Functional characterization of iron-sulfur clusters in brain iron homeostasis and its impact on Parkinson's Disease
SNCA (alpha-synuclein) is a protein whose function in the healthy brain is currently unknown. It is of great interest to Parkinson's researchers because it is a major constituent of Lewy bodies, protein clumps that are the pathological hallmark of Parkinson’s disease. To help clarify its function, I make use of Arabidopsis thaliana as a novel platform to confine this human PD (Parkinson's disease) related protein in a completely new environment to observe its behavior in order to obtain some new clue for analyzing its function. Some new results were obtained in the last stage of funding.
1. For Arabidopsis seeds which contain SNCA-ox (overexpression of SNCA), their germination are inhibited on media which contains high concentration of sucrose. This is some interesting discovery as no such observation was reported before. In the beginning, I thought that it is because of the osmotic stress resulting from high concentration of sucrose. However, same concentration of sorbitol in place of sucrose in the media did not produce similar phenomena. It looks like that high concentration of sucrose together with high amount of SNCA inhibit seeds germination. At the moment, I cannot explain the mechanism of inhibition. But this experiment will be repeated again to find out the threshold of sucrose concentration for germination inhibition. Considering the expression difference between different strains of transgenic plants, it might be difficult. I will also check whether the inhibited seeds can recover its germination once they are transferred to normal growth media.
2. In some transgenic Arabidopsis plants which produce high expression of SNCA-EYFP, massive aggregations of SNCA-EYFP are observed just like the aggregation formed in PD patients' brain and these plants are therefore named PD plants. As mentioned before, the seeds were collected from these PD plants, their germinated seedlings have been observed and similar aggregation can be found in seedlings as young as five days of germination. I further turned back to check the seeds and little aggregation can be detected in the cotyledon cells. Aggregation in general are FIRST observed in old tissues especially in old leaves and it is rarely found in young tissue. I reasoned that the very few aggregations formed in some old tissues would act as an induction factor (or seeds) that caused universal aggregation in whole plants including seeds. To confirm this speculation, I tried to extract some SNCA-EYFP aggregations from plants and to inject them into plants which also express high concentration of SNCA-EYFP but do not have aggregation, expecting to observe induced aggregation. Because of limitation of technology of injection, I have not observed this kind of induction yet. I will try to find an alternative method to import aggregated SNCA-EYFP in future.
3. I have extracted some SNCA-EYFP aggregations. I am considering to find a collaboration to use these pathological protein in animal models to test whether this aggregated protein can cause PD. If it is so, the biological security of these transgenic plants should be taken into account.
Manuscripts for two publications are in preparation.
Scientists have known for many years that alpha-synuclein and the aggregations it forms, Lewy bodies are a pathological hallmark of PD. The question is aroused: is alpha-synuclein accumulation a result of neurodegeneration in PD, or is it the cause of PD? If alpha-synuclein accumulation causes or enhances PD progression, how does it play a role in PD?
Using Arabidopsis as a naive model which does not contain endogenous SNCA, I have collected several interesting results which are helpful for answering these questions. High expression of SNCA do have harm effect on plant growth and germination. High concentration of sucrose seems to promote the negative effect of SNCA on germination inhibition. The mechanism is unclear but it is not because of the osmotic stress caused by high concentration as same concentration of sorbitol does not have the similar effect. To find out the mechanism that SNCA plus sucrose inhibits germination which help understand the role of SNCA in this process.
Can SNCA aggregation function as pathogen? From my experiment, the answer should be yes. I will improve the inject technology and try to clarify that aggregations of SNCA from one plants will work as in another plant to cause new aggregation.
PD plants provides novel insight into the biology and pathology of a-synclein
Using Arabidopsis as a novel platform, I expressed SNCA and its mutated forms in plant cell, in order to observe the performance of high concentration of this protein and mutated forms. Experiments did not only make PD plants but provide much exciting data, highlighting Arabidopsis as an invaluable tool for human disease research
A. Parkinson’s disease is the second most popular neurodegenerative disease after Alzheimer’s disease affecting millions of people in Europe. The hallmark of Parkinson’s disease is the aggregation of alpha-synuclein (in the form of Lewy bodies) in brain neurons especially dopaminergic neurons. I expressed alpha-synuclein (SNCA) and its three mutated forms: A30P, A53T, and E46K in Arabidopiss in high concentration to observe their performance in different micro-environments from neurons in order to provide novel insight into the function of this puzzling protein.
1. I obtained PD plants (Lewy bodies produced in the cell) from all the four different transgenic types of plants: plants with constitutive expression of wild type SNCA; plants with A30P mutated SNCA; plants with E46K mutated SNCA; and plants with A53T mutated SNCA.
2. Lewy bodies production is age-dependent. No aggregation was observed until the nearly half of the seeds have been set most probably due to the downfall of mitochondrial function.
3. To confirm this hypothesis, I applied hypoxia treatment to the young leaves with high expression of SNCA or its mutated forms but there is no aggregation at all. Certain time of this kind of treatment definitely caused the formation of Lewy bodies of SNCA indicating a very important effect of oxygen shortage on this type of pathology.
4. Lewy bodies were first shown in old leaves but later in young leaves they were also found, of which the possible reason was movement of aggregation from cell to cell, or from organ to organ.
5. The next generation of PD plants showed dramatic early onset of SNCA aggregation even at the very young seedlings, which explains, to some extent, the reason why familial human PD cases could be found at young age.
5. Lewy bodies, in the PD plants, are not solid, with many channels through them, which cannot be detected by immune-staining.
6. To observe the effect of high oxidative micro-environment on SNCA performance, I expressed SNCA targeted to the chloroplast in Arabidopsis. Sequencing result confirmed the accuracy of the plasmid used to transform Arabidopsis and transient expression revealed that SNCA was expressed and imported into the chloroplasts. However, in stably transformed plants, even if abundant snca RNA was detected, no SNCA accumulated in the chloroplasts at all, indicating an unknown degradation system working to remove SNCA from the chloroplasts. This result provided a potentially valuable degradation for removing surplus SNCA from human neurons.
7. SNCA targeted to the mitochondria by signal peptide also gave a surprise. Most of this protein was cleaved and western blot showed to strong bands. It is not clear whether this cleaved form of SNCA maintained the normal function. SNCA in the mitochondria also formed aggregation during the late stage of growth.
8. In most of the growth time, overexpression of SNCA or its mutated forms did not cause any special phenotypes. However, after flowering, a relatively high ratio of siliques did not set seeds indicating a lot of flowers are infertile. Microscope check revealed the failed development of most pollen. It is still under way to find out the causes behind this kind of failure.
B. Iron-sulfur cluster
Two kinds of iron-sulfur cluster biogenesis systems co-exist in Arabidopsis. I took advantage of yeast and plant systems to check the redundancy of these two systems. Results have been obtained, which are able to explain how to use one system to replace the other.
Iron-sulfur clusters in brain iron homeostasis and Parkinson's Disease
Functional characterization of iron-sulfur clusters in brain iron homeostasis and its impact on Parkinson's Disease: 1.Iron-sulfur cluster biogenesis, 2.Iron homeostasis, and 3.Iron and SNCA aggregation.
1.Generate all the necessary vectors for the whole project.
To check the possible interaction of HSCB, HSCA9 (homologue of HscA) with IRP1 or FBXL5, I have subcloned these gene cDNAs in BiFC (Bimolecular fluorescence complementation) vectors. HSCB gene presented some challenge for subcloning as its sequence contains both restriction sites that are needed for subcloning. So I modified its gene sequence accordingly to eliminate the restriction sites but maintained the same amino acid sequence.
Ferritin protein is the main iron storage protein in cells and its status plays a key role in controlling iron homeostasis. Iron-sulfur cluster biogenesis as a main consumer of iron should have a tight relationship with this protein. To check which element(s) of iron-sulfur cluster biogenesis system is interacting with ferritin to obtain iron from it, I have cloned all four ferritin genes from Arabidopsis and subcloned them in BiFC vectors. The corresponding BiFC plasmids for all the genes of iron-sulfur cluster biogenesis systems in Arabidopsis have also been made (more than 20 plasmids).
My primary experiment revealed that human Parkinson’s disease gene DJ-1 (Park7) might interact with cytosolic iron-sulfur cluster biogenesis systems. To further expand this discovery in Arabidopsis, I plan to check whether all three DJ-1 homologues of Arabidopsis can interact with iron-sulfur cluster biogenesis systems in the chloroplast and the cytosol. These three genes, AtDJ-1a, AtDJ-1b and AtDJ-1c, are also subcloned in BiFC vectors.
SNCA and its aggregation under iron stress condition is also a topic of my proposal. I have made three mutated forms of SNCA (A30P, A53T, E46K) and subcloned these three mutated forms and wild type SNCA gene in plasmids separately to overexpress them in plant cell or plasmids for BiFC experiment in plant cell or mammalian cell. Have also subcloned in BiFC vectors VMAT2 gene, which is supposed to be synaptic vesicle protein correlated with dopamine transportation.
Part of above genes such as HSCB, IRP1, FBXL5, SNCA, VMAT and DJ-1 have also been cloned in plasmids for YTH (yeast two hybrid) experiment, an alternative method to detect or confirm protein interactions.
2. Generate and maintain transgenic mammalian cells and zebrafish.
A well performed mammalian cell transfection and maintaining system has been established. As it is easier to transfect mammalian cells than to maintain transfected cells, the routine work is to subculture mammalian cell to maintain their active growth. Transfection can be readily done once the experiment is required. The plasmids that are used to transfect cells are prepared using kit to guarantee high quality and stored in freezer.
As Mr Mohammed Gebriel, who took charge of zebrafish maintenance system, has left the lab and the zebrafish raising was shut down. However, zebrafish can be obtained from a nearby market all year around. Furthermore, Dr Maria Doitsidou (who has just won Helse vest funding) brought a new model animal, C.elegans from Columbia University of USA. The collaboration with Dr Doitsidou has been fixed. Worm raising and subculture are going on well. I am subcloning several human genes in plasmids that specifically express these genes in dopaminergic neurons of worms to check their possible interactions in neuron cells.
3.Maintain transgenic Arabidopsis plants and observe SNCA aggregation in plant cell.
Work has been done to amplify the transgenic plant seeds and to screen for homozygous transgenic plants. These plants include: AtHscB-ox (overexpressing Arabidopsis HscB, knockout Arabidopsis HscB, AtHscA-ox (Arabidopsis HscA), AtIscU1-ox (Arabidopsis IscU1), AtDJ-1a-ox (Arabidopsis DJ-1 like gene a), AtDJ-1b (Arabidopsis DJ-1 like gene b), AtDJ-1c (Arabidopsis DJ-1 like gene c), Arabidospsis overepxressing human SNCA and Arabidopsis overexpressing SNCA three mutant forms (A30P, A53T or E46K) separately. Through the antibiotic resistance screening, homozygous plant seeds have been collected for further experiment use.
Aggregation of SNCA as a hallmark of Parkinson’s disease has been a focus of research but its mechanism is still unclear. Arabidopsis as a novel model might be able to provide novel information on this topci. Using the stable transgenic plant seedlings and transient expression leaves, I have done primary observation of SNCA in plant cell. I focused on the aggregation behavior of SNCA in plant cell under normal or different stress conditions. Interesting results have obtained which are shown below.
Figure 1. SNCA tagged with EYFP expressed in leaf cell, no aggregation.
Figure 2. SNCA aggregated under 10 uM FeCl2 treatment in leaf cell.
Figure 3. SNCA aggregated under 10uM CuCl2 in leaf cell.
Figure 4. SNCA aggregated under 10 uM H2O2 in leaf cell
Figure 5. SNCA aggregated under dehydration in root cell.
Figure 6. BiFC (Bimolecular fluorescence complementation) confirmed the interaction between human SNCA and VMAT2 in plant cell. SNCA is tagged with the N-terminal half of EYFP while VMAT2 is tagged with the C-terminal half of EYFP. Both of them are introduced into plant cell. The recovered green fluorescence indicates that both proteins interact with each. Otherwise, there will be no green fluorescence.
4. Analysis of iron-sulfur cluster biogenesis and determination of the localization of HSCB-HSPA9 in human and worm brain tissue.
Application for human brain tissue is ongoing and will take some time to obtain the material. The antibodies of HSCB and HSPA9 have been ordered recently.
Iron-sulfur cluster itself and its biogenesis process are sensitive to oxygen. However, chloroplast, a very oxidative organelle, accommodates a special iron-sulfur cluster biogenesis system (SUF system), which seems to be able to protect iron-sulfur cluster biogenesis from ultra-oxidative condition and safely transfer the cluster to apo-protein. On the contrary, the mitochondrial iron-sulfur cluster biogenesis system (ISC) lacks this ability. To test whether this SUF system can be introduced to mitochondria to replace ISC system, I did complementation experiments to check whether part elements of SUF can replace the counterparts of ISC. A conditionally lethal mutant yeast mutant was taken into account, of which two iron-sulfur scaffold proteins Isu1 and Isu2 are knocked out except that Isu2 is conditionally deleted upon adding certain chemical. It was used to screen for SUF elements that can take the part of two Isu genes under knockout condition for both Isu genes. Yeast does not have chloroplasts. The six different elements of SUF system (AtSufA, B, C, D, S or E) from Arabidopsis were introduced in this mutant singly or in different combination. The result showed that once the two genes, AtSufB and AtSufC, were transformed together into this mutant, it was readily rescued indicating that AtSufB-C can replace Isu1-Isu2 even though they do not show obvious similarity (Figure 7). AtSufB or AtSufC only, or combination with other elements, or other element combinations do not work. To check whether AtSufB-AtSufC can promote the rescued yeast mutant to perform better under stress conditions, I am testing the effect of different stress conditions especially oxidative stress on yeast growth and development.
Figure 7. Left: Yeast grown on galatose containing media; Middle: Yeast grown on glucose containing media; Right: Diagram of yeast scratched on media. 1.Wild type yeast strain W303A. 2.?Isu1-Isu2/Gal-Isu2 (Isu1 and Isu2 double knockout mutant with a copy of Isu2 controlled by Galatose induced promoter). 3. Double mutant with pGAD-AtSufB and pGBK-AtSufC. 4.Double mutant with pGAD-AtSufC and pGBK-AtSufB. 5.Double mutant with pGAD-AtSufD and pGBK-AtSufB. 6.Double mutant with pGAD-AtSufB and pGBK-AtSufD. 7. Double mutant with pGAD-AtSufD and pGBK-AtSufC. 8. Double mutant with pGAD-AtSufC and pGBK-AtSufD.
Further experiments have revealed that AtHscB of Arabidopsis can also be replaced by AtSufB, AtSufC or AtSufD separately (Figure 8). An AtSufD complemented HscB mutant grows better under oxidative conditions than the wild type (data now shown here). So my primary experiments suggested that the chloroplastidic SUF system, an oxygen-resistant machinery, can replace the mitochondrial ISC system.
Figure 8. A yeast Jac1 (HscB homologue) is lethal so it is maintained by special plasmid expressing a Jac1 gene. FOA can delete this plasmid so that Jac1 expression is removed and yeast dies. AtSufB, C or D are separately cloned in other kind of plasmid which cannot be deleted by FOA. As they can complement Jac1 so the yeast mutant transformed with these genes survives.
Parkinson disease protein DJ-1 binds metals and protects against metal-induced cytotoxicity.
J Biol Chem 2013 Aug 2;288(31):22809-20. Epub 2013 jun 21
The value of Arabidopsis research in understanding human disease states.
Curr Opin Biotechnol 2011 Apr;22(2):300-7. Epub 2010 des 6
Iron-sulfur clusters: biogenesis, molecular mechanisms, and their functional significance.
Antioxid Redox Signal 2011 Jul;15(1):271-307. Epub 2011 feb 3