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Mitochondrial alterations and tendency to apoptosis in peripheral blood cells from children with Down syndrome
FEBS Letters 581 (2007) 521–525
Mitochondrial alterations and tendency to apoptosis in peripheral blood cells from children with Down syndrome Erika Roata,1, Nicole Pradaa,b,1, Roberta Ferraresia, Chiara Giovenzanaa, Milena Nasia, Leonarda Troianoa, Marcello Pintia, Elisa Nemesa, Enrico Luglia, Ornella Biagionic, Mauro Mariottic, Luigi Ciaccid, Ugo Consolod, Fiorella Ballie, Andrea Cossarizzaa,* a
d
Chair of Immunology, Department of Biomedical Sciences, University of Modena and Reggio Emilia, via Campi 287, 41100 Modena, Italy b Department of Pathobiology, University of Palermo, via Tukory 211, 90134 Palermo, Italy c Department of Child Neuropsychiatry, AUSL Modena, via Cardarelli 45, 41100 Modena, Italy Department of Neurosciences, Head–Neck, Rehabilitation, Section of Dentistry and Maxillofacial Surgery, University of Modena and Reggio Emilia, via del Pozzo 71, 41100 Modena, Italy e Department of Pediatrics, University of Modena and Reggio Emilia, via del Pozzo 71, 41100 Modena, Italy Received 5 December 2006; accepted 26 December 2006 Available online 17 January 2007 Edited by Jesus Avila
Abstract Different types of cells from subjects with Down syndrome (DS) have an increased susceptibility to cell death. We have studied apoptosis and mitochondrial (mt) membrane potential (DWm) in peripheral blood mononuclear cells (PBMC) from DS children and age-matched healthy donors after in vitro treatment with apoptogenic molecules, along with mtDNA content. We found that PBMC from DS and healthy controls had a similar tendency to undergo apoptosis and a similar amount of mtDNA. However, in cells from DS subjects, mitochondria showed a higher loss of DWm, underlying the presence of an increasing susceptibility of these organelles to damaging agents. 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Down syndrome; Mitochondria; mtDNA; Apoptosis
1. Introduction Down syndrome (DS, trisomy 21) is the most frequent chromosomal abnormality in live births and the most frequent cause of mental retardation. This condition is associated with a number of manifestations including heart defects, type I diabetes mellitus, hypotonia, increased susceptibility to bacterial and viral infections and a precocious development of Alzheimer disease, as well as several defects of the acquired immune response [1–4]. Neurons from DS subjects display an increased susceptibility to apoptosis [5–7]. Fetal DS neurons are also characterized by high production of reactive oxygen species (ROS), that in turn involve mitochondrial death pathway, as shown by increased levels of bax, and cytoplasmic translocation of cytochrome c and apoptosis inducing factor (AIF) [5,8]. Deletions, mutations or replication abnormalities of mitochondrial (mt) DNA due to genetic defects, hypoxia, oxidative * Corresponding author. Fax: +39 0 59 2055426. E-mail address: [email protected] (A. Cossarizza). 1
E. Roat and N. Prada have equally contributed to this work.
stress or age can impair mitochondrial function resulting in energy depletion and increased susceptibility to apoptosis [9]. Many alterations have been described in mitochondria of DS subjects [10,11]. Astrocytes and fibroblasts show decreased mitochondrial redox activity and depolarization of mitochondrial membrane potential (DWm) [12]. This dysfunction could be responsible of a higher vulnerability to exogenous toxic insults, such as those provoked by ROS [5]. A decreased repair of oxidative damage in mtDNA from DS fibroblasts compared to normal fibroblasts has been reported, which leads to a greater oxidative damage in mtDNA [10]. Lee et al. also demonstrated an impairment in the levels of transcription of the mitochondrial gene ATPase6 in DS fetuses compared to control ones [13], which could affect the generation of ATP [11]. Peripheral blood mononuclear cells (PBMC) from DS subjects are characterized by several alterations including impairment in proliferative response to phytohemagglutinin [14], defective production of interleukin-2 [15] and a significant reduction in IL-1b secretion after activation with LPS [16]. Since some of these alterations could be related to a reduced mitochondria functionality, we analyzed different parameters related to mitochondrial functionality in PBMC from DS subjects. In particular, we quantified the content of mtDNA per cell and studied the behaviour of PBMC and mitochondria after exposure to different drugs, and compared these parameters to those obtained in healthy donors.
2. Materials and methods 2.1. Subjects We studied a total of 16 children with DS with a mean age of 5.1 years (range: 1–16), 11 males and 5 females; as controls, we studied a total of 24 karyotypically normal children with a mean age of 4.5 years (range: 1–10), 21 males and 3 females. All subjects were in healthy conditions, without relevant acute or chronic disease affecting the immune system. Informed consent was obtained by their parents. 2.2. Cell culture PBMC were isolated from a minimum of 5 ml freshly collected blood according to standard procedures. PBMC were plated at the density of 4 · 105 cells/ml in complete culture medium, i.e. RPMI
0014-5793/$32.00 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.12.058
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E. Roat et al. / FEBS Letters 581 (2007) 521–525
1640 supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM L -glutamine, 100 IU/ml penicillin, 100 lg/ml streptomycin (all from Gibco BRL, Life Tecnologies, Scotland) at 37 C in humidified atmosphere (5% CO2 in air). Cells were treated with Doxorubicin (Doxo), Daunomicin (Dauno) or Quercetin (Qu) (Sigma–Aldrich, St. Louis, USA), all at the final concentration of 100 lM for 24 and 48 h, and then analyzed by flow cytometry.
2.6. mtDNA quantification DNA was extracted from 5 · 106 PBMC using the QIAamp DNA Mini Kit (QIAGEN) according to the manufacturer’s protocols and stored at 20 C until use. Content of mtDNA per cell was measured by using Mitochondrial DNA qPCR kit (from GeneMoRe Italy srl, Modena, Italy) as described [19].
2.3. Detection of mitochondrial membrane potential (DWm) Cells were stained with the DWm-sensitive probe JC-1 (final concentration 10 lM), as described [17].
2.7. Statistical analysis Statistical analyses were performed by two-tail Student t-test, using the software Prism 3.03 under Windows XP. A P value <0.05 was considered significant.
2.4. Detection of DNA content in living cells Hoechst 33342 was used to measure DNA content in living, intact cells thanks to its capability to bind sequences of 3 AT base pairs [18]. When excited by UV, it emits at 455 nm and allows the quantification of DNA content. Cells were stained with 5 lM Hoechst 33342 at 37 C for 30 minutes in complete medium without FCS and immediately analyzed. Those with the typical hypodiploid peak were considered apoptotic. 2.5. Flow cytometry DWm and apoptosis were analyzed by a Cyflow ML (from Partec GmbH, Mu¨nster, Germany), equipped with a solid state blue laser (emitting at 488 nm, 200 mW, kept at 50 mW), a UV Mercury lamp HBO (100 long life, 100 W) and a red diode laser (635 nm, 25 mW). Data were analyzed by Partec Flomax 3.0 and WinMDI softwares, under Windows XP.
3. Results 3.1. Cells from DS patients show a higher loss of mitochondrial membrane potential (DWm) Fig. 1 shows a representative example of changes in DWm (upper panels) and apoptosis (lower panels) in Doxo-treated PBMC from a DS patient. As shown in Fig. 2, after 24 (upper panel) and 48 h (lower panel) of treatment Doxo, Dauno and Qu induced a significant loss of DWm in PBMC of both DS patients and healthy donors. No significant differences in the percentage of cells with depolarized DWm were present between the two groups after 24 h of treatment. However, after 48 h
Fig. 1. Cytofluorimetric analysis of PBMC from a DS patient. Representative example of cytofluorimetric analysis showing changes in DWm (upper panels) and apoptosis (lower panels) in PBMC from a DS child after 48 h of incubation with 100 lM doxorubicin.
E. Roat et al. / FEBS Letters 581 (2007) 521–525
523
mtDNA content
50 25
(copies per cell)
24 h 75
% of cells with low Δψm
75
HD DS
100
50
25
0 100
** 48 h
75
0
**
HD
*
50 25
DS
Fig. 4. Mitochondrial DNA content in PBMC from DS and healthy children. Mitochondrial DNA content is expressed as copies of mtDNA per cell in PBMC from DS and control healthy donors (HD). Values are expressed as means ± S.E.M. No statistical difference was present between the two groups.
0
CTRL
DOXO
DAUNO
QU
Fig. 2. Changes in DWm in cells from DS and healthy children. Percentage of PBMC from DS children and healthy donors (HD) with low DWm after 24 (upper panel) and 48 (lower panel) hours of incubation with doxorubicin (DOXO), daunomicin (DAUNO), quercetin (QU), or no stimulus (CTRL). Values are expressed as means ± S.E.M. HD vs. DS: *P < 0.05; **P < 0.005.
of incubation with Doxo, Dauno and Qu, PBMC from DS subjects showed a consistent increase in the percentage of cells with a relevant loss of DWm when compared to cells from control children (for Doxo: P = 0.008; Dauno: P = 0.004; Qu: P = 0.013). 3.2. Cells from DS do not display an increased tendency to undergo apoptosis As shown in Fig. 3, after 24 (upper panel) and 48 h (lower panel) of treatment Doxo, Dauno and Qu induced a consistent apoptosis in both DS and healthy subjects. However, even if a
HD DS
100
24 h
% of apoptotic nuclei
75 50 25 0 100
48 h 75 50 25 0
CTRL
DOXO
DAUNO
QU
Fig. 3. Apoptotic nuclei in DS and healthy children. Percentage of apoptotic nuclei in PBMC cultures from DS children and healthy donors (HD) after 24 (upper panel) and 48 (lower panel) hours of incubation with doxorubicin (DOXO), daunomicin (DAUNO), quercetin (QU), or no stimulus (CTRL). Values are expressed as means ± S.E.M. In no case comparison between DS and HD revealed a statistical significance.
higher tendency was noted in cells from DS patients after 48 h of culture, in no case the difference between the two groups of donors was significant. 3.3. mtDNA quantification We finally measured the amount of mtDNA in PBMC from DS patients and healthy children, and found that they had a comparable number of copies of mtDNA per cell (Fig. 4).
4. Discussion Changes in mitochondrial functionality and increased apoptosis have been described in patients affected by DS. These alterations have been found in neurons, whose degeneration is supposed to be responsible for the precocious onset of Alzheimer disease, which typically affects DS individuals after the age of 35–40 years [20], in whom b-amiloid is over-expressed [21]. Little is known on the presence of mitochondrial damages and tendency to undergo apoptosis in peripheral blood cells from DS individuals, who present profound alterations in the distribution of several lymphocyte subpopulations [22,23]. DS patients present a high risk of developing pathologies of the immune system, such as autoimmune diseases or leukaemia [24,25]. Complex changes of the normal cellular homeostasis exist that could explain, at least in part, these pathologies. On the one side, autoimmunity can result from a decreased apoptosis of potentially autoreactive lymphocytes that are generated during the process of intrathymic negative selection, or to the loss of peripheral tolerance by mature T lymphocytes. On the other hand, tumour cells, including leukemic lymphocytes, are often characterized by the increased expression and activity of anti-apoptotic genes, that are responsible for the increased resistance to a variety of apoptogenic stimuli. When mitochondria are involved in the apoptotic process, one of the possible events is a precocious loss of DWm, followed by the release of cytochrome c and AIF, leading to caspase activation, which in turn causes to the degradation of nuclear DNA. We analyzed the susceptibility to undergo apoptosis and changes in DWm in peripheral blood cells from children with DS, compared to age-matched healthy controls, either in basal conditions or in response to different agents (daunomycin,
524
doxorubicin), or to a natural flavonoid such as quercetin, that have a strong pro-apoptotic effect [26,27]. We found that, after 24 h of incubation, PBMC from DS and controls showed a comparable percentage of cells with low DWm. After 48 h, the treatment with toxic substances increased the number of cells with low DWm both in DS and control cells, but such effect was much stronger in DS. This indicates that mitochondria from DS patients had a higher sensitivity to damaging agents, in agreement with what found by other authors. Indeed, studies on astrocytes from DS subjects demonstrated a lower DWm and a decrease in mitochondrial redox activity compared to control cells [28]. This observation, along with others reporting an impairment in mitochondrial redox activity, could explain the lower DWm displayed by PBMC from DS subjects after incubation with apoptogenic substances. We found that PBMC from DS and healthy controls have a similar tendency to undergo apoptosis, even if DS mitochondria were more sensitive to several damaging agents. Thus, since DS cells with altered mitochondrial function do not undergo apoptosis, it might be hypothesized that DS patients tend to maintain damaged cells, or that they have a higher capacity to repair functional damages. It remains to be established if and how this phenomenon can be linked to the development of autoimmunity or neoplastic disorders. A decrease in mtDNA content leads to an impaired production of enzymes of the respiratory chain, with a consequent reduction in the production of ATP, and a subsequent alteration in cellular metabolism. Therefore, to investigate the possibility that an increased sensitivity of mitochondria to apoptogenic agents was due to alterations in the amount of mtDNA, we have quantified the number of copies of mtDNA per cell in PBMC from DS and controls. This represent a new information since, to our knowledge, no data are currently present in literature about analysis of mtDNA copy number in DS patients. Changes in mtDNA content from DS cells could be expected also because such alterations have been reported during the aging process and DS is characterized by precocious aging. We have found that all donors had the same amount of mtDNA, but it has to be underlined that the donors we could analyze were very young, and thus it might be that alterations in the synthesis and changes in the amount of mtDNA could be detectable only in older patients, after several years of exposure to ROS, causing mtDNA alterations. Moreover, most PBMC have a relatively short half life, as they are constantly renewed in the body; this could be a limiting factor in revealing differences in mtDNA synthesis. Thus, it would be extremely interesting to measure mtDNA in cells with a longer life, that are chronically exposed to mitochondrial damages. Acknowledgement: We kindly acknowledge all the children and their families who made this study possible. This paper has been supported by MURST-COFIN 2004 and by FIRB 2001 grants to A.C. We acknowledge Partec GmbH (Mu¨nster, Germany) and Space srl (Milan, Italy) for precious technical support, and GeneMoRe Italy srl (Modena, Italy) for quantitative analysis of mtDNA content.
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E. Roat et al. / FEBS Letters 581 (2007) 521–525 [2] Fong, C.T. and Brodeur, G.M. (1987) Down’s syndrome and leukemia: epidemiology, genetics, cytogenetics and mechanisms of leukemogenesis. Cancer Genet. Cytogenet. 28, 55–76. [3] Prada, N., Nasi, M., Troiano, L., Roat, E., Pinti, M., Nemes, E., Lugli, E., Ferraresi, R., Ciacci, L., Bretoni, D., Biagioni, O., Gibertoni, M., Cornia, C., Meschiari, L., Gramazio, E., Mariotti, M., Consolo, U., Balli, F. and Cossarizza, A. (2005) Direct analysis of thymic function in children with Down’s syndrome. Immun. Ageing 2, 4. [4] Navarro, C.L., Cau, P. and Levy, N. (2006) Molecular bases of progeroid syndromes. Hum. Mol. Genet. 15 Spec No 2 (Spec No 2), R151–R161. [5] Busciglio, J. and Yankner, B.A. (1995) Apoptosis and increased generation of reactive oxygen species in Down’s syndrome neurons in vitro. Nature 378, 776–779. [6] Teipel, S.J. and Hampel, H. (2006) Neuroanatomy of Down syndrome in vivo: a model of preclinical Alzheimer’s disease. Behav. Genet. 36, 405–415. [7] Chen, Y.Z. (2004) APP induces neuronal apoptosis through APPBP1-mediated downregulation of beta-catenin. Apoptosis 9, 415– 422. [8] Helguera, P., Pelsman, A., Pigino, G., Wolvetang, E., Head, E. and Busciglio, J. (2005) Ets-2 promotes the activation of a mitochondrial death pathway in Down’s syndrome neurons. J. Neurosci. 25, 2295–2303. [9] Zeviani, M. and Antozzi, C. (1997) Mitochondrial disorders. Mol. Hum. Reprod. 3, 133–148. [10] Druzhyna, N., Nair, R.G., LeDoux, S.P. and Wilson, G.L. (1998) Defective repair of oxidative damage in mitochondrial DNA in Down’s syndrome. Mutat. Res. 409, 81–89. [11] Lee, H.C., Yin, P.H., Lu, C.Y., Chi, C.W. and Wei, Y.H. (2000) Increase of mitochondria and mitochondrial DNA in response to oxidative stress in human cells. Biochem. J. 348 (Pt 2), 425–432. [12] Lenaz, G., Baracca, A., Fato, R., Genova, M.L. and Solaini, G. (2006) New insights into structure and function of mitochondria and their role in aging and disease. Antioxid. Redox. Signal. 8, 417–437. [13] Lee, S.H., Lee, S., Jun, H.S., Jeong, H.J., Cha, W.T., Cho, Y.S., Kim, J.H., Ku, S.Y. and Cha, K.Y. (2003) Expression of the mitochondrial ATPase6 gene and Tfam in Down syndrome. Mol. Cells 15, 181–185. [14] Hamurcu, Z.D.H., Patiroglu, T., Kumandas, S., Imamoglu, N., Ozkul, Y. and Donmez-Altuntas, H. (2006) Age-dependent decreases in mitogen-stimulation level and RNA content in peripheral blood mononuclear cells of down syndrome patients. Cytometry B Clin. Cytom. [15] Gerez, L., Madar, L., Arad, G., Sharav, T., Reshef, A., Ketzinel, M., Sayar, D., Silberberg, C. and Kaempfer, R. (1991) Aberrant regulation of interleukin-2 but not of interferon-gamma gene expression in Down syndrome (trisomy 21). Clin. Immunol. Immunopathol. 58, 251–266. [16] Park, E., Alberti, J., Mehta, P., Dalton, A., Sersen, E. and Schuller-Levis, G. (2000) Partial impairment of immune functions in peripheral blood leukocytes from aged men with Down’s syndrome. Clin. Immunol. 95, 62–69. [17] Cossarizza, A., Baccarani-Contri, M., Kalashnikova, G. and Franceschi, C. (1993) A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the Jaggregate forming lipophilic cation 5,5 0 ,6,6 0 -tetrachloro1,1 0 ,3,3 0 - tetraethylbenzimidazolcarbocyanine iodide (JC-1). Biochem. Biophys. Res. Commun. 197, 40–45. [18] Shapiro, H.M. (1995) Pratical Flow Cytometry, Wiley-Liss, New York. [19] Mussini, C., Pinti, M., Bugarini, R., Borghi, V., Nasi, M., Nemes, E., Troiano, L., Guaraldi, G., Bedini, A., Sabin, C., Esposito, R. and Cossarizza, A. (2005) Effect of treatment interruption monitored by CD4 cell count on mitochondrial DNA content in HIV-infected patients: a prospective study. Aids 19, 1627–1633. [20] Mann, D.M., Yates, P.O., Marcyniuk, B. and Ravindra, C.R. (1985) Pathological evidence for neurotransmitter deficits in Down’s syndrome of middle age. J. Ment. Defic. Res. 29 (Pt 2), 125–135. [21] Tanzi, R.E., Gusella, J.F., Watkins, P.C., Bruns, G.A., GeorgeHyslop, P.St., Van Keuren, M.L., Patterson, D., Pagan, S., Kurnit, D.M. and Neve, R.L. (1987) Amyloid beta protein gene:
E. Roat et al. / FEBS Letters 581 (2007) 521–525 cDNA, mRNA distribution, and genetic linkage near the Alzheimer locus. Science 235, 880–884. [22] Cossarizza, A., Monti, D., Montagnani, G., Ortolani, C., Masi, M., Zannotti, M. and Franceschi, C. (1990) Precocious aging of the immune system in Down syndrome: alteration of B lymphocytes, T-lymphocyte subsets, and cells with natural killer markers. Am. J. Med. Genet. Suppl. 7, 213–218. [23] Cossarizza, A., Ortolani, C., Forti, E., Montagnani, G., Paganelli, R., Zannotti, M., Marini, M., Monti, D. and Franceschi, C. (1991) Age-related expansion of functionally inefficient cells with markers of natural killer activity in Down’s syndrome. Blood 77, 1263–1270. [24] Satge, D., Sommelet, D., Geneix, A., Nishi, M., Malet, P. and Vekemans, M. (1998) A tumor profile in Down syndrome. Am. J. Med. Genet. 78, 207–216.
525 [25] Goldacre, M.J., Wotton, C.J., Seagroatt, V. and Yeates, D. (2004) Cancers and immune related diseases associated with Down’s syndrome: a record linkage study. Arch. Dis. Child. 89, 1014–1017. [26] Lee, T.J., Kim, O.H., Kim, Y.H., Lim, J.H., Kim, S., Park, J.W. and Kwon, T.K. (2006) Quercetin arrests G2/M phase and induces caspase-dependent cell death in U937 cells. Cancer Lett. 240, 234–242. [27] Lugli, E., Troiano, L., Ferraresi, R., Roat, E., Prada, N., Nasi, M., Pinti, M., Cooper, E.L. and Cossarizza, A. (2005) Characterization of cells with different mitochondrial membrane potential during apoptosis. Cytometry A 68, 28–35. [28] Busciglio, J., Pelsman, A., Wong, C., Pigino, G., Yuan, M., Mori, H. and Yankner, B.A. (2002) Altered metabolism of the amyloid beta precursor protein is associated with mitochondrial dysfunction in Down’s syndrome. Neuron 33, 677–688.
Mitochondrial alterations and tendency to apoptosis in peripheral blood cells from children with Down syndrome Erika Roata,1, Nicole Pradaa,b,1, Roberta Ferraresia, Chiara Giovenzanaa, Milena Nasia, Leonarda Troianoa, Marcello Pintia, Elisa Nemesa, Enrico Luglia, Ornella Biagionic, Mauro Mariottic, Luigi Ciaccid, Ugo Consolod, Fiorella Ballie, Andrea Cossarizzaa,* a
d
Chair of Immunology, Department of Biomedical Sciences, University of Modena and Reggio Emilia, via Campi 287, 41100 Modena, Italy b Department of Pathobiology, University of Palermo, via Tukory 211, 90134 Palermo, Italy c Department of Child Neuropsychiatry, AUSL Modena, via Cardarelli 45, 41100 Modena, Italy Department of Neurosciences, Head–Neck, Rehabilitation, Section of Dentistry and Maxillofacial Surgery, University of Modena and Reggio Emilia, via del Pozzo 71, 41100 Modena, Italy e Department of Pediatrics, University of Modena and Reggio Emilia, via del Pozzo 71, 41100 Modena, Italy Received 5 December 2006; accepted 26 December 2006 Available online 17 January 2007 Edited by Jesus Avila
Abstract Different types of cells from subjects with Down syndrome (DS) have an increased susceptibility to cell death. We have studied apoptosis and mitochondrial (mt) membrane potential (DWm) in peripheral blood mononuclear cells (PBMC) from DS children and age-matched healthy donors after in vitro treatment with apoptogenic molecules, along with mtDNA content. We found that PBMC from DS and healthy controls had a similar tendency to undergo apoptosis and a similar amount of mtDNA. However, in cells from DS subjects, mitochondria showed a higher loss of DWm, underlying the presence of an increasing susceptibility of these organelles to damaging agents. 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Down syndrome; Mitochondria; mtDNA; Apoptosis
1. Introduction Down syndrome (DS, trisomy 21) is the most frequent chromosomal abnormality in live births and the most frequent cause of mental retardation. This condition is associated with a number of manifestations including heart defects, type I diabetes mellitus, hypotonia, increased susceptibility to bacterial and viral infections and a precocious development of Alzheimer disease, as well as several defects of the acquired immune response [1–4]. Neurons from DS subjects display an increased susceptibility to apoptosis [5–7]. Fetal DS neurons are also characterized by high production of reactive oxygen species (ROS), that in turn involve mitochondrial death pathway, as shown by increased levels of bax, and cytoplasmic translocation of cytochrome c and apoptosis inducing factor (AIF) [5,8]. Deletions, mutations or replication abnormalities of mitochondrial (mt) DNA due to genetic defects, hypoxia, oxidative * Corresponding author. Fax: +39 0 59 2055426. E-mail address: [email protected] (A. Cossarizza). 1
E. Roat and N. Prada have equally contributed to this work.
stress or age can impair mitochondrial function resulting in energy depletion and increased susceptibility to apoptosis [9]. Many alterations have been described in mitochondria of DS subjects [10,11]. Astrocytes and fibroblasts show decreased mitochondrial redox activity and depolarization of mitochondrial membrane potential (DWm) [12]. This dysfunction could be responsible of a higher vulnerability to exogenous toxic insults, such as those provoked by ROS [5]. A decreased repair of oxidative damage in mtDNA from DS fibroblasts compared to normal fibroblasts has been reported, which leads to a greater oxidative damage in mtDNA [10]. Lee et al. also demonstrated an impairment in the levels of transcription of the mitochondrial gene ATPase6 in DS fetuses compared to control ones [13], which could affect the generation of ATP [11]. Peripheral blood mononuclear cells (PBMC) from DS subjects are characterized by several alterations including impairment in proliferative response to phytohemagglutinin [14], defective production of interleukin-2 [15] and a significant reduction in IL-1b secretion after activation with LPS [16]. Since some of these alterations could be related to a reduced mitochondria functionality, we analyzed different parameters related to mitochondrial functionality in PBMC from DS subjects. In particular, we quantified the content of mtDNA per cell and studied the behaviour of PBMC and mitochondria after exposure to different drugs, and compared these parameters to those obtained in healthy donors.
2. Materials and methods 2.1. Subjects We studied a total of 16 children with DS with a mean age of 5.1 years (range: 1–16), 11 males and 5 females; as controls, we studied a total of 24 karyotypically normal children with a mean age of 4.5 years (range: 1–10), 21 males and 3 females. All subjects were in healthy conditions, without relevant acute or chronic disease affecting the immune system. Informed consent was obtained by their parents. 2.2. Cell culture PBMC were isolated from a minimum of 5 ml freshly collected blood according to standard procedures. PBMC were plated at the density of 4 · 105 cells/ml in complete culture medium, i.e. RPMI
0014-5793/$32.00 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.12.058
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E. Roat et al. / FEBS Letters 581 (2007) 521–525
1640 supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM L -glutamine, 100 IU/ml penicillin, 100 lg/ml streptomycin (all from Gibco BRL, Life Tecnologies, Scotland) at 37 C in humidified atmosphere (5% CO2 in air). Cells were treated with Doxorubicin (Doxo), Daunomicin (Dauno) or Quercetin (Qu) (Sigma–Aldrich, St. Louis, USA), all at the final concentration of 100 lM for 24 and 48 h, and then analyzed by flow cytometry.
2.6. mtDNA quantification DNA was extracted from 5 · 106 PBMC using the QIAamp DNA Mini Kit (QIAGEN) according to the manufacturer’s protocols and stored at 20 C until use. Content of mtDNA per cell was measured by using Mitochondrial DNA qPCR kit (from GeneMoRe Italy srl, Modena, Italy) as described [19].
2.3. Detection of mitochondrial membrane potential (DWm) Cells were stained with the DWm-sensitive probe JC-1 (final concentration 10 lM), as described [17].
2.7. Statistical analysis Statistical analyses were performed by two-tail Student t-test, using the software Prism 3.03 under Windows XP. A P value <0.05 was considered significant.
2.4. Detection of DNA content in living cells Hoechst 33342 was used to measure DNA content in living, intact cells thanks to its capability to bind sequences of 3 AT base pairs [18]. When excited by UV, it emits at 455 nm and allows the quantification of DNA content. Cells were stained with 5 lM Hoechst 33342 at 37 C for 30 minutes in complete medium without FCS and immediately analyzed. Those with the typical hypodiploid peak were considered apoptotic. 2.5. Flow cytometry DWm and apoptosis were analyzed by a Cyflow ML (from Partec GmbH, Mu¨nster, Germany), equipped with a solid state blue laser (emitting at 488 nm, 200 mW, kept at 50 mW), a UV Mercury lamp HBO (100 long life, 100 W) and a red diode laser (635 nm, 25 mW). Data were analyzed by Partec Flomax 3.0 and WinMDI softwares, under Windows XP.
3. Results 3.1. Cells from DS patients show a higher loss of mitochondrial membrane potential (DWm) Fig. 1 shows a representative example of changes in DWm (upper panels) and apoptosis (lower panels) in Doxo-treated PBMC from a DS patient. As shown in Fig. 2, after 24 (upper panel) and 48 h (lower panel) of treatment Doxo, Dauno and Qu induced a significant loss of DWm in PBMC of both DS patients and healthy donors. No significant differences in the percentage of cells with depolarized DWm were present between the two groups after 24 h of treatment. However, after 48 h
Fig. 1. Cytofluorimetric analysis of PBMC from a DS patient. Representative example of cytofluorimetric analysis showing changes in DWm (upper panels) and apoptosis (lower panels) in PBMC from a DS child after 48 h of incubation with 100 lM doxorubicin.
E. Roat et al. / FEBS Letters 581 (2007) 521–525
523
mtDNA content
50 25
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24 h 75
% of cells with low Δψm
75
HD DS
100
50
25
0 100
** 48 h
75
0
**
HD
*
50 25
DS
Fig. 4. Mitochondrial DNA content in PBMC from DS and healthy children. Mitochondrial DNA content is expressed as copies of mtDNA per cell in PBMC from DS and control healthy donors (HD). Values are expressed as means ± S.E.M. No statistical difference was present between the two groups.
0
CTRL
DOXO
DAUNO
QU
Fig. 2. Changes in DWm in cells from DS and healthy children. Percentage of PBMC from DS children and healthy donors (HD) with low DWm after 24 (upper panel) and 48 (lower panel) hours of incubation with doxorubicin (DOXO), daunomicin (DAUNO), quercetin (QU), or no stimulus (CTRL). Values are expressed as means ± S.E.M. HD vs. DS: *P < 0.05; **P < 0.005.
of incubation with Doxo, Dauno and Qu, PBMC from DS subjects showed a consistent increase in the percentage of cells with a relevant loss of DWm when compared to cells from control children (for Doxo: P = 0.008; Dauno: P = 0.004; Qu: P = 0.013). 3.2. Cells from DS do not display an increased tendency to undergo apoptosis As shown in Fig. 3, after 24 (upper panel) and 48 h (lower panel) of treatment Doxo, Dauno and Qu induced a consistent apoptosis in both DS and healthy subjects. However, even if a
HD DS
100
24 h
% of apoptotic nuclei
75 50 25 0 100
48 h 75 50 25 0
CTRL
DOXO
DAUNO
QU
Fig. 3. Apoptotic nuclei in DS and healthy children. Percentage of apoptotic nuclei in PBMC cultures from DS children and healthy donors (HD) after 24 (upper panel) and 48 (lower panel) hours of incubation with doxorubicin (DOXO), daunomicin (DAUNO), quercetin (QU), or no stimulus (CTRL). Values are expressed as means ± S.E.M. In no case comparison between DS and HD revealed a statistical significance.
higher tendency was noted in cells from DS patients after 48 h of culture, in no case the difference between the two groups of donors was significant. 3.3. mtDNA quantification We finally measured the amount of mtDNA in PBMC from DS patients and healthy children, and found that they had a comparable number of copies of mtDNA per cell (Fig. 4).
4. Discussion Changes in mitochondrial functionality and increased apoptosis have been described in patients affected by DS. These alterations have been found in neurons, whose degeneration is supposed to be responsible for the precocious onset of Alzheimer disease, which typically affects DS individuals after the age of 35–40 years [20], in whom b-amiloid is over-expressed [21]. Little is known on the presence of mitochondrial damages and tendency to undergo apoptosis in peripheral blood cells from DS individuals, who present profound alterations in the distribution of several lymphocyte subpopulations [22,23]. DS patients present a high risk of developing pathologies of the immune system, such as autoimmune diseases or leukaemia [24,25]. Complex changes of the normal cellular homeostasis exist that could explain, at least in part, these pathologies. On the one side, autoimmunity can result from a decreased apoptosis of potentially autoreactive lymphocytes that are generated during the process of intrathymic negative selection, or to the loss of peripheral tolerance by mature T lymphocytes. On the other hand, tumour cells, including leukemic lymphocytes, are often characterized by the increased expression and activity of anti-apoptotic genes, that are responsible for the increased resistance to a variety of apoptogenic stimuli. When mitochondria are involved in the apoptotic process, one of the possible events is a precocious loss of DWm, followed by the release of cytochrome c and AIF, leading to caspase activation, which in turn causes to the degradation of nuclear DNA. We analyzed the susceptibility to undergo apoptosis and changes in DWm in peripheral blood cells from children with DS, compared to age-matched healthy controls, either in basal conditions or in response to different agents (daunomycin,
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doxorubicin), or to a natural flavonoid such as quercetin, that have a strong pro-apoptotic effect [26,27]. We found that, after 24 h of incubation, PBMC from DS and controls showed a comparable percentage of cells with low DWm. After 48 h, the treatment with toxic substances increased the number of cells with low DWm both in DS and control cells, but such effect was much stronger in DS. This indicates that mitochondria from DS patients had a higher sensitivity to damaging agents, in agreement with what found by other authors. Indeed, studies on astrocytes from DS subjects demonstrated a lower DWm and a decrease in mitochondrial redox activity compared to control cells [28]. This observation, along with others reporting an impairment in mitochondrial redox activity, could explain the lower DWm displayed by PBMC from DS subjects after incubation with apoptogenic substances. We found that PBMC from DS and healthy controls have a similar tendency to undergo apoptosis, even if DS mitochondria were more sensitive to several damaging agents. Thus, since DS cells with altered mitochondrial function do not undergo apoptosis, it might be hypothesized that DS patients tend to maintain damaged cells, or that they have a higher capacity to repair functional damages. It remains to be established if and how this phenomenon can be linked to the development of autoimmunity or neoplastic disorders. A decrease in mtDNA content leads to an impaired production of enzymes of the respiratory chain, with a consequent reduction in the production of ATP, and a subsequent alteration in cellular metabolism. Therefore, to investigate the possibility that an increased sensitivity of mitochondria to apoptogenic agents was due to alterations in the amount of mtDNA, we have quantified the number of copies of mtDNA per cell in PBMC from DS and controls. This represent a new information since, to our knowledge, no data are currently present in literature about analysis of mtDNA copy number in DS patients. Changes in mtDNA content from DS cells could be expected also because such alterations have been reported during the aging process and DS is characterized by precocious aging. We have found that all donors had the same amount of mtDNA, but it has to be underlined that the donors we could analyze were very young, and thus it might be that alterations in the synthesis and changes in the amount of mtDNA could be detectable only in older patients, after several years of exposure to ROS, causing mtDNA alterations. Moreover, most PBMC have a relatively short half life, as they are constantly renewed in the body; this could be a limiting factor in revealing differences in mtDNA synthesis. Thus, it would be extremely interesting to measure mtDNA in cells with a longer life, that are chronically exposed to mitochondrial damages. Acknowledgement: We kindly acknowledge all the children and their families who made this study possible. This paper has been supported by MURST-COFIN 2004 and by FIRB 2001 grants to A.C. We acknowledge Partec GmbH (Mu¨nster, Germany) and Space srl (Milan, Italy) for precious technical support, and GeneMoRe Italy srl (Modena, Italy) for quantitative analysis of mtDNA content.
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