay buffer (20 mM Tris-HCl pH 7.5, 1 mM ATP, 2 mM MgCl₂, and 0.1% bovine serum albumin containing 100 µM N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin). Fluorescence of cleaved 7-amino-4-methylcoumarinpeptides was monitored every 10 min at 37°C at 355 nm excitation and 460 nm emission using a Wallac 1420 multilabel counter (PerkinElmer Wallac, Gaithersburg, MD).

Western Blotting. Cytosolic proteins were separated by 10% or 12% SDS-polyacrylamide gel electrophoresis on Mini-Protean III electrophoresis device (Bio-Rad). After electrophoresis proteins were transferred to polyvinylidene difluoride membrane (GE Healthcare, Uppsala, Sweden) with a Mini-Protean II blotting cell (Bio-Rad) according to manufacturer's instructions and immunostained using rabbit polyclonal anti-proteasome 20S LMP7 (dilution 1:1000; Abcam, Cambridge, UK), rabbit polyclonal anti-proteasome 20S X (dilution 1:1000; Abcam), rabbit polyclonal anti-GLT-1 (dilution 1:1000; Calbiochem, La Jolla, CA), monoclonal anti--actin (dilution 1:4000, Sigma) or rabbit polyclonal anti-ubiquitin antibodies (dilution 1:1000; DakoCytomation Denmark A/S, Glostrup, Denmark) and horseradish peroxidase-labeled anti-mouse IgG (dilution 1:4000; GE Healthcare) or horseradish peroxidase-labeled anti-rabbit IgG (dilution 1:3000; GE Healthcare) secondary antibodies, followed by enhanced chemiluminescence detection (GE Healthcare). Quantifications were done on a STORM Imager (GE Healthcare) with ImageQuant software (GE Healthcare).

View larger version (32K): [in this window] [in a new window]   Fig. 1. PDTC treatment of ALS rats resulted in decreased survival. A, for all PDTC-treated TG animals, survival was 121 ± 21 days (gray line, n = 19) and 141 ± 12 days for TG animals that received plain H2O (black line, n = 12); **, p < 0.01. B, similarly, the onset of paralysis occurred earlier in PDTC-treated rats. Onset of paralysis occurred on average at 109 ± 22 days for TG PDTC (gray line) and 120 ± 14 days for untreated TG (black line) rats; **, p < 0.01. The disease duration from onset to the end stage was 12 ± 4 days for TG PDTC rats (C) and 11 ± 3 days for untreated TG rats (D). Weight gain for untreated TG animals compared with WT untreated (E) and PDTC-treated TG animals compared with untreated TG rats (F) did not show significant differences, but a decrease in weight was observed after the onset of disease, which occurred earlier in PDTC-treated rats compared with untreated animals.

Statistical Analyses. The results are presented as mean ± S.D. Differences between groups (^*, p < 0.05 was considered statistically significant) were determined by Student's t test (two-group comparisons) or with analysis of variance combined with Bonferroni posthoc test on SPSS software version 11.5 (SPSS Inc., Chicago, IL).

	Results

Top Abstract Materials and Methods Results Discussion References

 
PDTC Treatment Decreased Survival of G93A-SOD1 Transgenic Rats. PDTC treatment decreased the survival of G93A-SOD1 TG rats by 15% (140 ± 13 days in untreated group and 122 ± 21 days in PDTC group, p < 0.01) (Fig. 1A). Compared with untreated group of G93A-SOD1 TG rats, the onset of paralysis (Fig. 1B) occurred significantly earlier in PDTC-treated animals (109 ± 22 and 120 ± 14 days for PDTC TG and untreated TG, respectively; p < 0.01), whereas there was no difference in the duration of the disease from the onset to the end stage between PDTC- (12 ± 4 days) and vehicle-treated (11 ± 3 days) ALS rats (Fig. 1, C and D). Weight gain measured at or after the onset of the disease showed no significant differences between PDTC and untreated TG or WT rats (Fig. 1, E and F). The mean weight decreased earlier (weeks 13-14) in PDTC-treated TG mice because of the earlier onset of the disease in PDTC-treated group. We did not observe any significant signs of toxicity of the PDTC treatment, as judged by weight gain, consumption of drinking water, development of diarrhea, copper concentration of the liver, ataxia, overall locomotor activity, or immunohistochemical signs of increased gliosis or myelin loss in the ventral spinal cord.

The Effect of PDTC Was Not Mediated through NF-B Inhibition. NF-B activation may promote the expression of the genes that mediate inflammation or apoptosis and some genes that support survival. EMSA analysis of the spinal cord samples showed no differences in DNA binding activity of NF-B between PDTC and untreated groups (Fig. 2). However, although there was no statistically significant difference between any of groups, there was a trend toward increased DNA binding activity of NF-B in G93A-SOD1 TG rats.

View larger version (12K): [in this window] [in a new window]   Fig. 2. EMSA analysis of spinal cord samples showed no statistically significant differences in DNA binding activity of NF-B between PDTC and untreated groups; n = 5, results shown are mean ± S.D.

PDTC Treatment Increased the Levels of Ubiquitinated Proteins, Decreased Proteasome Activity, and Prevented the Immunoproteasome Induction in G93A-SOD1 TG Rats. As Cu-PDTC complexes may interfere with proteasomes, we investigated whether PDTC alters the amount of ubiquitinated proteins in the spinal cord. Immunoblotting revealed that the levels of ubiquitinated proteins were increased by 33% in the cytosolic fraction of the spinal cord of PDTC-treated TG rats at the end stage of the disease compared with untreated endstage animals (1507 ± 167 and 1136 ± 156, respectively; p < 0.05) (Fig. 3).

View larger version (26K): [in this window] [in a new window]   Fig. 3. Levels of ubiquitinated proteins increased significantly in PDTC-treated TG rats at the end stage of the disease (A) compared with untreated end-stage animals (*, p < 0.05), presymptomatics (**, p < 0.01), or WT rats (***, p < 0.001) (B); n = 5, results shown are mean ± S.D.

View larger version (19K): [in this window] [in a new window]   Fig. 4. The levels of immunoproteasome (LMP7) increased along with the disease progression in the spinal cord of G93A-SOD1 TG rats but not in the cortex. In G93A-SOD1 TG rats the levels of 20S LMP7 protein in the spinal cord was increased 6-fold between 8 and 16 weeks (**, p < 0.01) of age and reached 9-fold increase (***, p < 0.001) at the end stage (A and B). Levels of constitutive proteasome 20S X remained unchanged through the disease progression in the spinal cord (A and C); n = 4, results shown are mean ± S.D.

To investigate in detail whether PDTC alters the proteasome expression in the spinal cord, we used immunoblotting for 20S X and 20S LMP7, markers of constitutive and inducible proteasome, respectively. The level of 20S X was not changed in G93A-SOD1 TG rats at any time point of the disease progression (Fig. 4, A and C). Instead, the expression of immunoproteasome measured by immunoblotting for 20S LMP7, an inducible subunit, was strongly increased in the spinal cord but not in the cortex along with the disease progression of G93A-SOD1 TG rats (Fig. 4A). The amount of LMP7 protein was increased 6-fold between 8 and 16 weeks of age (334 ± 236 and 1905 ± 174, p < 0.01, when 8 weeks and 16 weeks were compared, respectively) and reached 9-fold increase at the end stage (3101 ± 603, p < 0.001) (Fig. 4B). PDTC treatment completely prevented the induction of 20S LMP7 at the end stage of G93A-SOD1 TG rats (1476 ± 479 and 2978 ± 1296, for PDTC treated and untreated, respectively; p < 0.05; Fig. 5, A and B), whereas no effect of PDTC on the constitutive proteasome subunit was detected. It is noteworthy that in WT animals, 20S LMP7 was barely detectable or undetectable in the cytosolic fraction (Fig. 5A), and PDTC had no effect on expression of this protein or 20S X in WT animals (Fig. 5A).

View larger version (18K): [in this window] [in a new window]   Fig. 5. PDTC led to decreased immunoproteasome levels and increased GLT-1 levels in TG rats at the end stage, whereas PDTC had no effect on the levels of constitutive proteasome, immunoproteasome, or GLT-1 in WT rats. PDTC treatment completely prevented the induction of 20S LMP7 at the end stage of G93A-SOD1 TG rats (A and B), whereas no effect of PDTC on the constitutive proteasome subunit was detected. A, it is noteworthy that in WT animals, 20S LMP7 was barely detectable or undetectable in the cytosolic fraction, and PDTC had no effect on the expression of this protein or 20S X in WT animals. PDTC also had an effect on the levels of astrocyte specific glutamate transporter (GLT-1). A and C, in the spinal cords of untreated TG rats, the levels of GLT-1 were decreased, whereas in PDTC-treated TG rats, the levels of GLT-1 were at the same levels as in WT rats. *, p < 0.05, n = 5, results shown are mean ± S.D.

The changes in immunoproteasome levels after PDTC treatment were not due to common reduction of astroglial functions, because PDTC also increased the levels of astrocyte-specific glutamate transporter (GLT-1). In the spinal cords of untreated TG rats, the levels of GLT-1 were decreased, whereas in PDTC-treated TG rats, the levels of GLT-1 were at the same levels as in WT rats (Fig. 5, A and C).

Immunoproteasome Induction in G93A-SOD1 TG Was Localized in Glia. Constitutive proteasome was expressed in cells throughout the gray matter in the ventral horn of the lumbar spinal cord in both PDTC-treated TG and WT rats and in untreated TG and WT rats (Fig. 6A). However, immunoproteasome was expressed only in untreated TG rats at the end stage (Fig. 6B). From the appearance, the cells showing immunoproteasome staining looked non-neuronal. Double-labeling immunohistochemistry with confocal imaging showed that the immunoproteasome 20S LMP7 was expressed in astrocytes and microglia (Fig. 7B), whereas proteasome 20S X was also expressed in neurons (Fig. 7A).

View larger version (49K): [in this window] [in a new window]   Fig. 6. 20S X and 20S LMP7 expression in lumbar spinal cord. A, 20S X, a marker for constitutive proteasome, was expressed throughout the gray matter in the ventral horn in both PDTC-treated TG and WT rats and in untreated TG and WT rats. B, 20S LMP7, an inducible -subunit of immunoproteasome, was expressed only in the spinal cords of the untreated TG rats in the end stage but not in the PDTC-treated or WT rats. Magnification, 100x.

View larger version (48K): [in this window] [in a new window]   Fig. 7. Expression of constitutive proteasome was localized in neurons and glia, whereas immunoproteasome was expressed only in glia. A, constitutive proteasome 20S X was expressed in astrocytes (stained with glial fibrillary acidic protein), neurons (stained with NeuN), and microglia (stained with CD68). B, immunoproteasome 20S LMP7 was expressed mainly in astrocytes and microglia. Arrows point to cells that have colocalized cell marker and proteasome staining. Arrowheads point to non-neuronal, presumably microglial cell. Double-staining with confocal imaging. Magnification, 400x.

	Discussion

Top Abstract Materials and Methods Results Discussion References

In agreement with the previous studies on mouse ALS models (Cheroni et al., 2005; Puttaparthi and Elliott, 2005), we observed that both increase in proteasome activity and induction of immunoproteasome selectively occur in the affected spinal cord tissue of a G93A-SOD1 TG rat model. Three catalytic subunits of the proteasome, namely 1, 2, and 5, have close homologs, LMP2, MECL-1, and LMP7, that are selectively induced under certain conditions, such as the treatment of cells with -interferon. This chance in subunit composition is believed to be involved in antigen processing/presentation and may influence catalytic specificity, at which different catalytic sites hydrolyze peptide bonds which may be relevant for immune response (DeMartino and Slaughter, 1999). It is noteworthy that we found that PDTC treatment strongly inhibited these ALS model-specific changes in proteasome. On the other hand, in line with previous reports showing that PDTC is a metal chelator and transports Cu²⁺ from the extracellular medium into the cell, we found that PDTC treatment increased copper concentration in the spinal cord of both G93A-SOD1 TG and WT rats. Considering that PDTC is also known as a proteasome inhibitor (Kim et al., 2004) and that increased Cu²⁺ concentration may be needed for the proteasome inhibitory activity of PDTC, it is likely that the detrimental effect of PDTC on G93A-SOD1 TG rats is mediated by the inhibition of immunoproteasome. This inhibition of immunoproteasome was also evidenced as increased levels of ubiquitinated proteins in PDTC-treated G93A-SOD1 TG rats. Because 20S X, a marker of constitutive proteasome, was not affected in G93A-SOD1 TG rats or by PDTC treatment, whereas 20S LMP7, an inducible -subunit of immunoproteasome, was induced selectively in astrocytes and microglia, our results suggest that immunoproteasome in non-neuronal cells plays a protective role in G93A-SOD1 TG rats.

In addition to its effect on proteasome activity, increased copper concentration may well trigger other cellular processes, which in animals overexpressing G93A-SOD1 could accelerate neurodegeneration. An oral, 15-day PDTC treatment at millimolar concentrations has been reported to increase Cu²⁺ concentration and levels of lipid peroxidation products in rat peripheral nerves (Calviello et al., 2005), implicating that at least in some rodent tissues, a long-term PDTC treatment may cause oxidative stress associated with increased Cu²⁺ concentrations. On the other hand, a 7-month PDTC treatment with the same protocol as in the present study significantly increases cortical copper concentrations in a TG mouse model of Alzheimer's disease and prevents the decrease in cognition without increasing oxidative stress in the brain (Malm et al., submitted for publication). Even though the induction of immunoproteasome was observed selectively in G93A-SOD1 TG rat spinal cords and PDTC prevented this induction without causing changes in other known targets of PDTC, we cannot exclude the possibility that the increased copper concentration enhances motor neuron degeneration in G93A-SOD1 TG rats also by other mechanisms in parallel with immunoproteasome inhibition. However, it should be noted that -lactams, such as ceftriaxone, which have been reported to be neuroprotective in models of ALS by increasing expression of glutamate transporter GLT-1 (Rothstein et al., 2005), are also metal chelators. PDTC resembles -lactams because it significantly increases the expression of GLT-1 and Cu²⁺ concentration in the spinal cord. We hypothesize that even though -lactams and PDTC might both be able to modulate GLT-1 and Cu²⁺ concentration, only PDTC but not -lactams inhibits immunoproteasome. Because induction of immunoproteasome may be a rather selective characteristic for ALS (models) compared with models of ischemia, trauma, and amyloid-accumulating diseases, inhibition of immunoproteasome alone in ALS models could increase the accumulation of ubiquitinated proteins, including ubiquitinated SOD1, which has been suggested to gain neurotoxic functions such as increased peroxidase activity.

PDTC is an established inhibitor of NF-B. Even though inhibition of NF-B has frequently been associated with tissue and cellular protection, inhibition of NF-B may also accelerate neurodegeneration because of the survivalsupporting role of some NF-B-regulated genes, such as manganese SOD and Bcl-2 (Mattson and Camandola, 2001). Even though we detected a trend for increased NF-B binding activity in the spinal cords of G93A-SOD1 TG rats, no statistically significant differences between any of the untreated/PDTC-treated TG and WT rat groups were observed. It is possible that in a long-term disease such as ALS, NF-B activity, even though being induced, is not maintained at so high levels, and on the other hand, oral PDTC administration may not allow achieving tissue concentrations as high as intraperitoneal administration does, and thereby does not result in efficient inhibition of NF-B. In addition, we cannot exclude the possibility that NF-B binding to DNA is increased at time points other than the presymptomatic (100 days) and end-stage time points studied here. Nevertheless, our experiments do not provide evidence for a central role of NF-B in ALS. In a previous study, NF-B immunoreactivity was found to be increased in astrocytes surrounding degenerating motor neurons (Migheli et al., 1997). Therefore, the possible antiapoptotic effects of NF-B may not be, at least, directly involved in neuronal survival in TG ALS models.

In summary, we report that PDTC, a multipotent drug providing protection in various animal models by inhibiting NF-B, acting as an antioxidant and activating Akt-GSK3 pathway, also induces GLT-1, a potential drug target in brain diseases. Regardless of these beneficial effects, oral daily treatment with PDTC at the dose of 50 mg/kg reduces the survival of G93A-SOD1 TG rats, possibly by preventing immunoproteasome activity in non-neuronal cells and thereby accelerating the formation of toxic SOD1-containing protein aggregates. The reduced survival of PDTC-treated G93A-SOD1 TG rats is likely not due to PDTC toxicity, because the onset of specific disease symptoms also occurred earlier, and we did not observe any side effects in PDTC-treated rats. Moreover, a similar treatment protocol is beneficial in a rodent model of Alzheimer's disease (Malm et al., submitted for publication), whereas PDTC treatment even at substantially lower doses (10 mg/kg) is not beneficial in a TG model of ALS (L. van den Bosch, T. Ahtoniemi, J. Kolstinaho, unpublished data). Immunoproteasome may be important for coping with the toxic consequences of mutant SOD1 in tissues affected by ALS.

	Footnotes

 
This work was supported by the Sigrid Juselius foundation.

ABBREVIATIONS: PDTC, pyrrolidine dithiocarbamate; ALS, amyotrophic lateral sclerosis; EMSA, electrophoretic mobility shift assay; NF-B, nuclear factor B; SOD, superoxide dismutase; WT, wild type; TG, transgenic; GLT, glutamate transporter.

Address correspondence to: Dr. Jari Koistinaho, Department of Neurobiology, A. I. Virtanen Institute for Molecular Sciences, University of Kuopio, P.O.B. 1627, FIN-70211 Kuopio, Finland. E-mail: jari.koistinaho{at}uku.fi

	References

Top Abstract Materials and Methods Results Discussion References

 
Alexianu ME, Kozovska M, and Appel SH (2001) Immune reactivity in a mouse model of familial ALS correlates with disease progression. Neurology 57: 1282-1289.[Abstract/Free Full Text]

Bruijn LI, Miller TM, and Cleveland DW (2004) Unraveling the mechanisms involved in motor neuron degeneration in ALS. Annu Rev Neurosci 27: 723-749.[CrossRef][Medline]

Calviello G, Filippi GM, Toesca A, Palozza P, Maggiano N, Nicuolo FD, Serini S, Azzena GB, and Galeotti T (2005) Repeated exposure to pyrrolidine-dithiocarbamate induces peripheral nerve alterations in rats. Toxicol Lett 158: 61-71.[CrossRef][Medline]

Cheng G, Whitehead SN, Hachinski V, and Cechetto DF (2006) Effects of pyrrolidine dithiocarbamate on beta-amyloid (25-35)-induced inflammatory responses and memory deficits in the rat. Neurobiol Dis 23: 140-151.[CrossRef][Medline]

Cheroni C, Peviani M, Cascio P, Debiasi S, Monti C, and Bendotti C (2005) Accumulation of human SOD1 and ubiquitinated deposits in the spinal cord of SOD1G93A mice during motor neuron disease progression correlates with a decrease of proteasome. Neurobiol Dis 18: 509-522.[CrossRef][Medline]

Cuzzocrea S, Chatterjee PK, Mazzon E, Dugo L, Serraino I, Britti D, Mazzullo G, Caputi AP, and Thiemermann C (2002) Pyrrolidine dithiocarbamate attenuates the development of acute and chronic inflammation. Br J Pharmacol 135: 496-510.[CrossRef][Medline]

DeMartino GN and Slaughter CA (1999) The proteasome, a novel protease regulated by multiple mechanisms. J Biol Chem 274: 22123-22126.[Free Full Text]

Drachman DB, Frank K, Dykes-Hoberg M, Teismann P, Almer G, Przedborski S, and Rothstein JD (2002) Cyclooxygenase 2 inhibition protects motor neurons and prolongs survival in a transgenic mouse model of ALS. Ann Neurol 52: 771-778.[CrossRef][Medline]

Elliott JL (2001) Cytokine upregulation in a murine model of familial amyotrophic lateral sclerosis. Brain Res Mol Brain Res 95: 172-178.[Medline]

Gong YH, Parsadanian AS, Andreeva A, Snider WD, and Elliott JL (2000) Restricted expression of G86R Cu/Zn superoxide dismutase in astrocytes results in astrocytosis but does not cause motoneuron degeneration. J Neurosci 20: 660-665.[Abstract/Free Full Text]

Gurney ME, Cutting FB, Zhai P, Doble A, Taylor CP, Andrus PK, and Hall ED (1996) Benefit of vitamin E, riluzole, and gabapentin in a transgenic model of familial amyotrophic lateral sclerosis. Ann Neurol 39: 147-157.[CrossRef][Medline]

Hayakawa M, Miyashita H, Sakamoto I, Kitagawa M, Tanaka H, Yasuda H, Karin M, and Kikugawa K (2003) Evidence that reactive oxygen species do not mediate NF-kappaB activation. EMBO (Eur Mol Biol Organ) J 22: 3356-3366.[CrossRef][Medline]

Helenius M, Hanninen M, Lehtinen SK, and Salminen A (1996) Changes associated with aging and replicative senescence in the regulation of transcription factor nuclear factor-kappa B. Biochem J 318 (Pt 2):603-608.

Howland DS, Liu J, She Y, Goad B, Maragakis NJ, Kim B, Erickson J, Kulik J, DeVito L, Psaltis G, et al. (2002) Focal loss of the glutamate transporter EAAT2 in a transgenic rat model of SOD1 mutant-mediated amyotrophic lateral sclerosis (ALS). Proc Natl Acad Sci USA 99: 1604-1609.[Abstract/Free Full Text]

Kiaei M, Petri S, Kipiani K, Gardian G, Choi DK, Chen J, Calingasan NY, Schafer P, Muller GW, Stewart C, et al. (2006) Thalidomide and lenalidomide extend survival in a transgenic mouse model of amyotrophic lateral sclerosis. J Neurosci 26: 2467-2473.[Abstract/Free Full Text]

Kim I, Kim CH, Kim JH, Lee J, Choi JJ, Chen ZA, Lee MG, Chung KC, Hsu CY, and Ahn YS (2004) Pyrrolidine dithiocarbamate and zinc inhibit proteasomedependent proteolysis. Exp Cell Res 298: 229-238.[CrossRef][Medline]

Koh SH, Lee YB, Kim KS, Kim HJ, Kim M, Lee YJ, Kim J, Lee KW, and Kim SH (2005) Role of GSK-3beta activity in motor neuronal cell death induced by G93A or A4V mutant hSOD1 gene. Eur J Neurosci 22: 301-309.[CrossRef][Medline]

Kriz J, Nguyen MD, and Julien JP (2002) Minocycline slows disease progression in a mouse model of amyotrophic lateral sclerosis. Neurobiol Dis 10: 268-278.[CrossRef][Medline]

La Rosa G, Cardali S, Genovese T, Conti A, Di Paola R, La Torre D, Cacciola F, and Cuzzocrea S (2004) Inhibition of the nuclear factor-kappaB activation with pyrrolidine dithiocarbamate attenuating inflammation and oxidative stress after experimental spinal cord trauma in rats. J Neurosurg Spine 1: 311-321.[Medline]

Lino MM, Schneider C, and Caroni P (2002) Accumulation of SOD1 mutants in postnatal motoneurons does not cause motoneuron pathology or motoneuron disease. J Neurosci 22: 4825-4832.[Abstract/Free Full Text]

Liu SF, Ye X, and Malik AB (1999) Inhibition of NF-kappaB activation by pyrrolidine dithiocarbamate prevents In vivo expression of proinflammatory genes. Circulation 100: 1330-1337.

Matsui N, Kasajima K, Hada M, Nagata T, Senga N, Yasui Y, Fukuishi N, and Akagi M (2005) Inhibition of NF-kappaB activation during ischemia reduces hepatic ischemia/reperfusion injury in rats. J Toxicol Sci 30: 103-110.[CrossRef][Medline]

Mattson MP and Camandola S (2001) NF-kappaB in neuronal plasticity and neurodegenerative disorders. J Clin Investig 107: 247-254.[Medline]

Messina S, Bitto A, Aguennouz M, Minutoli L, Monici MC, Altavilla D, Squadrito F, and Vita G (2006) Nuclear factor kappa-B blockade reduces skeletal muscle degeneration and enhances muscle function in Mdx mice. Exp Neurol 198: 234-241.[CrossRef][Medline]

Migheli A, Piva R, Atzori C, Troost D, and Schiffer D (1997) c-Jun, JNK/SAPK kinases and transcription factor NF-kappa B are selectively activated in astrocytes, but not motor neurons, in amyotrophic lateral sclerosis. J Neuropathol Exp Neurol 56: 1314-1322.[Medline]

Mulder DW, Kurland LT, Offord KP, and Beard CM (1986) Familial adult motor neuron disease: amyotrophic lateral sclerosis. Neurology 36: 511-517.[Abstract]

Nguyen MD, Julien JP, and Rivest S (2001) Induction of proinflammatory molecules in mice with amyotrophic lateral sclerosis: no requirement for proapoptotic interleukin-1 beta in neurodegeneration. Ann Neurol 50: 630-639.[CrossRef][Medline]

Nurmi A, Goldsteins G, Narvainen J, Pihlaja R, Ahtoniemi T, Grohn O, and Koistinaho J (2006) Antioxidant pyrrolidine dithiocarbamate activates Akt-GSK signaling and is neuroprotective in neonatal hypoxia-ischemia. Free Radic Biol Med 40: 1776-1784.[CrossRef][Medline]

Nurmi A, Lindsberg PJ, Koistinaho M, Zhang W, Juettler E, Karjalainen-Lindsberg ML, Weih F, Frank N, Schwaninger M, and Koistinaho J (2004a) Nuclear factor-kappaB contributes to infarction after permanent focal ischemia. Stroke 35: 987-991.[Abstract/Free Full Text]

Nurmi A, Vartiainen N, Pihlaja R, Goldsteins G, Yrjanheikki J, and Koistinaho J (2004b) Pyrrolidine dithiocarbamate inhibits translocation of nuclear factor kappa-B in neurons and protects against brain ischaemia with a wide therapeutic time window. J Neurochem 91: 755-765.[CrossRef][Medline]

Pramatarova A, Laganiere J, Roussel J, Brisebois K, and Rouleau GA (2001) Neuronspecific expression of mutant superoxide dismutase 1 in transgenic mice does not lead to motor impairment. J Neurosci 21: 3369-3374.[Abstract/Free Full Text]

Puttaparthi K and Elliott JL (2005) Non-neuronal induction of immunoproteasome subunits in an ALS model: possible mediation by cytokines. Exp Neurol 196: 441-451.[CrossRef][Medline]

Reisinger EC, Kern P, Ernst M, Bock P, Flad HD, and Dietrich M (1990) Inhibition of HIV progression by dithiocarb. German DTC Study Group. Lancet 335: 679-682.[CrossRef][Medline]

Rosen DR, Siddique T, Patterson D, Figlewicz DA, Sapp P, Hentati A, Donaldson D, Goto J, O'Regan JP, Deng HX, et al. (1993) Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature (Lond) 362: 59-62.[CrossRef][Medline]

Rothstein JD, Patel S, Regan MR, Haenggeli C, Huang YH, Bergles DE, Jin L, Dykes Hoberg M, Vidensky S, Chung DS, et al. (2005) Beta-lactam antibiotics offer neuroprotection by increasing glutamate transporter expression. Nature (Lond) 433: 73-77.[CrossRef][Medline]

Schreck R, Meier B, Mannel DN, Droge W, and Baeuerle PA (1992) Dithiocarbamates as potent inhibitors of nuclear factor kappa B activation in intact cells. J Exp Med 175: 1181-1194.[Abstract/Free Full Text]

Tortarolo M, Veglianese P, Calvaresi N, Botturi A, Rossi C, Giorgini A, Migheli A, and Bendotti C (2003) Persistent activation of p38 mitogen-activated protein kinase in a mouse model of familial amyotrophic lateral sclerosis correlates with disease progression. Mol Cell Neurosci 23: 180-192.[CrossRef][Medline]

Van Den Bosch L, Tilkin P, Lemmens G, and Robberecht W (2002) Minocycline delays disease onset and mortality in a transgenic model of ALS. Neuroreport 13: 1067-1070.[CrossRef][Medline]

Wu AS, Kiaei M, Aguirre N, Crow JP, Calingasan NY, Browne SE, and Beal MF (2003) Iron porphyrin treatment extends survival in a transgenic animal model of amyotrophic lateral sclerosis. J Neurochem 85: 142-150.[Medline]

Zhu S, Stavrovskaya IG, Drozda M, Kim BY, Ona V, Li M, Sarang S, Liu AS, Hartley DM, Wu du C, et al. (2002) Minocycline inhibits cytochrome c release and delays progression of amyotrophic lateral sclerosis in mice. Nature (Lond) 417: 74-78.[CrossRef][Medline]

This Article Abstract Full Text (PDF) All Versions of this Article: mol.106.028415v1 71/1/30    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Ahtoniemi, T. Articles by Koistinaho, J. Search for Related Content PubMed PubMed Citation Articles by Ahtoniemi, T. Articles by Koistinaho, J.