J of Proteomics | April 20, 2007 | vol. 103 | no.10 Development and implementation of a novel interactome platform in studies on neurodegeneration. Ewelina Maliszewska-Cyna, Kelly Markham, Gerold Schmitt-Ulms Centre for Research in Neurodegenerative Diseases (CRND), Proteomics Unit Tanz Neuroscience Building University of Toronto _________________________________________________________ Background Neurodegenerative disorders Neurodegenerative diseases are among the major scourges of modern society. To a certain degree, these diseases represent deficits frequently associated with the experience of senescence, such as forgetfulness, loss of dexterity and deterioration in strength and locomotor functions (1). These disorders have been termed a “silent epidemic”, because they have been inadequately recognized by the public and, more importantly, because of their late onset leading to predominant involvement of the elderly, who have low public visibility because of their social isolation. The most common disorders in this category are Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis (2). In addition, there exists a large collection of less common neurodegenerative disorders. Together, they represent an enormous burden on both patients and their families. These diseases also constitute an increasing economic burden on society. In spite of the increasing prevalence of neurodegeneration, their nature remains an enigma. Where is the line to be drawn between a disease and the normal progression of senescence associated with the natural degradation of neurons? What is the mechanism by which functionally related neurons die surrounded by other regions that are spared? Is there more than one cause for each of the relatively clear defined syndromes? Is there a shared pathogenesis or a clinical overlap among the neurodegenerative disorders? These are only some of the many issues that face researchers today. Substantial advances have taken place in the field of neuroscience that have had direct relevance to neurodegeneration. Molecular biology, immunohistochemistry, clinical physiology, and pharmacology have all provided an insight into our understanding of these degenerative processes (3). In some areas, these developments generated practical benefits leading to the establishment of treatment methods, whereas in other instances the knowledge has remained frustratingly theoretical. Amyloid hypothesis of Alzheimer’s disease One of best known neurodegenerative disorders is Alzheimer’s disease (AD) and the proposed amyloid hypothesis has become the main focus of AD research (4). Amyloid β- peptide (Aβ) is recognized as the primary component of the neuritic plaques of AD patient’s brain tissue (Fig.1). Consequently, identification of mutations in the gene coding for the Aβ precursor protein (APP) illustrated that APP mutations contribute to the formation of Aβ deposits, also called the amyloid fibrils. It is well known that most of the mutations occur within or in close proximity to the APP gene, that is normally cleaved by proteases called the α-, β- and γ-secretases (5). In the absence of mutations in the APP gene, the function of α-secretase is being favored, whereas in the mutated APP gene it is the β- and γ-secretases that are predominantly active. Furthermore, APP mutations internal to the Aβ sequence elevate the self-aggregation of Aβ into amyloid fibrils – the hallmark of Alzheimer’s disease (2). In addition to the mutations in the APP gene, there are other genes associated with AD, the presenilin family being the most
Transcript
J of Proteomics | April 20, 2007 | vol. 103 | no.10
Development and implementation of a novel interactome platform in studies on neurodegeneration.
Ewelina Maliszewska-Cyna, Kelly Markham, Gerold Schmitt-Ulms
Centre for Research in Neurodegenerative Diseases (CRND), Proteomics Unit Tanz Neuroscience Building University of Toronto
_________________________________________________________ Background Neurodegenerative disorders Neurodegenerative diseases are among the major scourges of modern society. To a certain degree, these diseases represent deficits frequently associated with the experience of senescence, such as forgetfulness, loss of dexterity and deterioration in strength and locomotor functions (1). These disorders have been termed a “silent epidemic”, because they have been inadequately recognized by the public and, more importantly, because of their late onset leading to predominant involvement of the elderly, who have low public visibility because of their social isolation. The most common disorders in this category are Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis (2). In addition, there exists a large collection of less common neurodegenerative disorders. Together, they represent an enormous burden on both patients and their families. These diseases also constitute an increasing economic burden on society.
In spite of the increasing prevalence of neurodegeneration, their nature remains an enigma. Where is the line to be drawn between a disease and the normal progression of senescence associated with the natural degradation of neurons? What is the mechanism by which functionally related neurons die surrounded by other regions that are spared? Is there more than one cause for each of the relatively clear defined syndromes? Is there a shared pathogenesis or a clinical overlap among the neurodegenerative disorders?
These are only some of the many issues that face researchers today. Substantial
advances have taken place in the field of neuroscience that have had direct relevance to neurodegeneration. Molecular biology, immunohistochemistry, clinical physiology, and pharmacology have all provided an insight into our understanding of these degenerative processes (3). In some areas, these developments generated practical benefits leading to the establishment of treatment methods, whereas in other instances the knowledge has remained frustratingly theoretical. Amyloid hypothesis of Alzheimer’s disease One of best known neurodegenerative disorders is Alzheimer’s disease (AD) and the proposed amyloid hypothesis has become the main focus of AD research (4). Amyloid β-peptide (Aβ) is recognized as the primary component of the neuritic plaques of AD patient’s brain tissue (Fig.1). Consequently, identification of mutations in the gene coding for the Aβ precursor protein (APP) illustrated that APP mutations contribute to the formation of Aβ deposits, also called the amyloid fibrils. It is well known that most of the mutations occur within or in close proximity to the APP gene, that is normally cleaved by proteases called the α-, β- and γ-secretases (5). In the absence of mutations in the APP gene, the function of α-secretase is being favored, whereas in the mutated APP gene it is the β- and γ-secretases that are predominantly active. Furthermore, APP mutations internal to the Aβ sequence elevate the self-aggregation of Aβ into amyloid fibrils – the hallmark of Alzheimer’s disease (2).
In addition to the mutations in the APP gene, there are other genes associated with AD, the presenilin family being the most
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prominent one. Mutations in the PSs interfere with the cleavage of APP causing the overproduction of the most destructive amyloidogenic peptide, the Aβ-42 (5). Presenilin 1 (PS1) and presenilin 2 (PS2) are incorporated into protein complexes destined for maturation within the ER and Golgi apparatus. The maturation involves endoproteolysis of PSs into N- and C-terminal fragments, and the recruitment of additional components into the complex, all of these processes being essential for normal activity of secretases. Consequently, mutation within PS1
or PS2 causes the abnormal maturation and results in the disruption of the protein complex function.
The formation of tau protein neurofibrillary tangles constitutes another hallmark of AD pathology. Studies have shown that tau tangles are being deposited only after changes in Aβ metabolism and initial plaque formation has occurred. Therefore, cerebral Aβ accumulation is the primary influence in AD patients and the rest of the disease process, including tau protein
neurofibrillary tangles formation - an imbalance between Aβ production and Aβ clearance.
Knowledge of molecular and biochemical processes behind Alzheimer’s disease allows our research team to pursue interactome studies. Our objective is to carefully analyze AD bait interactomes, which involves co-immunoprecipitation (Co-IP) studies. Co-IP has its disadvantages, mostly associated with the interference from antibody bands in gel analysis. In those cases, where several proteins may be co-precipitated with the target, presence of the co-eluted antibody heavy and light chains (25 and 50 kDa bands in reducing SDS-PAGE gel) in the preparation can obscure the results. The ideal situation would be to conduct the Co-IP without contamination of the eluted antigen with the antibody. With this potential interference eliminated, only the co-precipitated proteins will be present and detected on a gel. One such method has been reported by Burckstummer et al., who suggest tandem affinity purification (TAP), a generic two-step affinity purification protocol that enables the isolation of protein complexes under close-to-physiological conditions for subsequent analysis by mass spectrometry (6). It is because of the above reasons that protein tagging with the TAP system would become a method of choice in our laboratory for these types of experiments. Cell culture models of neurodegeneration. Neurospheres Cultures of purified cell populations obtained from animal live tissue are an invaluable model for the study of the nervous system. The goal of such a culture is to establish a cell population that retains its cytoarchitecture, maintains maturation and differentiation patterns and preserves its original function. Cell cultures provide the following advantages: 1.Control over the environment, such as nutrients, ions, temperature, gas phases and cofactors. 2. Isolation from modulators, such as hormonal, humoral and metabolic influences, normally present in the body. 3. Direct accessibility to the cells allowing for visual observation of morphology and cell dynamics. 4. Rapid preparation for morphological techniques, immunocyto- chemistry assays and biochemical analyses (7). In addition, protein tagging in an entire organism poses great difficulties, hence a cell
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culture is a preferred model to study manipulated cell systems.
All of these characteristics make neural cell culture one of the most efficient and versatile experimental models available for studying the possible role of various physical and chemical agents involved in neuronal loss associated with neurodegenerative disorders. Cancerous cells and neuroblastoma cells in particular, are used by many research centres to study neurodegeneration, as those cells have the capacity to proliferate indefinitely (8). Nevertheless, there are limitations to this model associated with little control over their cell cycle and proliferation rates. As a result, our lab explores other possible models.
One such model is the neuronal stem cells system (NSCs). This is the undifferentiated pool of cells of the nervous system with the ability to proliferate, self-renew and generate a large number of differentiated progeny cells (9). Data also indicate that stem cells are not only functioning during embryonic development, but also throughout our lives. Reynolds and Weiss (1992) were the first to isolate NSCs (10). In vitro, NSCs form aggregates, or neurospheres (NS), in the presence of epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF) (Fig. 2). Neurospheres are in fact a heterogeneous mixture of stem and progenitor cells, however, over time, there are only nestin-positive NSCs that retain the ability to form neurospheres and, more importantly, retain their multipotentiality over extended periods of time.
Although many researchers report that NSCs are indeed a useful tool, there are certain caveats associated with this model. One of them comes into play when one desires to deliver a gene of interest into neurospheres (9, 10). It appears that widely used transfection methods, with the use of such reagents as Lipofectamine 2000, are not applicable when handling NS. Because of the relatively slow growth rate of NS, a considerable amount of transfected signal disappears before cells achieve the desired titer. Moreover, because NSCs aggregate into NS and later on grow in suspension, LF2000 must be constantly present in the medium, which proves to be toxic and leads to cell death. Davidson et al. succeeded in adeno-viral mediated gene transfer into
nestin-positive NSCs while preserving their potential for self-renewal and proliferation (10) It is for this reason that adenoviral transfection is used instead of the standard LF2000 method when delivering the gene of interest into neurospheres (10, 11). In view of the above evidences, it seems plausible to create a stable pool of neuronal stem cells that would serve as a model to study neurodegeneration involved in Alzheimer’s disease. The objective of my project consisted of two parts. Firstly, the development and maintenance of a stable culture of neurospheres was to be achieved. Secondly, optimization of a transfection method using lentivirus was necessary in order to establish a stable line of transfected cells expressing a gene of interest. An application of this tool to study the expression of PS1 and PS2 proteins was also explored. Materials and Methods
Neurospheres culture in T75 flasks Procedure for tissue extraction was adapted from Weiss et al. with few modifications (8). Whole brains were removed and cleared of meninges from mouse embryos at embryonic day 14 (Charles River Laboratories, CD-1 strain) (Fig.3). Tissue wastriturated with fire polished pipette in 3 ml cold PBS and centrifuged at 1500 rpm for 10 min (Napco
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2028R). Supernatant was removed and cells were resuspended in 1 ml DMEM/F-12 medium supplemented with 6% glucose G-7021), 2mM L-glutamine (Sigma, G-3202), 3mM sodium bicarbonate (Sigma, S-5761), 5mM HEPES (Sigma, H-4034), 30 nM sodium selenite (Sigma, S-9133) and hormone mix containing 25ug/ml insulin (Sigma, I-5500), 100 ug/ml transferring (Sigma, T-2252), 20nM progesterone (Sigma, P-6149) and 60uM putrescine (Sigma, P-7505). 20 ng/ml of hEGF (PeproTech, 315-09), 100 units of penicillin and 100ug/ml of streptomycin (Gibco, 15140-122) was also added to the medium. Finally, cells were plated at a density of 0.75x105 cells/ml in T75 flasks containing 10ml of medium. Cells were cultured in the incubator at 37oC and 5% CO2 and passaged every 7 days. To test for the presence of NS, an aliquot of cells was collected during each passage and tested for the presence of nestin using monoclonal antibody and 12.5% SDS PAGE
gel. Primary and secondary antibodies and their respective dilutions are given in Table 1. Cell count and viability Cell density was determined before each passage using a hemocytometer and viability was determined using the standard trypan blue test (Sigma, cat # T8154). Cell counts were performed in duplicate as necessary. Spherical aggregates were gently dissociated mechanically and a sample was taken for cell count. Lentiviral transfection and transduction of HEK 293T cells One day before the experiment, cells were plated to achieve a cell density of 2x106/100mm dish. Calcium-phosphate precipitate, or CPP (CalphosPM Mammalian Transfection Kit, Clontech, 631312) was added to a plasmid mixture containing the envelope (pMd2G plasmid) and packaging (pPAX2
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plasmid) fractions of lentivirus as well as pWPI transfer insert containing the GFP sequence (Table 2). Plasmid mixture of 700ul was combined with 700ul of 2 x HEPES buffered saline (HBS) and incubated for 20 min. at room temp. The mixture was added dropwise to the cells for overnight incubation in 37oC and 5%CO2. After the media change, cells were incubated overnight. The media containing viral supernatant was collected the following day. A new series of HEK 293T cells was plated in two 12-well plates to achieve 50% confluency the following day. On the next day, a second sample of viral supernatant was collected, combined with the previous one in the quick seal tubes and centrifuged at 121000 x g for 2 hours at 4oC (Beckman SW32ti) to concentrate the viral sample. Viral pellet was then resuspended in 300ul of growth media. Fresh HEK 293T cells were transduced at different dilutions of viral supernatant (10ul – undiluted as well as 1:10 and 1:100 dilutions) and left for a 48 hour incubation period. Finally, the cells were examined under the ultraviolet microscope for determination of transfection and transduction efficiency. Results Neurospheres We isolated neural stem cells from an embryonic day 14 mouse brain and maintained them in EGF supplemented medium. Earlier work showed that cells propagated in EGF or bFGF displayed characteristics of stem cells, that is, they were self-renewable and multipotent (7, 8). Over a period of 7 days, single cells formed multicellular aggregates with a morphology consistent with that of neurospheres grown in suspension (Fig. 2). After optimizing the medium components, there was an almost 10-fold increase in cell density over the period of three weeks (Table 3). After the fourth week, the cell density and viability started to decrease rapidly. Cells from collected neurospheres were tested for the expression of the progenitor cell marker, nestin, (Fig. 4). Lentiviral transduction After successful transfection, HEK 293T cells were transduced at different dilutions of viral supernatant. The results are outlined in Figure 5. Transduction achieved the highest yield
when no dilution was made and cells were transduced with 10ul of viral suspension per each well (Fig. 5 B, D). Conversely, transducing cells with diluted viral material (1:100) resulted in a very low transduction rate. At the same time, the transduction rate remained unchanged when used cells were grown attached to the plates (Fig. 5A, B) or in suspension culture (Fig. 5C, D). Discussion The objective of this study was to design a robust and efficient tool for the purpose of interactome studies. One aspect of such studies is a requirement of a large quantity of protein-starting material, and it is for this reason that the neurosphere culture system requires a substantial expansion. Many researchers reported high yields of NS when these were cultured in spinner flasks, also called bioreactors (7, 12, 13). Because NS grow in suspension, upscaling the cell system into spinner flasks of size up to 1L does not pose any disadvantages. When placed on the Thermolyne Cell-Gro magnetic stirrer (speed
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at 100rpm), cells achieve high density while retaining their characteristics of stem cells, as reported by Sen et al. (12). Relatively slow growth rate and a requirement of sustained low cell density are other reasons why large volume of starting material is plausible in order to obtain enough material for interactome studies coupled with mass spectrometry analyses. Even though the period of robust NS culture achieved in our laboratory is shorter than that reported in the literature (3, 6, 9), it is sufficient for the transfection to be coupled along with the interactome studies. Nevertheless, if one would like to preserve this culture system for prolonged periods of time, one would have to consider adding a second
growth factor. It has been reported that although initially NS cells grow well in EGF-alone, at later stages, they change their requirements in such a way that supplementation with both EGF and bFGF is required (14, 15). Even though Lentiviral experiments have been conducted on the HEK 293T cell line, we infer that the same technique and conditions can be applied to a NS system as well. What is of concern to this system is to what degree the biology of neurospheres be influenced by the viral transduction and by the presence of a foreign genome in the cell system. Because of these reasons and in order to avoid overexpressing the protein of interest, fairly low levels of transduction is desirable.
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Moreover, the influence of the virus on the activity and functioning of examined protein remains unknown. More studies involving protein cleavage patterns that would further decide on choice of the protein tagging system are necessary. As this new interactome platform is being developed, one can implement this tool to study bait interactomes of Alzheimer’s disease. The protein of interest to our lab is the TAP-tagged PS1 and PS2. It has been reported by Burckstummer et al. that the application of TAP procedure resulted in a tenfold increase in protein-complex yield and improved the specificity of the procedure. Therefore, the TAP purification system coupled with the Lentiviral transduction tool has a large potential to obtain large amounts of the intact protein complexes as well as generate enough material for mass spectrometry analysis. Figure 6 outlines the details of the TAP-tagged experimental design. If successful, this system may prove to be of more use than currently used, yet not always reliable, antibodies.
Future perspectives It would be worthwhile to implement this novel interactome platform into research on TAP-PS1 and TAP-PS2 inserts. In addition, the research would take full advantage of many transgenic animal models that are currently available at CRND. Insights gained from interactome analyses of Tg systems might lead to novel therapeutic and diagnostic methods of Alzheimer’s disease or simply to a better understanding of processes governing neurodegeneration. Acknowledgements The authors thank Yu Bai for technical assistance with Lentiviral work as well as Rasanjala Weerasekera for valuable suggestions regarding molecular biological techniques. This study was supported by a grant from the Canadian Institutes of Health Research (CIHR), operating since Sept. 15, 2003. References ______________________________________
1. Gottlieb DI 2002 Large-scale sources of neural stem cells. Annu. Rev. Neurosci. 25:381-407
2. Selkoe DJ 2004 Cell biology of protein misfolding: the examples of Alzheimer’s and Parkinson’s diseases. Nature cell biol. 6(11):1054-1060
3. Morshead CM and van der Kooy D2001 A new ‘spin’ on neural stem cells? Curr. Opin. Neurobiol. 11(1):59-65
4. Hardy J and Selkoe DJ 2002 The amyloid hypothesis of Alzheimer’s disease. Science 297(5580):353-356
5. Rogaeva E The solved and unsolved mysteries of the genetics of early-onset Alzheimer’s disease. Neuromolec. Med. 2(1):33-42
6. Burckstummer et al. 2006 An efficient tandem affinity purification procedure for interaction proteomics in mammalian cells. Nat. mechanics 3(12):1013-1019
7. Kornblum HI et al. 2001 A genetic analysis of neural progenitor differentiation. Neuron 29(2):325-339
8. Vescovi AL et al. 1999 Extended serial passaging of mammalian neural stem cells in suspension bioreactors. Biotech & bioeng. 65(5):589-599
9. Kallos MS et al. 2004 Cell cycle kinetics of expanding populations of eural stemcells and progenitor cells in vitro. Biotech & Bioeng. 88(3):332-347.
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10. Reynolds BA and Weiss S 1992 Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255(5052):1707-1710
11. Davidson BL et al. 2001 Viral-mediated gene transfer to mouse primary neural progenitor cells. Mol.. Ther. 5(1):16-24
12. Giri RK et al. 2005 Prion infection of mouse neurospheres. PNAS 103(10):3875-3880
13. Sen A et al.. 2002 Passaging protocols for mammalian neural stem cells in suspension bioreactor. Biotechnol. Prog. 18(2):337-345
14. Sen A et al. 2004 New tissue dissociation protocol for scaled-up production of neural stem cells in suspension bioreactors. Tis. Eng. 10(5/6):904-913
15. Tropepe V et al. 1999 Distinct neural stem cells proliferate in response to EGF and FGF in the developing mouse telencephalon. Devel. Biol. 208:166-188
