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-Synuclein induces apoptosis by altered expression in human peripheral lymphocyte in Parkinson’s disease

SEONGHAN KIM^*,1, BEOM S. JEON^,1, CHAEJEONG HEO^*, PIL SEON IM^*, TAE-BEOM AHN^,, JI-HEUI SEO^*, HYE-SUN KIM^*, CHEOL HYOUNG PARK^*, SE HOON CHOI^*, SEO-HYUN CHO^*, WANG JAE LEE and YOO-HUN SUH^*,2

^* Department of Pharmacology, College of Medicine, National Creative Research Initiative Center for Alzheimer’s Dementia and Neuroscience Research Institute, MRC, Seoul National University;
Department of Neurology, College of Medicine, Clinical Research Institute and MRC, Seoul National University Hospital;
Department of Neurology, Kyung Hee University Hospital, Seoul; and
Department of Anatomy, College of medicine, Seoul National University, Seoul, Republic of Korea

² Correspondence: Department of Pharmacology, College of Medicine, National Creative Research Initiative Center for Alzheimer’s Dementia and Neuroscience Research Institute, MRC, Seoul National University, Seoul 110-799, Republic of Korea. E-mail: yhsuh{at}plaza.snu.ac.kr

SPECIFIC AIMS

This work was performed to investigate whether peripheral blood mononuclear cells (PBMCs) show altered -synuclein (-SN) expression in Parkinson’s disease (PD) patients and to assess its functions, particularly with respect to peripheral immune abnormalities in PD patients. The pathological state in the central nervous system (CNS) may reflect the immune system with -SN acting as a mediator. We used an in vitro transfection approach to determine how -SN mediates lymphocyte apoptosis. We aimed to assess functions of -SN in the peripheral immune system of PD patients and their relation to its functions with the CNS.

PRINCIPAL FINDINGS

1. Altered expression of -SN in PBMCs of PD patients
To determine whether -SN plays a role in the peripheral immune system, we examined differential gene expression of -SN in PBMC obtained from idiopathic Parkinson’s disease (IPD) patients, multiple system atrophy (MSA) patients, and healthy controls and found that PBMCs of those with neurodegenerative disorders showed enhanced -SN gene expression vs. healthy controls (Fig. 1 A). The IPD group showed a 43.5% (P<0.001) increase in -SN gene expression in PBMCs vs. healthy controls; MSA patients, displayed a smaller increase, 23.5% (P<0.05). These increased levels of -SN mRNA were reflected by protein expression determined by Western blot (Fig. 1B ). As to mRNA levels, IPD groups expressed 38.2% (P<0.001) more -SN than the control group. However, the MSA group showed no significant difference from controls. These alterations in -SN levels showed no gender-related differences. The majority of IPD patients had been treated with levodopa(L-dopa) before this study Thus, we evaluated the effect of L-dopa on -SN gene expression. However, no significant change was detected between L-dopa-treated and untreated groups (Fig. 1C ). We conclude that L-dopa medication neither affects -SN expression nor reverses the effects of -SN expression.

View larger version (29K): [in this window] [in a new window]   Figure 1. A) PBMC from healthy controls (n=101), MSA patients (n=34), and IPD patients (n=151) were analyzed by semiquantitative RT-PCR for -SN expression. Data represent mean ±SE. *P < 0.05 and **P < 0.001 compared with control, respectively. B) Western blot for -SN expression in PBMC of controls (n=31), MSA (n=10), and IPD (n=43). Data represent mean ±SE. *P < 0.001 and **P < 0.05. C) Effect of L-dopa treatment. -SN expression levels were reevaluated. Untreated (n=24) and L-dopa-treated group (n=127) were compared with control (n=101). *P < 0.001. D) Age dependency of -SN expression. Age groups consisted of 30 s (CTL, n=20 and IPD, n=21), 40 s (CTL, n=23; IPD, n=28), 50 s (CTL, n=32; IPD, n=29), and 60 s (CTL, n=26; IPD, n=46). *P < 0.001. E) Each lymphocyte subset was analyzed for its -SN expression level. *P < 0.001 and **P < 0.01 compared with control, respectively. The n value for each sample was 8 (except T cell of IPD, n=13).

We investigated the relationship between -SN expression and age. From age 30 to the 60s, IPD samples displayed similar levels of increased -SN expression. Differences between IPD and controls were retained for each age group, but the gap was reduced with increasing age (Fig. 1D ).

To confirm involvement of each lymphocyte subset on PBMC -SN expression, isolated B cells, T cells, monocytes, and NK cells were analyzed. All lymphocyte subsets of IPD showed higher -SN than controls except for monocytes, whose -SN expressions was similar to those of controls (Fig. 1E ). As the most abundant lymphocyte population in PBMCs is T cells (70%), it can be speculated that increased -SN in PBMC of IPD is predominantly due to T lymphocytes.

2. -Synuclein induced apoptosis
We sought to determine whether increased -SN expression in PD patients was related to immune regulation, especially glucocorticoid-sensitive apoptosis. To investigate enhanced proapoptotic activity due to glucocorticoid (GC), we performed XTT assays after adding 1 µM of dexamethasone (DEX, synthetic glucocorticoid) to PBMCs for 16 h. The IPD group showed significantly more cell death than healthy controls; this apoptotic cell death displayed age dependency, as did -SN expression in PBMCs (Fig. 1D ). All IPD age groups showed DEX-sensitive apoptosis with no significant difference among them. However, controls showed increased DEX-susceptible apoptotic cell death in ages up to the 50s. Analysis of DEX sensitivities of lymphocyte subsets showed the same patterns as -SN expression levels. Combining our data with previous data (Fig. 1D, E ), it can be speculated that DEX-susceptible apoptosis may be related to -SN contents. Pearson correlation analysis showed a close correlation between -SN levels and DEX-induced cell deaths in IPD patients (r=0.76, P<0.001 by regression analysis). These data suggest that -SN expression levels might contribute to dexamethasone sensitivity of cells showing proapoptotic activity.

To confirm -SN expression-related apoptosis, the multiple myeloma cell line IM-9 was transfected with three forms of -SN: wild-type (WT), Ala30Pro (A30P), or Ala53Thr (A53T). Upon -SN transfection, apoptosis was induced by -SN overexpression alone (even without DEX treatment). The two mutant forms of -SN induced a much higher incidence of cell death (A30P, 52.1±2.3%; A53T, 49.1±2.7%; P<0.001) than the vector control transfectant. The WT induced cell death vs. the control (25.7±1.8%, P<0.01); cell death was confirmed to be apoptotic by flow cytometric analysis of annexin V-FITC staining. We performed the same experiment with an -SN-transfected human acute monocytic leukemia cell line, THP-1, and identified a similar pattern of apoptosis induction.

3. Caspase activation by -SN overexpression
Two main caspase activation pathways, receptor-mediated sequential activation of caspase-8 and cytochrome c-dependent nonreceptor mediated caspase-9 activation, are known to be involved in DEX-induced apoptosis. To determine which type of caspase pathway functions in -SN induced apoptosis, we analyzed caspase cleavages of -SN transfectants with or without DEX treatment (Fig. 2 ). After 5 h of 1 µM of DEX, activated caspase-8 and -9 were detected by immunoblotting. These results suggest that -SN overexpression mediates both caspase activation pathways, but the exact order of caspase activation is a subject of debate. DEX treatment showed a synergistic effect on -SN-induced apoptosis by enhancing caspase cleavage >2-fold vs. nontreated -SN transfectants. DEX treatment accelerated caspase activation as determined by detection of caspase cleavage after 2 h of DEX treatment.

View larger version (27K): [in this window] [in a new window]   Figure 2. -SN transfected IM-9 cells were treated with/without DEX. Cell lysates were analyzed by Western blot to identify caspase cleavages of caspase-8 and -9. ß-Tubulin was tested as an internal standard. Data reveal representative results for 8 independent experiments showing similar results.

4. -SN induced glucocorticoid receptor and CD95 (Fas) expression
Compared with the vector control transfectant, WT and mutant forms of -SN-transfected IM-9 contained more GR protein (WT, 4.2-fold; A30P, 5.9-fold; A53T, 6.4-fold).

-SN-induced caspase-8 activation suggests a possible involvement of CD95 (Fas) and CD95L (FasL), which act together in receptor-mediated apoptosis. Overexpression of WT, A30P, or A53T -SNs up-regulated CD95 gene expression 1.6-, 2.2-, and 2.0-fold, respectively, vs. the vector-only control. No induction of CD95L gene expression was detected in any -SN transfectants. Enhanced CD95 expression, as membranous surface protein, was confirmed by flow cytometric analysis. Overexpression of -SN up-regulated membranous surface CD95 21.25% (WT), 22.34% (A30P), and 24.66% (A53T) vs. the vector control; DEX treatment also up-regulated CD95 much more than in untreated groups, by 24.34%, 25.81%, and 27.45%, respectively. These data suggest that -SN up-regulated CD95 expression, which enhanced caspase-8 activation.

5. Increased reactive oxygen species (ROS) production by -SN
ROS involvement in -SN-induced lymphocyte apoptosis was measured using 2',7'-dichlorofluorescein diacetate. Each -SN transfectants induced ROS production, more evident in mutant forms than in WT. WT -SN produced 24.5% more than control, and the two mutants, A30P and A53T, produced 52% and 37.2% more, respectively.

CONCLUSIONS AND SIGNIFICANCE

To find a link between the CNS and the peripheral immune system in Parkinson’s disease, we investigated the role of -SN in the peripheral immune system of PD patients. -SN was significantly up-regulated in IPD patients at mRNA and protein levels vs. non-IPD controls. These alterations in -SN level were neither gender related nor affected by L-dopa medication in the peripheral system. Different IPD age groups maintained increased -SN levels similar to age-matched healthy controls. Nothing was known about the interrelation between the CNS and the peripheral immune system with respect to -SN expression. -SN expression patterns observed in this study provide evidence that there is a correlation between onset of PD and peripheral expression of -SN. Thus, the pathological state of the CNS may be reflected in the immune system by a mediator such as -SN. However, Hoehn and Yahr stage analysis showed no distinct correlation between PD stage and -SN expression. Thus, altered -SN expression may be due mainly to disease onset and aging. We thought it likely that -SN might be related via an aspect of apoptosis induction. We propose explanations of some of the immune abnormalities found in PD patients and speculate that there may be a close link between the CNS and the peripheral immune system. Differences in levels of -SN in patients and controls imply the possibility of its application as a diagnostic biomarker for early-onset Parkinson’s disease (i.e., before the age of 60).

We found that mutant forms of -SN (A30P and A53T) were more effective than WT at inducing apoptosis even without DEX treatment and that the overexpression enhanced DEX susceptibility. GR expression levels in cytoplasm were found to be closely correlated with the extent of GR-mediated DEX responses. In our study, -SN overexpression induced cytoplasmic expression of GR, which might be attributed to DEX-sensitive apoptosis. Many studies have reported that caspase activation plays crucial roles in DEX-induced apoptosis. Compared with DEX-induced caspase activation, -SN overexpression could activate caspase-8 and -9. Caspase-8 is activated by receptor triggering, then may be mediated by FADD/caspase complex formation. CD95-CD95L is one of the death receptor ligand systems crucial in apoptosis. In this study, overexpressing -SN up-regulated membranous CD95 (Fas), more highly enhanced in mutant forms than in WT. Up-regulation of CD95 was additively affected by DEX treatment. ROS was found to play a putative role in the cell death mechanism in PD in this order: A30P>A53T>wild-type -SN.

A recent report detailed the identification of -SN in the plasma of PD patients presumed to have been released from the CNS system. Based on our results, it can be suggested that -SN may be released from the peripheral system and that lymphocytes are the major sources of -SN. Moreover, a haploinsufficiency of -SN mutations in PBMCs was reported to have contributed to disease progression in familial PD. Such advances in our understanding of the functions of -SN in the peripheral immune system may provide the basis for further studies aimed at analyzing the link between the immune system and neurodegenerative disorders such as Parkinson’s disease. With advances in knowledge, we can expect to develop more effective therapeutic strategies by combinatorial approaches based on the peripheral immune system and the CNS.

View larger version (15K): [in this window] [in a new window]   Figure 3. Schematic diagram showing possible roles of -SN in apoptosis of peripheral lymphocytes. -SN induces GR expression that interacts with more GC making more susceptible to cell death. Pathways caspase-8 and -9 can be activated. CD95 (Fas) is up-regulated by -SN, then directed to activate caspase-8. ROS production by -SN is involved in this apoptosis mechanism.

FOOTNOTES

¹ These authors contributed equally to this work.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1917fje;

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