Neurobiology of Aging 35 (2014) 1656e1659
Contents lists available at ScienceDirect
Neurobiology of Aging journal homepage: www.elsevier.com/locate/neuaging
Brief communication
Age-associated changes of cerebrospinal fluid amyloid-b and tau in cynomolgus monkeys Feng Yue a, b, Chunling Lu c, Yi Ai d, Piu Chan a, b, Zhiming Zhang d, * a
Department of Neurobiology Beijing Institute of Geriatrics, Xuanwu Hospital of Capital Medical University, Beijing, China Key Laboratory on Neurodegenerative Disease of Ministry of Education and Key Laboratory on Parkinson’s Disease of Beijing, Beijing, China c WinconTheraCells Biotechnologies Co, LTD, Nanning, Guangxi, China d Department of Anatomy and Neurobiology, University of Kentucky College of Medicine, Lexington, KY, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 October 2013 Received in revised form 23 January 2014 Accepted 27 January 2014 Available online 31 January 2014
Nonhuman primates (NHPs) are useful for the study of age-associated changes in the brain as a model that is biologically closely related to humans. For example, with age, all NHPs analyzed to date, develop b-amyloid (Ab) plaques as seen in humans. Nevertheless, it is still unclear if NHPs have human-like ageassociated changes in Ab and tau protein in cerebrospinal fluid. The present study was an attempt to specifically address these issues. Cerebrospinal fluid levels of Ab and phosphorylated tau were measured in 37 and 22 cynomolgus monkeys, respectively, with ages ranging from 4 to 22-year-old. The result from the present study revealed significant age-associated declines in Ab42 levels but not in Ab40 and phosphorylated tau levels. This finding appears to parallel changes seen with human aging, in which decreased levels of Ab42 can be seen in normal older adults, and supporting that cynomolgus monkeys would be a useful model for studying age-related neurologic disorders associated with Alzheimer-like cerebral proteopathy. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: b-amyloid p-Tau Cynomolgus monkey Age CSF
1. Introduction Cerebrospinal fluid (CSF) biomarkers can be measured repeatedly in vivo. Thus, they have been recognized as key elements of research criteria for the preclinical phase of Alzheimer’s disease (AD). In humans, clinical evidence suggests that lower concentration of CSF Ab42 and higher concentration of total Tau protein (t-Tau) can be used to distinguish AD patients from cognitively normal age-matched controls. It has been presumed that the decrease of CSF Ab42 reflects its aggregation and deposition in the brain parenchyma; whereas the increase of CSF tau reflects its extracellular release after neuronal degeneration and neurofibrillary tangle formation (for reviews see Jack et al., 2010; Musiek and Holtzman, 2012; Wang et al., 2013). These markers have also been used to predict the conversion of mild cognitive impairment to AD (Brys et al., 2009; Buchhave et al., 2012). The amyloid precursor protein (APP) is cleaved into b-amyloid (Ab) peptides of different lengths (Ab40 and Ab42), each with different neurotoxic properties. Ab42 is the major component of neuronal plaques and is more prone to aggregation than Ab40. * Corresponding author at: Department of Anatomy and Neurobiology, University of Kentucky, Chandler Medical Center, 48 Whitney-Hendrickson Bldg, Lexington, KY 40536-0098, USA. Tel.: þ859 257 6032; fax: þ859 257 3625. E-mail address: [email protected] (Z. Zhang). 0197-4580/$ e see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2014.01.139
However, Ab40 is the most abundant form of Ab found in CSF and is found at approximately 10-fold higher levels than Ab42 (Galasko, 1998; Hulstaert et al., 1999; Wiltfang et al., 2002). Clinical studies have suggested that CSF Ab42 to Ab40 ratio could improve accuracy of diagnosis of AD as well as help the CSF differentiate AD from Parkinson’s disease with dementia and dementia with Lewy bodies (Lewczuk et al., 2004; Nutu et al., 2013). Nonhuman primates, including cynomolgus monkeys (Macaca fascicularis), have been widely used in AD-related preclinical studies because of its relatively long life span and high levels of sentience (Darusman et al., 2013; Kimura et al., 2005). As nontransgenic animals, nonhuman primates can supply the natural spatiotemporal profiles of Ab and tau pathologies presented in living brains. Despite increasing investigations of Ab and tau pathology in brain tissue of nonhuman primate (for a review see Heuer et al., 2012), it is still not known whether old cynomolgus monkeys would also have similar age-related changes in CSF biomarker signature. The present study was designed to specifically address these questions. 2. Methods 2.1. Animals A total of 37 cynomolgus monkeys (M. fascicularis) used in this study were purchased from the same vendor (Guangxi Grandforest
F. Yue et al. / Neurobiology of Aging 35 (2014) 1656e1659
A
Intergroup differences were evaluated by a 1-factor analysis of variance followed by Bonferroni multiple comparison tests. The relationships between age, levels of beta-amyloid, and tau protein were subsequently analyzed by linear regression. A value of p < 0.05 was considered significant in all analyses. All statistical analyses were conducted using Prism 5 GraphPad Software (San Diego, CA, USA).
15 10 5 0
4-6 Yrs
12-19 Yrs
B
CSF levels of Aβ 40 500 400 300 200 100 0
4-6 Yrs
12-19 Yrs
> 21 Yrs
Groups
3. Results
CSF levels of p-tau
C 100 80
pg/ml
A significant age-associated decline was found only in Ab42 (21-year-old group (Fig. 1A). However, there was only a slight decline in Ab40 levels (Fig. 1B) and virtually no change across groups in p-tau levels (Fig. 1C). Additionally, the age-associated decline in CSF Ab42 was significantly (r2 ¼ 0.17, p ¼ 0.018) correlated between age and CSF Ab42 concentration (Fig. 2A). By contrast, only a minor age-related change was seen in the CSF levels of Ab40 (Fig. 2B) and no correlation was seen between age and CSF levels of p-tau (Fig. 2C). It should be noted that the same CSF samples used to determine Ab concentrations were also analyzed for endogenous p-tau protein but only in 22 of 37 monkeys because of unavailability of CSF from the other 15 animals. Another notable observation in the study was that the individual variance within groups was much smaller for the animals in the 4 to 6-year-old group than for the other 2 age groups (Fig. 1C). The individual range in the youngest group was from 24.6 to 39.5 (pg/mL; standard deviation [SD] ¼ 8.37); whereas the levels ranged from 14.9 to 53.3 (pg/mL; SD ¼ 15.8) in the 12 to 19-year-old group, and from 9.3 to 87.5 (pg/mL; SD ¼ 22.9) in the >21-year-old group. However, the mean was virtually equal in the 3 different groups
> 21 Yrs
Groups
ng/ml
2.3. Statistical analysis
*
*
20
2.2. Immunoassays for CSF biomarker analysis All samples were obtained by lumbar puncture through the L3/ L4 or L4/L5 interspace and were stored at 80 C until analysis. Following procedures described elsewhere (Darusman et al., 2013; Wang et al., 2013); levels of CSF Ab40 or Ab42 were measured by a sandwich enzyme-linked immunosorbent assay (ELISA) (antibodies and standards were gifts from Tongi Medical School, Wuhan, China). The levels of p-tau in CSF were determined using an INNOTEST ELISA kit (Innogenetic N.V, Gent, Belgium). All samples were run in duplicate.
CSF levels of Aβ 42 25
ng/ml
Scientific Primate Company, Ltd, Guangxi, China) including 29 females and 8 males with age ranging from 4 to 22-year-old. Because 1 year of cynomolgus monkey is equal to 3 years of human life (Weinbauer et al., 2008), they were equivalent to 12 to 66-year-old humans. All animals were individually housed and maintained on a 12-hour light and/or 12-hour dark cycle with water available ad libitum. Toys, a mirror and food supplements were routinely provided to promote psychological well being for all tested animals. All procedures used in this study were fully reviewed and approved by the Animal Care and Use Committee and performed at the primate facility of Wincon TheraCells Biotechnologies Company in Nanning Guangxi China. The facility is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.
1657
60 40 20 0
4-6 Yrs
12-19 Yrs
> 21 Yrs
Groups Fig. 1. Intergroup analyses of Ab42, Ab40, and p-tau levels. Significant (* p < 0.05) differences were seen between experimental groups for CSF levels of Ab42 (A) but not for CSF levels of Ab40 (B), and p-tau (C). The error bars indicate the mean SEM. Abbreviations: Ab, b-amyloid; CSF, cerebrospinal fluid; p-tau, phosphorylated tau; SEM, standard error of the mean.
1658
F. Yue et al. / Neurobiology of Aging 35 (2014) 1656e1659
A
Correlation between age and Aβ 42 30
r2=0.17; P=0.0108
4. Discussion
ng/ml
20
10
0
0
5
10
15
20
25
Age
Correlation between age and Aβ 40
B 500
r2=0.03; P=0.29
ng/ml
400 300 200 100 0
0
5
10
15
20
25
Age
C
Correlation between age and p-tau
r2=0.003; P=0.81
100
pg/ml
80 60 40 20 0
0
5
10
15
(31.8 in 4 to 6-year-old, 27.9 in 12 to 19-year-old, and 30.8 in >21year-old group).
20
25
Age Fig. 2. Linear regression analyses with the 95% confidence band of the best-fit line. A significant negative correlation (r2 ¼ 0.17, p ¼ 0.0108) was found between age and the levels of Ab42 (A), whereas only a very weak correlation was observed in the levels of Ab40 (B), and no correlation was seen in the levels of p-tau (C). Abbreviations: Ab, b-amyloid; p-tau, phosphorylated tau.
Animal models have played a major role in defining critical disease-related mechanisms and exploring novel potential therapeutic approaches in neurodegenerative diseases like AD (Heuer et al., 2012). Here, for the first time, we report significant correlations between age and the decline of CSF Ab42 levels but not Ab40 and p-tau levels in cynomolgus monkeys. These results are in agreement with prior postmortem studies in aged cynomolgus monkeys in which senile plaques, a characteristic feature of AD, spontaneously form in the cortex (Kimura et al., 2005; Oikawa et al., 2010). Thus, in vivo studies, plus postmortem analysis of Alzheimerlike pathology, further support the value of aged cynomolgus monkeys in aging research. In addition, b-APP, which can be cleaved into amyloid-b peptides, is ubiquitously expressed in neurons of all macaque species examined to date; including rhesus, cynomolgus, and other Old World species (Martin et al., 1991, 1994). Also, APP695 (the 695 amino acid isoform) is completely homologous between human and rhesus monkeys and cynomolgus monkeys (Podlisny et al., 1991). The activity of the b-amyloid cleaving enzyme-1 (b-secretase, or BACE-1), which contributes to the liberation of Ab from APP, increases with age as seen in humans (Fukumoto et al., 2004). Thus, the results from the present study provide new and important insight on how closely age-related changes in CSF biomarkers in aged cynomolgus monkeys parallel changes seen in humans. To date, biochemistry and immunohistochemistry have been widely used in studying age-related changes of intracellular Ab in cynomolgus monkeys (Kimura et al., 2005; Nakamura et al., 1998; Oikawa et al., 2010). These studies demonstrated the ageassociated accumulation of insoluble Ab in the neocortical and hippocampal regions of animals as young as mid-20s. Levels of Ab were extremely high in those of advanced age (>30-year-old) and accumulation of p-tau present in the same regions (Oikawa et al., 2010). Although intracellular Ab42 was observed in cortical neurons in aged monkeys, Ab40 levels remained virtually the same regardless of age (Kimura et al., 2005). In the present study, we found an age-associated decrease in Ab42 levels in the CSF with little change in Ab40 levels. Interestingly, the age-associated changes in CSF levels seen in the present study were supported by a recent study also using young and aged cynomolgus monkeys (Darusman et al., 2013). In the study, Darusman et al. (2013) found that the Ab42 concentration in serum of the aged monkeys was significantly lower than in the younger subjects, while the CSF t-tau (total tau) and p-tau did not significantly differ between the groups. Results from both studies (the present and aforementioned studies) indicate that changes in these CSF biomarkers appear to parallel changes in serum, which suggests that levels of serum Ab42 and other biomarkers may reflect changes in CSF in nonhuman primates. It is hypothesized that decreases in CSF levels of Ab42 are because of the aggregations and deposition in the brain parenchyma, which in turn lead to increased levels of intracellular Ab42 (for a review see Jack et al., 2010). Based on the current assumptions, an accumulation of Ab-oligomers during the early, preclinical stage of AD may trigger a cascade of events including neuronal loss and atrophy within cortical and subcortical regions and in turn, may lead to cognitive declines. As mentioned previously, the decrease of CSF Ab42 levels seen in the present study suggests increases of depositions of b-amyloid in the brain, which may indicate
F. Yue et al. / Neurobiology of Aging 35 (2014) 1656e1659
that beginning of the molecular process of AD pathologies occur in aged cynomolgus monkeys (for a review see Nakayama et al., 2004). In conclusion, for the first time, quantitative ELISAs revealed age-associated changes in CSF levels of Ab42 but not Ab40 and ptau in cynomolgus monkeys. The result is supported by age-related changes of Ab in brain tissue (Kimura et al., 2005) and in serum (Darusman et al., 2013). A further comparative study of ageassociated changes between human and nonhuman primate could yield more informative clues to the unique human predisposition to AD. Acknowledgements This study was supported by grants from the National Natural Science Foundation of China (31240043), State High-Tech Development Plan of Ministry of Sciences and Technology of China (2012AA020703, 2012AA02A514), the National Basic Research Development Program of China (2011CB504101), and the Department of Anatomy and Neurobiology, University of Kentucky College of Medicine. The authors want to thank Dr Richard Grondin and Ms. April Evans for reviewing the manuscript. References Brys, M., Pirraglia, E., Rich, K., Rolstad, S., Mosconi, L., Switalski, R., GlodzikSobanska, L., De Santi, S., Zinkowski, R., Mehta, P., Pratico, D., Saint Louis, L.A., Wallin, A., Blennow, K., de Leon, M.J., 2009. Prediction and longitudinal study of CSF biomarkers in mild cognitive impairment. Neurobiol. Aging 30, 682e690. Buchhave, P., Minthon, L., Zetterberg, H., Wallin, A.K., Blennow, K., Hansson, O., 2012. Cerebrospinal fluid levels of beta-amyloid 1-42, but not of tau, are fully changed already 5 to 10 years before the onset of Alzheimer dementia. Arch. Gen. Psychiatry 69, 98e106. Darusman, H.S., Sajuthi, D., Kalliokoski, O., Jacobsen, K.R., Call, J., Schapiro, S.J., Gjedde, A., Abelson, K.S., Hau, J., 2013. Correlations between serum levels of beta amyloid, cerebrospinal levels of tau and phospho tau, and delayed response tasks in young and aged cynomolgus monkeys (Macaca fascicularis). J. Med. Primatol. 42, 137e146. Fukumoto, H., Rosene, D.L., Moss, M.B., Raju, S., Hyman, B.T., Irizarry, M.C., 2004. Beta-secretase activity increases with aging in human, monkey, and mouse brain. Am. J. Pathol. 164, 719e725. Galasko, D., 1998. CSF tau and Abeta42: logical biomarkers for Alzheimer’s disease? Neurobiol. Aging 19, 117e119. Heuer, E., Rosen, R.F., Cintron, A., Walker, L.C., 2012. Nonhuman primate models of Alzheimer-like cerebral proteopathy. Curr. Pharm. Des. 18, 1159e1169.
1659
Hulstaert, F., Blennow, K., Ivanoiu, A., Schoonderwaldt, H.C., Riemenschneider, M., De Deyn, P.P., Bancher, C., Cras, P., Wiltfang, J., Mehta, P.D., Iqbal, K., Pottel, H., Vanmechelen, E., Vanderstichele, H., 1999. Improved discrimination of AD patients using beta-amyloid(1-42) and tau levels in CSF. Neurology 52, 1555e1562. Jack Jr., C.R., Knopman, D.S., Jagust, W.J., Shaw, L.M., Aisen, P.S., Weiner, M.W., Petersen, R.C., Trojanowski, J.Q., 2010. Hypothetical model of dynamic biomarkers of the Alzheimer’s pathological cascade. Lancet Neurol. 9, 119e128. Kimura, N., Yanagisawa, K., Terao, K., Ono, F., Sakakibara, I., Ishii, Y., Kyuwa, S., Yoshikawa, Y., 2005. Age-related changes of intracellular Abeta in cynomolgus monkey brains. Neuropathol. Appl. Neurobiol. 31, 170e180. Lewczuk, P., Esselmann, H., Otto, M., Maler, J.M., Henkel, A.W., Henkel, M.K., Eikenberg, O., Antz, C., Krause, W.R., Reulbach, U., Kornhuber, J., Wiltfang, J., 2004. Neurochemical diagnosis of Alzheimer’s dementia by CSF Abeta42, Abeta42/Abeta40 ratio and total tau. Neurobiol. Aging 25, 273e281. Martin, L.J., Pardo, C.A., Cork, L.C., Price, D.L., 1994. Synaptic pathology and glial responses to neuronal injury precede the formation of senile plaques and amyloid deposits in the aging cerebral cortex. Am. J. Pathol. 145, 1358e1381. Martin, L.J., Sisodia, S.S., Koo, E.H., Cork, L.C., Dellovade, T.L., Weidemann, A., Beyreuther, K., Masters, C., Price, D.L., 1991. Amyloid precursor protein in aged nonhuman primates. Proc. Natl. Acad. Sci. U.S.A 88, 1461e1465. Musiek, E.S., Holtzman, D.M., 2012. Origins of Alzheimer’s disease: reconciling cerebrospinal fluid biomarker and neuropathology data regarding the temporal sequence of amyloid-beta and tau involvement. Curr. Opin. Neurol. 25, 715e720. Nakamura, S., Nakayama, H., Goto, N., Ono, F., Sakakibara, I., Yoshikawa, Y., 1998. Histopathological studies of senile plaques and cerebral amyloidosis in cynomolgus monkeys. J. Med. Primatol. 27, 244e252. Nakayama, H., Uchida, K., Doi, K., 2004. A comparative study of age-related brain pathologyeare neurodegenerative diseases present in nonhuman animals? Med Hypoth. 632, 198e202. Nutu, M., Zetterberg, H., Londos, E., Minthon, L., Nagga, K., Blennow, K., Hansson, O., Ohrfelt, A., 2013. Evaluation of the cerebrospinal fluid amyloid-beta1-42/ amyloid-beta1-40 ratio measured by alpha-LISA to distinguish Alzheimer’s disease from other dementia disorders. Dement Geriatr. Cogn. Disord. 36, 99e110. Oikawa, N., Kimura, N., Yanagisawa, K., 2010. Alzheimer-type tau pathology in advanced aged nonhuman primate brains harboring substantial amyloid deposition. Brain Res. 1315, 137e149. Podlisny, M.B., Tolan, D.R., Selkoe, D.J., 1991. Homology of the amyloid beta protein precursor in monkey and human supports a primate model for beta amyloidosis in Alzheimer’s disease. Am. J. Pathol. 138, 1423e1435. Wang, L., Brier, M.R., Snyder, A.Z., Thomas, J.B., Fagan, A.M., Xiong, C., Benzinger, T.L., Holtzman, D.M., Morris, J.C., Ances, B.M., 2013. Cerebrospinal fluid Abeta42, phosphorylated Tau181, and resting-state functional connectivity. JAMA Neurol. 70, 1242e1248. Weinbauer, G.F., Niehoff, M., Niehaus, M., Srivastav, S., Fuchs, A., Van Esch, E., Cline, J.M., 2008. Physiology and endocrinology of the ovarian cycle in macaques. Toxicol. Pathol. 36 (7S), 7Se23S. Wiltfang, J., Esselmann, H., Bibl, M., Smirnov, A., Otto, M., Paul, S., Schmidt, B., Klafki, H.W., Maler, M., Dyrks, T., Bienert, M., Beyermann, M., Ruther, E., Kornhuber, J., 2002. Highly conserved and disease-specific patterns of carboxyterminally truncated Abeta peptides 1-37/38/39 in addition to 1-40/42 in Alzheimer’s disease and in patients with chronic neuroinflammation. J. Neurochem. 81, 481e496.
Contents lists available at ScienceDirect
Neurobiology of Aging journal homepage: www.elsevier.com/locate/neuaging
Brief communication
Age-associated changes of cerebrospinal fluid amyloid-b and tau in cynomolgus monkeys Feng Yue a, b, Chunling Lu c, Yi Ai d, Piu Chan a, b, Zhiming Zhang d, * a
Department of Neurobiology Beijing Institute of Geriatrics, Xuanwu Hospital of Capital Medical University, Beijing, China Key Laboratory on Neurodegenerative Disease of Ministry of Education and Key Laboratory on Parkinson’s Disease of Beijing, Beijing, China c WinconTheraCells Biotechnologies Co, LTD, Nanning, Guangxi, China d Department of Anatomy and Neurobiology, University of Kentucky College of Medicine, Lexington, KY, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 October 2013 Received in revised form 23 January 2014 Accepted 27 January 2014 Available online 31 January 2014
Nonhuman primates (NHPs) are useful for the study of age-associated changes in the brain as a model that is biologically closely related to humans. For example, with age, all NHPs analyzed to date, develop b-amyloid (Ab) plaques as seen in humans. Nevertheless, it is still unclear if NHPs have human-like ageassociated changes in Ab and tau protein in cerebrospinal fluid. The present study was an attempt to specifically address these issues. Cerebrospinal fluid levels of Ab and phosphorylated tau were measured in 37 and 22 cynomolgus monkeys, respectively, with ages ranging from 4 to 22-year-old. The result from the present study revealed significant age-associated declines in Ab42 levels but not in Ab40 and phosphorylated tau levels. This finding appears to parallel changes seen with human aging, in which decreased levels of Ab42 can be seen in normal older adults, and supporting that cynomolgus monkeys would be a useful model for studying age-related neurologic disorders associated with Alzheimer-like cerebral proteopathy. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: b-amyloid p-Tau Cynomolgus monkey Age CSF
1. Introduction Cerebrospinal fluid (CSF) biomarkers can be measured repeatedly in vivo. Thus, they have been recognized as key elements of research criteria for the preclinical phase of Alzheimer’s disease (AD). In humans, clinical evidence suggests that lower concentration of CSF Ab42 and higher concentration of total Tau protein (t-Tau) can be used to distinguish AD patients from cognitively normal age-matched controls. It has been presumed that the decrease of CSF Ab42 reflects its aggregation and deposition in the brain parenchyma; whereas the increase of CSF tau reflects its extracellular release after neuronal degeneration and neurofibrillary tangle formation (for reviews see Jack et al., 2010; Musiek and Holtzman, 2012; Wang et al., 2013). These markers have also been used to predict the conversion of mild cognitive impairment to AD (Brys et al., 2009; Buchhave et al., 2012). The amyloid precursor protein (APP) is cleaved into b-amyloid (Ab) peptides of different lengths (Ab40 and Ab42), each with different neurotoxic properties. Ab42 is the major component of neuronal plaques and is more prone to aggregation than Ab40. * Corresponding author at: Department of Anatomy and Neurobiology, University of Kentucky, Chandler Medical Center, 48 Whitney-Hendrickson Bldg, Lexington, KY 40536-0098, USA. Tel.: þ859 257 6032; fax: þ859 257 3625. E-mail address: [email protected] (Z. Zhang). 0197-4580/$ e see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2014.01.139
However, Ab40 is the most abundant form of Ab found in CSF and is found at approximately 10-fold higher levels than Ab42 (Galasko, 1998; Hulstaert et al., 1999; Wiltfang et al., 2002). Clinical studies have suggested that CSF Ab42 to Ab40 ratio could improve accuracy of diagnosis of AD as well as help the CSF differentiate AD from Parkinson’s disease with dementia and dementia with Lewy bodies (Lewczuk et al., 2004; Nutu et al., 2013). Nonhuman primates, including cynomolgus monkeys (Macaca fascicularis), have been widely used in AD-related preclinical studies because of its relatively long life span and high levels of sentience (Darusman et al., 2013; Kimura et al., 2005). As nontransgenic animals, nonhuman primates can supply the natural spatiotemporal profiles of Ab and tau pathologies presented in living brains. Despite increasing investigations of Ab and tau pathology in brain tissue of nonhuman primate (for a review see Heuer et al., 2012), it is still not known whether old cynomolgus monkeys would also have similar age-related changes in CSF biomarker signature. The present study was designed to specifically address these questions. 2. Methods 2.1. Animals A total of 37 cynomolgus monkeys (M. fascicularis) used in this study were purchased from the same vendor (Guangxi Grandforest
F. Yue et al. / Neurobiology of Aging 35 (2014) 1656e1659
A
Intergroup differences were evaluated by a 1-factor analysis of variance followed by Bonferroni multiple comparison tests. The relationships between age, levels of beta-amyloid, and tau protein were subsequently analyzed by linear regression. A value of p < 0.05 was considered significant in all analyses. All statistical analyses were conducted using Prism 5 GraphPad Software (San Diego, CA, USA).
15 10 5 0
4-6 Yrs
12-19 Yrs
B
CSF levels of Aβ 40 500 400 300 200 100 0
4-6 Yrs
12-19 Yrs
> 21 Yrs
Groups
3. Results
CSF levels of p-tau
C 100 80
pg/ml
A significant age-associated decline was found only in Ab42 (21-year-old group (Fig. 1A). However, there was only a slight decline in Ab40 levels (Fig. 1B) and virtually no change across groups in p-tau levels (Fig. 1C). Additionally, the age-associated decline in CSF Ab42 was significantly (r2 ¼ 0.17, p ¼ 0.018) correlated between age and CSF Ab42 concentration (Fig. 2A). By contrast, only a minor age-related change was seen in the CSF levels of Ab40 (Fig. 2B) and no correlation was seen between age and CSF levels of p-tau (Fig. 2C). It should be noted that the same CSF samples used to determine Ab concentrations were also analyzed for endogenous p-tau protein but only in 22 of 37 monkeys because of unavailability of CSF from the other 15 animals. Another notable observation in the study was that the individual variance within groups was much smaller for the animals in the 4 to 6-year-old group than for the other 2 age groups (Fig. 1C). The individual range in the youngest group was from 24.6 to 39.5 (pg/mL; standard deviation [SD] ¼ 8.37); whereas the levels ranged from 14.9 to 53.3 (pg/mL; SD ¼ 15.8) in the 12 to 19-year-old group, and from 9.3 to 87.5 (pg/mL; SD ¼ 22.9) in the >21-year-old group. However, the mean was virtually equal in the 3 different groups
> 21 Yrs
Groups
ng/ml
2.3. Statistical analysis
*
*
20
2.2. Immunoassays for CSF biomarker analysis All samples were obtained by lumbar puncture through the L3/ L4 or L4/L5 interspace and were stored at 80 C until analysis. Following procedures described elsewhere (Darusman et al., 2013; Wang et al., 2013); levels of CSF Ab40 or Ab42 were measured by a sandwich enzyme-linked immunosorbent assay (ELISA) (antibodies and standards were gifts from Tongi Medical School, Wuhan, China). The levels of p-tau in CSF were determined using an INNOTEST ELISA kit (Innogenetic N.V, Gent, Belgium). All samples were run in duplicate.
CSF levels of Aβ 42 25
ng/ml
Scientific Primate Company, Ltd, Guangxi, China) including 29 females and 8 males with age ranging from 4 to 22-year-old. Because 1 year of cynomolgus monkey is equal to 3 years of human life (Weinbauer et al., 2008), they were equivalent to 12 to 66-year-old humans. All animals were individually housed and maintained on a 12-hour light and/or 12-hour dark cycle with water available ad libitum. Toys, a mirror and food supplements were routinely provided to promote psychological well being for all tested animals. All procedures used in this study were fully reviewed and approved by the Animal Care and Use Committee and performed at the primate facility of Wincon TheraCells Biotechnologies Company in Nanning Guangxi China. The facility is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.
1657
60 40 20 0
4-6 Yrs
12-19 Yrs
> 21 Yrs
Groups Fig. 1. Intergroup analyses of Ab42, Ab40, and p-tau levels. Significant (* p < 0.05) differences were seen between experimental groups for CSF levels of Ab42 (A) but not for CSF levels of Ab40 (B), and p-tau (C). The error bars indicate the mean SEM. Abbreviations: Ab, b-amyloid; CSF, cerebrospinal fluid; p-tau, phosphorylated tau; SEM, standard error of the mean.
1658
F. Yue et al. / Neurobiology of Aging 35 (2014) 1656e1659
A
Correlation between age and Aβ 42 30
r2=0.17; P=0.0108
4. Discussion
ng/ml
20
10
0
0
5
10
15
20
25
Age
Correlation between age and Aβ 40
B 500
r2=0.03; P=0.29
ng/ml
400 300 200 100 0
0
5
10
15
20
25
Age
C
Correlation between age and p-tau
r2=0.003; P=0.81
100
pg/ml
80 60 40 20 0
0
5
10
15
(31.8 in 4 to 6-year-old, 27.9 in 12 to 19-year-old, and 30.8 in >21year-old group).
20
25
Age Fig. 2. Linear regression analyses with the 95% confidence band of the best-fit line. A significant negative correlation (r2 ¼ 0.17, p ¼ 0.0108) was found between age and the levels of Ab42 (A), whereas only a very weak correlation was observed in the levels of Ab40 (B), and no correlation was seen in the levels of p-tau (C). Abbreviations: Ab, b-amyloid; p-tau, phosphorylated tau.
Animal models have played a major role in defining critical disease-related mechanisms and exploring novel potential therapeutic approaches in neurodegenerative diseases like AD (Heuer et al., 2012). Here, for the first time, we report significant correlations between age and the decline of CSF Ab42 levels but not Ab40 and p-tau levels in cynomolgus monkeys. These results are in agreement with prior postmortem studies in aged cynomolgus monkeys in which senile plaques, a characteristic feature of AD, spontaneously form in the cortex (Kimura et al., 2005; Oikawa et al., 2010). Thus, in vivo studies, plus postmortem analysis of Alzheimerlike pathology, further support the value of aged cynomolgus monkeys in aging research. In addition, b-APP, which can be cleaved into amyloid-b peptides, is ubiquitously expressed in neurons of all macaque species examined to date; including rhesus, cynomolgus, and other Old World species (Martin et al., 1991, 1994). Also, APP695 (the 695 amino acid isoform) is completely homologous between human and rhesus monkeys and cynomolgus monkeys (Podlisny et al., 1991). The activity of the b-amyloid cleaving enzyme-1 (b-secretase, or BACE-1), which contributes to the liberation of Ab from APP, increases with age as seen in humans (Fukumoto et al., 2004). Thus, the results from the present study provide new and important insight on how closely age-related changes in CSF biomarkers in aged cynomolgus monkeys parallel changes seen in humans. To date, biochemistry and immunohistochemistry have been widely used in studying age-related changes of intracellular Ab in cynomolgus monkeys (Kimura et al., 2005; Nakamura et al., 1998; Oikawa et al., 2010). These studies demonstrated the ageassociated accumulation of insoluble Ab in the neocortical and hippocampal regions of animals as young as mid-20s. Levels of Ab were extremely high in those of advanced age (>30-year-old) and accumulation of p-tau present in the same regions (Oikawa et al., 2010). Although intracellular Ab42 was observed in cortical neurons in aged monkeys, Ab40 levels remained virtually the same regardless of age (Kimura et al., 2005). In the present study, we found an age-associated decrease in Ab42 levels in the CSF with little change in Ab40 levels. Interestingly, the age-associated changes in CSF levels seen in the present study were supported by a recent study also using young and aged cynomolgus monkeys (Darusman et al., 2013). In the study, Darusman et al. (2013) found that the Ab42 concentration in serum of the aged monkeys was significantly lower than in the younger subjects, while the CSF t-tau (total tau) and p-tau did not significantly differ between the groups. Results from both studies (the present and aforementioned studies) indicate that changes in these CSF biomarkers appear to parallel changes in serum, which suggests that levels of serum Ab42 and other biomarkers may reflect changes in CSF in nonhuman primates. It is hypothesized that decreases in CSF levels of Ab42 are because of the aggregations and deposition in the brain parenchyma, which in turn lead to increased levels of intracellular Ab42 (for a review see Jack et al., 2010). Based on the current assumptions, an accumulation of Ab-oligomers during the early, preclinical stage of AD may trigger a cascade of events including neuronal loss and atrophy within cortical and subcortical regions and in turn, may lead to cognitive declines. As mentioned previously, the decrease of CSF Ab42 levels seen in the present study suggests increases of depositions of b-amyloid in the brain, which may indicate
F. Yue et al. / Neurobiology of Aging 35 (2014) 1656e1659
that beginning of the molecular process of AD pathologies occur in aged cynomolgus monkeys (for a review see Nakayama et al., 2004). In conclusion, for the first time, quantitative ELISAs revealed age-associated changes in CSF levels of Ab42 but not Ab40 and ptau in cynomolgus monkeys. The result is supported by age-related changes of Ab in brain tissue (Kimura et al., 2005) and in serum (Darusman et al., 2013). A further comparative study of ageassociated changes between human and nonhuman primate could yield more informative clues to the unique human predisposition to AD. Acknowledgements This study was supported by grants from the National Natural Science Foundation of China (31240043), State High-Tech Development Plan of Ministry of Sciences and Technology of China (2012AA020703, 2012AA02A514), the National Basic Research Development Program of China (2011CB504101), and the Department of Anatomy and Neurobiology, University of Kentucky College of Medicine. The authors want to thank Dr Richard Grondin and Ms. April Evans for reviewing the manuscript. References Brys, M., Pirraglia, E., Rich, K., Rolstad, S., Mosconi, L., Switalski, R., GlodzikSobanska, L., De Santi, S., Zinkowski, R., Mehta, P., Pratico, D., Saint Louis, L.A., Wallin, A., Blennow, K., de Leon, M.J., 2009. Prediction and longitudinal study of CSF biomarkers in mild cognitive impairment. Neurobiol. Aging 30, 682e690. Buchhave, P., Minthon, L., Zetterberg, H., Wallin, A.K., Blennow, K., Hansson, O., 2012. Cerebrospinal fluid levels of beta-amyloid 1-42, but not of tau, are fully changed already 5 to 10 years before the onset of Alzheimer dementia. Arch. Gen. Psychiatry 69, 98e106. Darusman, H.S., Sajuthi, D., Kalliokoski, O., Jacobsen, K.R., Call, J., Schapiro, S.J., Gjedde, A., Abelson, K.S., Hau, J., 2013. Correlations between serum levels of beta amyloid, cerebrospinal levels of tau and phospho tau, and delayed response tasks in young and aged cynomolgus monkeys (Macaca fascicularis). J. Med. Primatol. 42, 137e146. Fukumoto, H., Rosene, D.L., Moss, M.B., Raju, S., Hyman, B.T., Irizarry, M.C., 2004. Beta-secretase activity increases with aging in human, monkey, and mouse brain. Am. J. Pathol. 164, 719e725. Galasko, D., 1998. CSF tau and Abeta42: logical biomarkers for Alzheimer’s disease? Neurobiol. Aging 19, 117e119. Heuer, E., Rosen, R.F., Cintron, A., Walker, L.C., 2012. Nonhuman primate models of Alzheimer-like cerebral proteopathy. Curr. Pharm. Des. 18, 1159e1169.
1659
Hulstaert, F., Blennow, K., Ivanoiu, A., Schoonderwaldt, H.C., Riemenschneider, M., De Deyn, P.P., Bancher, C., Cras, P., Wiltfang, J., Mehta, P.D., Iqbal, K., Pottel, H., Vanmechelen, E., Vanderstichele, H., 1999. Improved discrimination of AD patients using beta-amyloid(1-42) and tau levels in CSF. Neurology 52, 1555e1562. Jack Jr., C.R., Knopman, D.S., Jagust, W.J., Shaw, L.M., Aisen, P.S., Weiner, M.W., Petersen, R.C., Trojanowski, J.Q., 2010. Hypothetical model of dynamic biomarkers of the Alzheimer’s pathological cascade. Lancet Neurol. 9, 119e128. Kimura, N., Yanagisawa, K., Terao, K., Ono, F., Sakakibara, I., Ishii, Y., Kyuwa, S., Yoshikawa, Y., 2005. Age-related changes of intracellular Abeta in cynomolgus monkey brains. Neuropathol. Appl. Neurobiol. 31, 170e180. Lewczuk, P., Esselmann, H., Otto, M., Maler, J.M., Henkel, A.W., Henkel, M.K., Eikenberg, O., Antz, C., Krause, W.R., Reulbach, U., Kornhuber, J., Wiltfang, J., 2004. Neurochemical diagnosis of Alzheimer’s dementia by CSF Abeta42, Abeta42/Abeta40 ratio and total tau. Neurobiol. Aging 25, 273e281. Martin, L.J., Pardo, C.A., Cork, L.C., Price, D.L., 1994. Synaptic pathology and glial responses to neuronal injury precede the formation of senile plaques and amyloid deposits in the aging cerebral cortex. Am. J. Pathol. 145, 1358e1381. Martin, L.J., Sisodia, S.S., Koo, E.H., Cork, L.C., Dellovade, T.L., Weidemann, A., Beyreuther, K., Masters, C., Price, D.L., 1991. Amyloid precursor protein in aged nonhuman primates. Proc. Natl. Acad. Sci. U.S.A 88, 1461e1465. Musiek, E.S., Holtzman, D.M., 2012. Origins of Alzheimer’s disease: reconciling cerebrospinal fluid biomarker and neuropathology data regarding the temporal sequence of amyloid-beta and tau involvement. Curr. Opin. Neurol. 25, 715e720. Nakamura, S., Nakayama, H., Goto, N., Ono, F., Sakakibara, I., Yoshikawa, Y., 1998. Histopathological studies of senile plaques and cerebral amyloidosis in cynomolgus monkeys. J. Med. Primatol. 27, 244e252. Nakayama, H., Uchida, K., Doi, K., 2004. A comparative study of age-related brain pathologyeare neurodegenerative diseases present in nonhuman animals? Med Hypoth. 632, 198e202. Nutu, M., Zetterberg, H., Londos, E., Minthon, L., Nagga, K., Blennow, K., Hansson, O., Ohrfelt, A., 2013. Evaluation of the cerebrospinal fluid amyloid-beta1-42/ amyloid-beta1-40 ratio measured by alpha-LISA to distinguish Alzheimer’s disease from other dementia disorders. Dement Geriatr. Cogn. Disord. 36, 99e110. Oikawa, N., Kimura, N., Yanagisawa, K., 2010. Alzheimer-type tau pathology in advanced aged nonhuman primate brains harboring substantial amyloid deposition. Brain Res. 1315, 137e149. Podlisny, M.B., Tolan, D.R., Selkoe, D.J., 1991. Homology of the amyloid beta protein precursor in monkey and human supports a primate model for beta amyloidosis in Alzheimer’s disease. Am. J. Pathol. 138, 1423e1435. Wang, L., Brier, M.R., Snyder, A.Z., Thomas, J.B., Fagan, A.M., Xiong, C., Benzinger, T.L., Holtzman, D.M., Morris, J.C., Ances, B.M., 2013. Cerebrospinal fluid Abeta42, phosphorylated Tau181, and resting-state functional connectivity. JAMA Neurol. 70, 1242e1248. Weinbauer, G.F., Niehoff, M., Niehaus, M., Srivastav, S., Fuchs, A., Van Esch, E., Cline, J.M., 2008. Physiology and endocrinology of the ovarian cycle in macaques. Toxicol. Pathol. 36 (7S), 7Se23S. Wiltfang, J., Esselmann, H., Bibl, M., Smirnov, A., Otto, M., Paul, S., Schmidt, B., Klafki, H.W., Maler, M., Dyrks, T., Bienert, M., Beyermann, M., Ruther, E., Kornhuber, J., 2002. Highly conserved and disease-specific patterns of carboxyterminally truncated Abeta peptides 1-37/38/39 in addition to 1-40/42 in Alzheimer’s disease and in patients with chronic neuroinflammation. J. Neurochem. 81, 481e496.
