Dimethyl sulfoxide delays PrPsc accumulation and disease symptoms in prion-infected hamsters

Brain Research 983 (2003) 137–143 www.elsevier.com / locate / brainres

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Dimethyl sulfoxide delays PrP sc accumulation and disease symptoms in prion-infected hamsters Gideon M. Shaked a , Roni Engelstein a , Inbal Avraham a , Esther Kahana b , Ruth Gabizon a , * a

Department of Neurology, The Agnes Ginges Center for Human Neurogenetics, Hadassah University Hospital, Jerusalem 91120, Israel b Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Shiva, Israel Accepted 22 May 2003

Abstract PrP Sc , an aberrantly folded protein, is the only identified component of the prion, an agent causing fatal neurodegenerative diseases such as scrapie and bovine spongiform encephalopathy. Dimethyl sulfoxide (DMSO) has been shown to reduce the accumulation of PrP Sc in scrapie-infected (ScN2a) cells, and to inhibit its aggregation in vitro. In humans, DMSO was used successfully in the treatment of various peripheral amyloidotic diseases. Here we show that administration of DMSO to scrapie-infected hamsters significantly prolonged disease incubation time, as well as delayed the accumulation of PrP Sc in Syrian hamster brains. Interestingly, administration of DMSO to scrapie sick hamsters resulted in increased clearance of protease-resistant PrP in their urine. We conclude that although DMSO by itself may not be sufficient to cure prion diseases, it may be considered as a component in a ‘cocktail’ drug approach for these disorders. Also, urine PrP testing should be considered for the assessment of treatment efficacy.  2003 Elsevier B.V. All rights reserved. Keywords: Dimethyl sulfoxide; PrP sc accumulation; Prion; Hamster; Scrapie; Bovine spongiform encephalopathy; Creutzfeldt–Jakob disease

1. Introduction Prion diseases are fatal neurodegenerative diseases which include, among others, Creutzfeldt–Jakob disease (CJD) in humans, as well as scrapie and bovine spongiform encephalopathy (BSE) in animals [27]. PrP Sc , a post-translationally modified conformational isoform of a normal protein, PrP C [23] is the only identified component of the infectious agent, denominated prion [25]. During the disease, PrP Sc accumulates in the brains of infected humans and animals [28]. Thereby, an important objective in the treatment of prion diseases should be to reduce the accumulation of PrP Sc in brain cells. Several approaches can be envisioned to achieve this goal. One possibility is to inhibit the conversion of PrP C to PrP Sc , most probably by stabilizing the a-helical structure of PrP C [7,17]. Another possibility would be to reduce the total amount of expressed PrP C . This may be achieved either by using neutralizing antibodies against the normal prion protein or by molecular methods such as anti sense PrP *Corresponding author. Tel.: 1972-2-677-7858; fax: 1972-2-6429441. E-mail address: [email protected] (R. Gabizon). 0006-8993 / 03 / $ – see front matter  2003 Elsevier B.V. All rights reserved. doi:10.1016 / S0006-8993(03)03045-2

mRNA [7,18]. Yet another possibility could be to inhibit the aggregation of PrP Sc once it is formed [8,34]. Reduced aggregation may result in increased clearance of the pathogenic protein from the brain, and thereby diminish the interaction of PrP Sc with targets in as yet unaffected brain cells. A large array of reagents has been shown to affect the accumulation of PrP Sc in ScN2a cells [4]. These cells have been used extensively as an in vitro model for prion diseases [5,10]. Among these reagents, Congo Red, DMSO, Pentosan Sulfate, Quinacrine, anti PrP antibodies, amphothericin [2,3] as well as many others [6,9,13,20,26,33,38,40]. While for some reagents a mechanism of anti prion activity was suggested, for others their way of action remains to be established. For example, we have shown that while DMSO could not solubilize preformed PrP Sc aggregates, it effectively inhibited the aggregation of PrP Sc molecules that occurs when membranes from scrapie-infected hamsters were extracted by detergents [34]. DMSO has been proven to be effective in the treatment of several peripheral amyloid diseases [19,31,32]. In addition, DMSO was shown to block the formation of an intermediate high in b-sheet content, which is a controlling

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step in the process of Ab peptide self assembly into amyloid plaques [37]. Interestingly, following a single dose of DMSO, the urine of human amyloidotic patients contained fibrils, which could be then stained with Congo Red [30]. This suggests that DMSO might either inhibit the formation or enhance solubilization of amyloid fibrils so that amyloidotic proteins are mobilized from the target tissue and eliminated by the kidneys. In this work, we tested the effect of DMSO on the length of prion disease incubation time in hamsters infected with prions, as well as on the accumulation of PrP Sc in the brains of these animals. Our results show that administration of DMSO to hamsters incubating scrapie significantly increased disease incubation time. In parallel, the accumulation rate of PrP Sc in the brains of the animals was decreased. In addition, DMSO administration markedly increased the excretion of UPrP Sc in the urine of the treated animals. This may suggest that PrP Sc molecules that were cleared from the brain due to reduced aggregation may subsequently be excreted in urine.

2. Materials and methods

2.1. Brain homogenates Hamster brains were homogenized in 10 volumes of homogenization buffer (10 mM Tris–HCl, pH 7.5, 300 mM sucrose). Following centrifugation (2000 rpm, 15 min, 4 8C), the supernatants were aliquoted and frozen (280 8C).

2.2. In vivo prion experiments Syrian hamsters, 4 weeks old, were inoculated with 50 ml of a 1 / 100 scrapie brain homogenate (of the 263 K stain) and examined daily for scrapie-associated symptoms as in Shaked et al. [34]. For the intracerebrally (i.c.) inoculated animals, infectivity titers were calculated from incubation time according to Prusiner et al. [29]. DMSO was administered via drinking water as described. Water bottles in DMSO groups were capped with cork because this solvent rapidly dissolves rubber [22]. Drinking solution was replaced daily and administered ad libitum. Since the concentration of DMSO in the water was 7.5% and each hamster consumed about 30 ml of water daily, we conclude that the daily dose per animal was on average 0.25 g DMSO.

2.3. Statistical analysis Titer values for i.c. inoculated, or incubation period (days) for intraperitoneally (i.p.) inoculated hamsters were statistically analyzed by one-way analysis of variance (ANOVA) and the non-parametric Kruskal–Wallis test, followed by a comparison of all groups versus control

group (Dunn’s method). In addition to prion disease symptoms, animal weight was also followed up periodically. Weight measurements were statistically analyzed by the unpaired t-test method.

2.4. Urine samples Hamster urine samples were collected, dialyzed, and further processed for SDS–PAGE Western blot according to Shaked et al. [36].

3. Results

3.1. DMSO administration prolongs scrapie incubation time in hamsters DMSO was added to the drinking water of Syrian hamsters inoculated either intracerebrally (i.c.) or intraperitoneally (i.p.) with scrapie brain homogenate, as described in Section 2. The DMSO concentrations used were based on those reported previously in the literature [21,22]. The DMSO–water solution was administered to the infected or control hamsters for various periods of time. For i.c. inoculated animals: (1) from 0 days post infection (dpi) to disease; (2) 0–14 dpi; (3) 14 dpi to disease; and (4) 55 dpi to disease. Intraperitoneally inoculated animals were treated from 0, 28, and 55 dpi to disease. Scrapie incubation time (days), as well as titer values (infectivity) are presented in Table 1. Statistical analysis were performed as described (Section 2). The results described in Table 1 indicate that administration of DMSO to hamsters infected with prions may significantly increase disease incubation time when compared to that observed in untreated hamsters (Log ID 50 5 6.0). This was true for groups treated with DMSO either from 0–end (Log ID 50 54.7) or from 14–end (Log ID 50 5 4.5). No significant effect was observed following DMSO treatment for 0–14 dpi (Log ID 50 56.5) as well as for those treated from 55 dpi to end (Log ID 50 56.2). DMSO was similarly administered to hamsters inoculated intraperitoneally with prions. In these experiments (Table 2), only animals treated with DMSO from 55 dpi to disease showed a significant increase in scrapie incubation time (median life span5136.8) as compared to controls (median life span5121). For the animals treated 0–end and 28–end, the effect was only marginal (median life span5127.6, 126.6, respectively), and not statistically significant. No significant difference could be appreciated in the length of the disease (symptoms to death) between treated and untreated groups (about 10–15 days). However, it should be taken into account that disease length is very short in the 263 K hamster scrapie model. In addition, for ethical reasons, we are required by our institution to euthanize animals when they show difficulties in obtaining food and water. The different effect of DMSO on the scrapie incubation

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Table 1 DMSO administration to i.c inoculated hamsters DMSO treatment (dpi)

No. of animals

Incubation period (days)a

Standard error (days)

Infectivity (Log ID 50 )

Control 0–end 14–end 0–14 55–end

20 10 10 5 5

93 101 107 90 91

1.4 3. 7 1.6 1.6 5.3

6.0 4.7 4.5 6.5 6.2

Variance from control group (P,0.05) 1 1 – –

DMSO was orally administered to i.c. scrapie-infected hamsters for different periods of time during disease incubation. Results are presented both as incubation time to disease and as titers. Titer calculation and statistical analysis are described in Section 2. a Incubation period means the number of days elapsed from prion inoculation to clinical symptoms.

time of i.c. as compared to i.p. scrapie-inoculated hamsters can be attributed to several mechanisms. DMSO probably affects the aggregation process in vivo at its first stages, when large PrP Sc aggregates are still not apparent in brain tissue. Following i.c. inoculation of hamster prions, a detectable amount of PrP Sc is apparent in the brain at 55 days of incubation. This is not the case after i.p. inoculation, which requires about 70 days of incubation before PrP Sc can be detected [11,12,39]. This suggests that DMSO treatment should start before a significant amount of PrP Sc is accumulated in the brains of the infected animals. In addition, there may be a physiological conflict between the beneficial anti-prion effect of DMSO, to its adverse effects, such as weight loss (see below). Since the i.p. incubation time is considerably longer than i.c. incubation time, the adverse effects of long-term administration of DMSO should be more apparent in the i.p. experiments. This suggest there may be a ‘therapeutic window’ in prion disease incubation time for the in vivo anti-prion DMSO activity, in which the conversion of PrP C to PrP Sc has already been established but the number of aggregates is still minimal. Such window could be different for each way of infection. Since it has been shown that following i.p. inoculation [11], PrP Sc accumulates first in peripheral tissues, such as the spleen, it is important to test whether DMSO can inhibit disease if administered only for the first 50 days following i.p. inoculation, thereby inhibiting neuroinvation. Table 2 DMSO administration to i.p inoculated hamsters DMSO treatment (dpi)

No. of animals

Incubation period a (days)

Standard error

Control 0–end 28–end 55–end

5 5 5 5

122 127.6 126.6 136.8

2.25 0.98 1.94 3.69

Variance from control group (P,0.05) – – 1

DMSO was orally administered to i.p. scrapie-infected hamsters for different periods of time during disease incubation. Results are presented as incubation time (days). Statistical analysis are described in Section 2. a Incubation period means the number of days elapsed from prion inoculation to severe clinical symptoms.

3.2. Long-term administration of DMSO resulted in weight loss Scrapie-infected and control hamsters drinking DMSO from 0 dpi to disease, display a considerable weight loss (P,0.001) at all inspected time points (30, 60, 90 dpi), when compared to animals to which DMSO was not administered. This suggests that the weight loss is indeed induced by DMSO and is independent from the effects of the prion disease incubation (Fig. 1a). DMSO may indeed cause systemic effects such as gastro-intestinal disturbances [1,15]. When DMSO was administered to the scrapie-infected animals later in the incubation period (55 dpi), the overall weight loss was reduced, as compared to long-term administration of the drug (Fig. 1b). We speculate that the apparent reduced anti-prion effect following long-term administration of DMSO is at least partially due to the toxic effects observed following the administration of this dose of the drug.

3.3. DMSO treatment delays the accumulation of PrP Sc in infected hamsters To determine whether DMSO administration affects PrP Sc brain accumulation, three scrapie-infected hamsters treated with or without DMSO were sacrificed at several time points during the incubation time. Similar samples of all brains were homogenized, digested with Proteinase K (PK) and immunoblotted with aPrP mAb 3F4. Brains were collected from i.c. inoculated animals at 55, 67, 78 and 105 dpi for the DMSO-treated animals and at 55, 67, 78 and 90 dpi for control hamsters (Fig. 2). PrP Sc was clearly visible at 55dpi for the untreated group but this was not the case for the scrapie-infected hamsters receiving the DMSO treatment. Comparable PrP Sc concentrations were found only following appearance of disease symptoms, practically close to the death of the animals. Interestingly, no difference was observed in the PrP Sc band pattern in the brains of DMSO treated and untreated animals, suggesting that despite the different kinetics of the disease, DMSO did not alter the strain of prions affecting the animals (Fig. 3).

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Fig. 1. DMSO treatment-induced weight loss in hamsters. (a) Normal and scrapie-infected hamsters were inoculated i.p. or i.c. with scrapie-infected hamster brain homogenate. To some infected and control animals DMSO was administered as described in Section 2. Weights (g) were measured at 30, 60, 90 dpi, and statistical values calculated as described in the Section 2. (b) Scrapie-infected hamsters were treated with DMSO for several periods of time during disease incubation; 0, 28, or 55 dpi to end for i.p. inoculated hamsters, and 0, 14, or 55 dpi to end for i.c. inoculated hamsters. Weights were measured at 30, 60, 90 dpi, and statistical values calculated as described in Section 2.

3.4. DMSO administration enhances the appearance of UPrP Sc in the urine of scrapie-infected animals DMSO was already shown to induce the release into urine of other amyloidogenic proteins [16]. We have recently found that a PrP isoform can be identified in the

urine of scrapie-infected hamsters, as well as in the urine of other animals and humans affected with prion diseases [36]. To investigate whether DMSO may affect the accumulation of PK-resistant UPrP in the urine of scrapie-infected Syrian hamsters, we administered DMSO for 6 days to a

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Fig. 4. DMSO increases the clearance of UPrP Sc into the urine of scrapie sick hamsters. Scrapie sick hamsters were treated with a 7.5% DMSO solution in their drinking water for 6 days. Urine samples were collected daily and immediately frozen. At the end of the experiment all samples were thawed and processed for the detection of UPrP Sc . Time points; 0, scrapie sick hamsters prior to treatment; 1, 3, 6 days of treatment; 7, 8 consecutive days after cessation of treatment.

Fig. 2. DMSO administration inhibits PrP sc accumulation. Syrian hamsters inoculated intracerebrally with scrapie brain homogenate, were treated with or without DMSO in the drinking water (14 dpi–end) as described above. Animals were sacrificed at specific time points in the incubation time and comparable samples of brain homogenates were immunoblotted for protease-resistant PrP with aPrP mAb 3F4. Days of incubation were 55, 67, 78 dpi and 105 dpi for the DMSO-treated animals or 90 dpi for control hamsters.

group of i.c. inoculated hamsters that have already shown first symptoms of disease. Urine was collected before, during, and after this period. As can be seen in Fig. 4, DMSO administration resulted in a considerable increase in PK-resistant UPrP levels. Once the DMSO treatment was terminated, the levels of PK-resistant PrP in urine went back to the original baseline within 2 days. These results are consistent with our in vitro results [34],

Fig. 3. DMSO administration does not affect the pattern of proteaseresistant PrP. Membranes were prepared from brains of terminally sick hamsters belonging to the diverse treatment groups, digested in the presence of proteinase K and subsequently similar samples were immunoblotted with aPrP mAb 3F4. As can be seen in the figure, all brains present similar patterns and concentrations of PrP Sc .

suggesting DMSO may inhibit the aggregation of the PrP Sc formed in the brain and thereby accelerate the clearance of the newly formed pathological protein. However, since DMSO could not cure the disease, at least in the experimental setup presented here, it seems that, at these conditions, the rate of PrP Sc synthesis was larger than the rate of PrP Sc clearance.

4. Discussion We have shown here that DMSO administration to scrapie-infected hamsters prolonged prion disease incubation time. Concomitantly, the rate of PrP Sc accumulation in the brain was reduced. In addition, DMSO administration increased the concentration of protease-resistant PrP in the urine of the treated animals, suggesting that inhibition of PrP Sc aggregation resulted in increased clearance of the pathogenic protein. DMSO has been shown before to reduce PrP Sc accumulation in ScN2a cells and to inhibit PrP Sc aggregation in vitro [35,40]. DMSO has been available for medical investigation since 1963. Although It was approved by the FDA only for the symptomatic treatment of interstitial cystitis, it has shown beneficial effects also for leukemia and cancer, cerebral ischemia, head trauma and infection [32]. DMSO was also given as a continuous treatment to human amyloidotic patients (7–16 months, 7–15 g / day). As opposed to the severe weight loss observed in the experiments described in this manuscript, mild nausea and an unpleasant breath odor were the patients’ main concern [31]. DMSO administration resulted in significant clinical improvement that was maintained as long as the drug was administered [31]. This suggests that, as in our experiments, DMSO inhibits aggregation and thereby enhances clearance but cannot inhibit the basic amyloidotic process. It is believed that prion disease pathogenesis includes several biochemical steps [28]. First, the synthesis of PrP C , followed by its encounter with PrP Sc . The next step is the

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establishment of the conversion process of PrP C into PrP Sc , and then the accumulation of PrP Sc in the brain cells, which is probably the main damage-producing step [7,14,41]. An effective treatment for prion diseases should inhibit as many of these steps as possible. The ideal treatment would therefore consist of a cocktail comprising several reagents, each inhibiting a different step in prion disease pathogenesis. To this effect, it is important to study the mechanism of action, in vivo and in vitro, of each of the anti-prion agents identified in cell culture and other experiments. DMSO, which we have shown to inhibit the aggregation of PrP Sc , may constitute a component in such a cocktail. We have also shown here that the route of infection, which determines incubation time and probably other factors affecting disease pathogenesis, such as involvement of peripheral tissues, may also determine treatment efficacy. This emphasizes yet again how long and complicated is the way between in vitro experiments on cells and in vivo treatment in humans. This is true even for a drug such as DMSO, which has been previously used in the treatment of other diseases. The poor results obtained so far in the treatment of neurodegenerative diseases suggests that future studies should focus on the development of prophylactic treatment for at-risk individuals. When patients are diagnosed with prion or any other neurodegenerative disease, they already exert great irreversible brain damage, which may be impossible to reverse regardless of the quality of the treatment. It is therefore important to concentrate on the study of long-term and non-toxic treatments that can be administered to either mutation carriers of genetic prion diseases or to individuals at risk of developing vCJD, rather than to terminal patients. A good example is flupirtine, which is already in clinical trials [24]. This approach requires not only the development of effective prophylactic reagents, but is also dependent on the presence of a method for early diagnosis of prion diseases, so that the individuals at risk can be identified as early as possible. Our preliminary results in humans and other species suggest urine testing may be useful in early diagnosis. Since DMSO administration resulted in a significant increase in UPrP secretion, it will be important to investigate whether urine testing may also serve as an indication for the efficacy of anti-prion treatment.

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