- Email: [email protected]
No effect of anti-leprosy drugs in the prevention of Alzheimer’s disease and β-amyloid neurotoxicity
Journal of the Neurological Sciences 165 (1999) 28–30
No effect of anti-leprosy drugs in the prevention of Alzheimer’s disease and b-amyloid neurotoxicity Masumi Endoh
a,b ,
*, Tatsuhide Kunishita a , Takeshi Tabira a
a
b
Department of Demyelinating Disease and Aging, National Institute of Neuroscience, NCNP, Kodaira, Tokyo 187 -8502, Japan Department of Bioregulation, Leprosy Research Center, National Institute of Infectious Diseases, 4 -2 -1 Aoba, Higashimurayama, Tokyo 189 -0002, Japan Received 14 April 1998; received in revised form 16 February 1999; accepted 23 February 1999
Abstract There is continuing controversy as to whether or not anti-leprosy drugs prevent Alzheimer’s disease (AD). Therefore, we examined the effect of anti-leprosy drugs on the prevalence of AD in leprosy patients, and also investigated the effect of anti-leprosy drugs on amyloid b-protein (Ab)-induced neurotoxicity in vitro. The present study suggests that anti-leprosy treatments do not prevent the onset of AD. None of our data found anti-leprosy drugs (dapsone, rifampicin, clofazimine, minomycin or ofloxacin) had any effect on Ab neurotoxicity. It is now important to examine the infection of Mycobacterium leprae in the central nervous system to clarify the reason for the low prevalence of senile dementia, and low frequency of Ab deposition in leprosy patients. 1999 Elsevier Science B.V. All rights reserved. Keywords: Anti-leprosy drug; Dementia; Amyloid b-protein; Neurotoxicity; Alzheimer’s disease
1. Introduction An epidemiological study by McGeer et al. [1] indicated that Japanese leprosy patients had a significantly low prevalence of senile dementia, if they had been under anti-leprosy treatment with 4,49-diaminodiphenyl sulfone (dapsone) and closely related drugs for the preceding 5 years. The overall prevalence of senile dementia was 2.9% in 1410 patients who were continuously on anti-leprosy treatment, compared with 6.25% in 1761 untreated patients. Although another report failed to confirm a preventive effect of anti-leprosy treatment on Alzheimer’s disease (AD) [2], McGeer et al. [3] suggest that dapsone prevents AD because of its anti-inflammatory properties. In agreement with the epidemiological study by McGeer et al. [1], we reported that the frequency of senile plaques and *Corresponding author. Tel.: 181-42-391-8211; fax: 181-42-3949092. E-mail address: [email protected] (M. Endoh)
b-amyloid (Ab) deposition was significantly lower in brains of elderly leprosy patients than controls [4]. This report suggested that the low level of Ab deposition may be due to the effect of anti-leprosy drugs, and it can be speculated that anti-leprosy drugs may prevent Ab aggregation [5] and neurotoxicity, resulting in the low frequency of senile plaques in brains of leprosy patients. The present report aims to resolve the controversy regarding the effect of anti-leprosy drugs on the low prevalence of AD in leprosy patients, and to investigate the effect of antileprosy drugs on Ab-induced neurotoxicity in vitro.
2. Materials and methods We obtained the medication records from 196 elderly patients with leprosy, who were in the National Leprosarium Tama Zensho-en, Tokyo, Japan. Of the 196 patients, 163 (83.2%) had lepromatous leprosy, 29 (14.8%) had tuberculoid leprosy, and the remaining pa-
0022-510X / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0022-510X( 99 )00057-X
M. Endoh et al. / Journal of the Neurological Sciences 165 (1999) 28 – 30
tients had indeterminate leprosy. For all patients, the degree of dementia was defined on the basis of Karasawa’s criteria for judging senility, which are well correlated with other well-known dementia rating scales such as Hasegawa’s Dementia Rating Scales and Mental Status Questionnaire [6]. Statistical analysis was done by the 232 x 2 test. Relative risks (odds ratios; OR) and 95% confidence intervals (CI) were computed. Ab1-40 peptide (BACHEM) and Ab1-42 peptide (Research Biochemicals International) were used for in vitro study. Ab1-40 peptide was preincubated in PBS at 378C for 7 days either alone or in combination with anti-leprosy drugs (dapsone, rifampicin, clofazimine, minomycin or ofloxacin) at a drug concentration from 1 mg / ml to 100 mg / ml. The method for primary neuronal culture was as described previously [7]. Briefly, cells from the cerebrum of BALB / c mice on the 14th embryonic day were minced and treated with trypsin and suspended in serum-free defined medium. Cells were seeded in a poly-D-lysinecoated 24-well plate (BIOCOAT, Becton Dickinson) at a density of 5310 4 cells / cm 2 and cultured for 5 days. Then, mixtures of anti-leprosy drugs and Ab1-42 peptide (10 mM) or preincubated Ab1-40 peptide (10 mM) solution were added to the cell culture. After 4 days of co-culture, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay was performed to assess the cell viability.
3. Results and discussion Table 1 shows the prevalence of dementia related to the status of medication in leprosy patients over the age of 70. One hundred and ten patients were under continuous or intermittent treatment mainly dapsone, while 86 patients were not treated with anti-leprosy drugs for at least 10 years prior to the study. The prevalence of dementia was 16.4% in treated cases (continuous or intermittent treatment), and that of untreated cases was 19.8%. The difference was not significant (OR50.79, 95% CI50.53– Table 1 Prevalence of dementia among leprosy patients treated and untreated with anti-leprosy drugs Age range
Cases treated for 10 years a
Cases not treated for 10 years a
Total n
Total n
Demented n
70–74 75–79 80–84 $85 Overall $70 a
%
Demented n
%
46 35 17 12
4 7 2 5
8.7 20.8 11.8 41.7
24 21 20 21
1 1 5 10
4.2 4.8 25.0 47.6
110
18
16.4
86
17
19.8
The duration prior to this study.
29
1.84; P50.668), nor was the apparent increased prevalence of dementia in younger (,age 80) treated patients (OR5 3.37, 95% CI51.87–5.97; P50.091). More lepromatous patients were treated with anti-leprosy drugs (58.3%) than tuberculoid patients (3.5%). However, the prevalence of dementia was similar in tuberculoid patients (20.7%) and lepromatous patients (17.8%, data not shown). Therefore, we concluded that anti-leprosy treatment did not prevent the onset of AD in patients with leprosy. Next, we examined the effect of anti-leprosy drugs on the neurotoxicity of Ab1-40 or 1-42 peptide using cultured mouse cerebral neurons. Four days after the addition of anti-leprosy drugs alone, Ab peptide alone, or a combination of drugs and Abpeptide, cell viability was assessed by the colorimetric MTT assay, which estimates the reducing activity of mitochondrial dehydrogenase and is an early induction of damage leading to cell death. As shown in Fig. 1, no toxic or trophic effects were observed in exposure of neuronal cells to 100 mg / ml dapsone, rifampicin and clofazimine (columns 2–4), which are used world wide in clinical application as multi-drug therapy (MDT) for leprosy patients recommended by WHO. On the other hand, typical reduction of cell viability was observed in exposure of neuronal cells to 10 mM Ab1-42 peptide alone (column 5 in Fig. 1). Dapsone, rifampicin and clofazimine did not improve the reduced neuronal cell viability induced by Ab peptide at any drug concentrations we used (columns 6–8 in Fig. 1 shows results of 100 mg / ml drug concentration). Minomycin and ofloxacin, which are considered new clinical applications for treatment of leprosy, did not show any effect on Ab neurotoxicity (data not shown). In vitro studies using Ab1-40 had shown similar results. We conclude that anti-leprosy drugs do not prevent the onset of AD, and they do not have any effect on Ab neurotoxicity. There is an interesting finding, however, that treatment appears to be associated with increased prevalence of dementia in younger (,age 80) treated patients. It can thus be speculated that leprosy treatment is associated with delayed onset of AD. These results contrast with those reported by McGeer et al. [1]. One of the reasons for conflicting results may be difference in duration of studies of medication in patients. In addition to our previous studies regarding genetic factors [8,9], the reason for the low incidence of senile dementia in leprosy patients and for the low frequency of Ab deposition in brains of leprosy patients is not yet clear. It is well recognized that the central nervous system of mammals is isolated from the infection of Mycobacterium leprae (M. leprae), which is the causative agent for leprosy. However, our recent study clarified that the M. leprae gene was detected in the brain of M. leprae-infected mice by PCR (manuscripts in preparation). This evidence indicates that M. leprae (or a fragment) crosses the blood–brain barrier in leprosy patients and this may lower the prevalence of senile dementia or frequency of Ab deposition in leprosy pa-
30
M. Endoh et al. / Journal of the Neurological Sciences 165 (1999) 28 – 30
Fig. 1. Effect of anti-leprosy drugs on neurotoxicity induced by Ab1-42 peptide (Ab) on cerebral primary neurons of mice. No anti-leprosy drugs (DDS, dapsone; RFP, rifampicin; CLF, clofazimine) showed toxic or trophic effects at any drug concentration when they were preincubated alone or in combination with Ab and added to the neuronal cultures. Values represented the mean6S.D. (n54). As indicated on the y-axis, percent cell survival values in the MTT assays were normalized to the assay of control (untreated neuronal cells).
tients. Therefore, it is now important to examine the infection of M. leprae in the central nervous system in leprosy patients.
Acknowledgements We thank Dr Masako Namisato at the Tama Zensho-en for providing the clinical information of the leprosy patients. This work was partially supported by a Grant-inAid for Scientific Research, the Ministry of Education, Science, Sports and Culture, Japan.
References [1] McGeer PL, Harada N, Kimura H, McGeer EG, Schulzer M. Prevalence of dementia amongst elderly Japanese with leprosy: apparent effect of chronic drug therapy. Dementia 1992;3:146–9. [2] Goto M, Kimura T, Hagio S, Ueda K, Kitajima S, Tokunaga H et al. Neuropathological analysis of dementia in a Japanese leprosarium. Dementia 1995;6:157–61.
[3] McGeer PL, Schulzer M, McGeer EG. Arthritis and anti-inflammatory agents as possible protective factors for Alzheimer’s disease: a review of 17 epidemiologic studies. Neurology 1996;47:425–32. [4] Chui D-H, Tabira T, Izumi S, Koya G, Ogata J. Decreased bamyloid and increased abnormal tau deposition in the brain of aged patients with leprosy. Am J Pathol 1994;145:771–5. [5] Tomiyama T, Asano S, Suwa Y, Morita T, Kataoka K, Mori H et al. Rifampicin prevents the aggregation and neurotoxicity of amyloid b protein in vitro. Biochem Biophys Res Commun 1994;204:76–83. [6] Otsuka T, Shimonaka Y, Maruyama S, Nakazato K, Kitamura T, Yaguchi K et al. A new screening test for dementia. Jpn J Psychiatry Neurol 1988;42:223–9. [7] Endoh M, Kunishita T, Tabira T. Thioredoxin from activated macrophages as a trophic factor for central cholinergic neurons in vitro. Biochem Biophys Res Commun 1993;192:760–5. [8] Endoh M, Ueki A, Takahashi K, Yamanaka Y, Izumi S, Tabira T. Significantly increased frequency of apolipoprotein E ´4 allele in elderly non-demented leprosy patients. Neurosci Lett 1996;207:206– 8. [9] Endoh M, Ueki A, Takahashi K, Yamanaka Y, Izumi S, Tabira T. Alpha-1-antichymotrypsin is not associated with the increased frequency of apolipoprotein E ´4 allele in elderly non-demented leprosy patients. Dement Geriatr Cogn Disord 1998;9:26–8.
No effect of anti-leprosy drugs in the prevention of Alzheimer’s disease and b-amyloid neurotoxicity Masumi Endoh
a,b ,
*, Tatsuhide Kunishita a , Takeshi Tabira a
a
b
Department of Demyelinating Disease and Aging, National Institute of Neuroscience, NCNP, Kodaira, Tokyo 187 -8502, Japan Department of Bioregulation, Leprosy Research Center, National Institute of Infectious Diseases, 4 -2 -1 Aoba, Higashimurayama, Tokyo 189 -0002, Japan Received 14 April 1998; received in revised form 16 February 1999; accepted 23 February 1999
Abstract There is continuing controversy as to whether or not anti-leprosy drugs prevent Alzheimer’s disease (AD). Therefore, we examined the effect of anti-leprosy drugs on the prevalence of AD in leprosy patients, and also investigated the effect of anti-leprosy drugs on amyloid b-protein (Ab)-induced neurotoxicity in vitro. The present study suggests that anti-leprosy treatments do not prevent the onset of AD. None of our data found anti-leprosy drugs (dapsone, rifampicin, clofazimine, minomycin or ofloxacin) had any effect on Ab neurotoxicity. It is now important to examine the infection of Mycobacterium leprae in the central nervous system to clarify the reason for the low prevalence of senile dementia, and low frequency of Ab deposition in leprosy patients. 1999 Elsevier Science B.V. All rights reserved. Keywords: Anti-leprosy drug; Dementia; Amyloid b-protein; Neurotoxicity; Alzheimer’s disease
1. Introduction An epidemiological study by McGeer et al. [1] indicated that Japanese leprosy patients had a significantly low prevalence of senile dementia, if they had been under anti-leprosy treatment with 4,49-diaminodiphenyl sulfone (dapsone) and closely related drugs for the preceding 5 years. The overall prevalence of senile dementia was 2.9% in 1410 patients who were continuously on anti-leprosy treatment, compared with 6.25% in 1761 untreated patients. Although another report failed to confirm a preventive effect of anti-leprosy treatment on Alzheimer’s disease (AD) [2], McGeer et al. [3] suggest that dapsone prevents AD because of its anti-inflammatory properties. In agreement with the epidemiological study by McGeer et al. [1], we reported that the frequency of senile plaques and *Corresponding author. Tel.: 181-42-391-8211; fax: 181-42-3949092. E-mail address: [email protected] (M. Endoh)
b-amyloid (Ab) deposition was significantly lower in brains of elderly leprosy patients than controls [4]. This report suggested that the low level of Ab deposition may be due to the effect of anti-leprosy drugs, and it can be speculated that anti-leprosy drugs may prevent Ab aggregation [5] and neurotoxicity, resulting in the low frequency of senile plaques in brains of leprosy patients. The present report aims to resolve the controversy regarding the effect of anti-leprosy drugs on the low prevalence of AD in leprosy patients, and to investigate the effect of antileprosy drugs on Ab-induced neurotoxicity in vitro.
2. Materials and methods We obtained the medication records from 196 elderly patients with leprosy, who were in the National Leprosarium Tama Zensho-en, Tokyo, Japan. Of the 196 patients, 163 (83.2%) had lepromatous leprosy, 29 (14.8%) had tuberculoid leprosy, and the remaining pa-
0022-510X / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0022-510X( 99 )00057-X
M. Endoh et al. / Journal of the Neurological Sciences 165 (1999) 28 – 30
tients had indeterminate leprosy. For all patients, the degree of dementia was defined on the basis of Karasawa’s criteria for judging senility, which are well correlated with other well-known dementia rating scales such as Hasegawa’s Dementia Rating Scales and Mental Status Questionnaire [6]. Statistical analysis was done by the 232 x 2 test. Relative risks (odds ratios; OR) and 95% confidence intervals (CI) were computed. Ab1-40 peptide (BACHEM) and Ab1-42 peptide (Research Biochemicals International) were used for in vitro study. Ab1-40 peptide was preincubated in PBS at 378C for 7 days either alone or in combination with anti-leprosy drugs (dapsone, rifampicin, clofazimine, minomycin or ofloxacin) at a drug concentration from 1 mg / ml to 100 mg / ml. The method for primary neuronal culture was as described previously [7]. Briefly, cells from the cerebrum of BALB / c mice on the 14th embryonic day were minced and treated with trypsin and suspended in serum-free defined medium. Cells were seeded in a poly-D-lysinecoated 24-well plate (BIOCOAT, Becton Dickinson) at a density of 5310 4 cells / cm 2 and cultured for 5 days. Then, mixtures of anti-leprosy drugs and Ab1-42 peptide (10 mM) or preincubated Ab1-40 peptide (10 mM) solution were added to the cell culture. After 4 days of co-culture, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay was performed to assess the cell viability.
3. Results and discussion Table 1 shows the prevalence of dementia related to the status of medication in leprosy patients over the age of 70. One hundred and ten patients were under continuous or intermittent treatment mainly dapsone, while 86 patients were not treated with anti-leprosy drugs for at least 10 years prior to the study. The prevalence of dementia was 16.4% in treated cases (continuous or intermittent treatment), and that of untreated cases was 19.8%. The difference was not significant (OR50.79, 95% CI50.53– Table 1 Prevalence of dementia among leprosy patients treated and untreated with anti-leprosy drugs Age range
Cases treated for 10 years a
Cases not treated for 10 years a
Total n
Total n
Demented n
70–74 75–79 80–84 $85 Overall $70 a
%
Demented n
%
46 35 17 12
4 7 2 5
8.7 20.8 11.8 41.7
24 21 20 21
1 1 5 10
4.2 4.8 25.0 47.6
110
18
16.4
86
17
19.8
The duration prior to this study.
29
1.84; P50.668), nor was the apparent increased prevalence of dementia in younger (,age 80) treated patients (OR5 3.37, 95% CI51.87–5.97; P50.091). More lepromatous patients were treated with anti-leprosy drugs (58.3%) than tuberculoid patients (3.5%). However, the prevalence of dementia was similar in tuberculoid patients (20.7%) and lepromatous patients (17.8%, data not shown). Therefore, we concluded that anti-leprosy treatment did not prevent the onset of AD in patients with leprosy. Next, we examined the effect of anti-leprosy drugs on the neurotoxicity of Ab1-40 or 1-42 peptide using cultured mouse cerebral neurons. Four days after the addition of anti-leprosy drugs alone, Ab peptide alone, or a combination of drugs and Abpeptide, cell viability was assessed by the colorimetric MTT assay, which estimates the reducing activity of mitochondrial dehydrogenase and is an early induction of damage leading to cell death. As shown in Fig. 1, no toxic or trophic effects were observed in exposure of neuronal cells to 100 mg / ml dapsone, rifampicin and clofazimine (columns 2–4), which are used world wide in clinical application as multi-drug therapy (MDT) for leprosy patients recommended by WHO. On the other hand, typical reduction of cell viability was observed in exposure of neuronal cells to 10 mM Ab1-42 peptide alone (column 5 in Fig. 1). Dapsone, rifampicin and clofazimine did not improve the reduced neuronal cell viability induced by Ab peptide at any drug concentrations we used (columns 6–8 in Fig. 1 shows results of 100 mg / ml drug concentration). Minomycin and ofloxacin, which are considered new clinical applications for treatment of leprosy, did not show any effect on Ab neurotoxicity (data not shown). In vitro studies using Ab1-40 had shown similar results. We conclude that anti-leprosy drugs do not prevent the onset of AD, and they do not have any effect on Ab neurotoxicity. There is an interesting finding, however, that treatment appears to be associated with increased prevalence of dementia in younger (,age 80) treated patients. It can thus be speculated that leprosy treatment is associated with delayed onset of AD. These results contrast with those reported by McGeer et al. [1]. One of the reasons for conflicting results may be difference in duration of studies of medication in patients. In addition to our previous studies regarding genetic factors [8,9], the reason for the low incidence of senile dementia in leprosy patients and for the low frequency of Ab deposition in brains of leprosy patients is not yet clear. It is well recognized that the central nervous system of mammals is isolated from the infection of Mycobacterium leprae (M. leprae), which is the causative agent for leprosy. However, our recent study clarified that the M. leprae gene was detected in the brain of M. leprae-infected mice by PCR (manuscripts in preparation). This evidence indicates that M. leprae (or a fragment) crosses the blood–brain barrier in leprosy patients and this may lower the prevalence of senile dementia or frequency of Ab deposition in leprosy pa-
30
M. Endoh et al. / Journal of the Neurological Sciences 165 (1999) 28 – 30
Fig. 1. Effect of anti-leprosy drugs on neurotoxicity induced by Ab1-42 peptide (Ab) on cerebral primary neurons of mice. No anti-leprosy drugs (DDS, dapsone; RFP, rifampicin; CLF, clofazimine) showed toxic or trophic effects at any drug concentration when they were preincubated alone or in combination with Ab and added to the neuronal cultures. Values represented the mean6S.D. (n54). As indicated on the y-axis, percent cell survival values in the MTT assays were normalized to the assay of control (untreated neuronal cells).
tients. Therefore, it is now important to examine the infection of M. leprae in the central nervous system in leprosy patients.
Acknowledgements We thank Dr Masako Namisato at the Tama Zensho-en for providing the clinical information of the leprosy patients. This work was partially supported by a Grant-inAid for Scientific Research, the Ministry of Education, Science, Sports and Culture, Japan.
References [1] McGeer PL, Harada N, Kimura H, McGeer EG, Schulzer M. Prevalence of dementia amongst elderly Japanese with leprosy: apparent effect of chronic drug therapy. Dementia 1992;3:146–9. [2] Goto M, Kimura T, Hagio S, Ueda K, Kitajima S, Tokunaga H et al. Neuropathological analysis of dementia in a Japanese leprosarium. Dementia 1995;6:157–61.
[3] McGeer PL, Schulzer M, McGeer EG. Arthritis and anti-inflammatory agents as possible protective factors for Alzheimer’s disease: a review of 17 epidemiologic studies. Neurology 1996;47:425–32. [4] Chui D-H, Tabira T, Izumi S, Koya G, Ogata J. Decreased bamyloid and increased abnormal tau deposition in the brain of aged patients with leprosy. Am J Pathol 1994;145:771–5. [5] Tomiyama T, Asano S, Suwa Y, Morita T, Kataoka K, Mori H et al. Rifampicin prevents the aggregation and neurotoxicity of amyloid b protein in vitro. Biochem Biophys Res Commun 1994;204:76–83. [6] Otsuka T, Shimonaka Y, Maruyama S, Nakazato K, Kitamura T, Yaguchi K et al. A new screening test for dementia. Jpn J Psychiatry Neurol 1988;42:223–9. [7] Endoh M, Kunishita T, Tabira T. Thioredoxin from activated macrophages as a trophic factor for central cholinergic neurons in vitro. Biochem Biophys Res Commun 1993;192:760–5. [8] Endoh M, Ueki A, Takahashi K, Yamanaka Y, Izumi S, Tabira T. Significantly increased frequency of apolipoprotein E ´4 allele in elderly non-demented leprosy patients. Neurosci Lett 1996;207:206– 8. [9] Endoh M, Ueki A, Takahashi K, Yamanaka Y, Izumi S, Tabira T. Alpha-1-antichymotrypsin is not associated with the increased frequency of apolipoprotein E ´4 allele in elderly non-demented leprosy patients. Dement Geriatr Cogn Disord 1998;9:26–8.