1Department of Neuropathology, Medical School Düsseldorf, Germany, 2Center of Behavioral Neuroscience, University of Düsseldorf
Bader, V., Ottis, P., Pum, M., Huston, J. P., Korth, C. Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species. J. Vis. Exp. (66), e4132, doi:10.3791/4132 (2012).
Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases Alzheimer's disease (Aβ, tau), Parkinson's Disease (α-synuclein), Huntington's disease (polyglutamine, huntingtin), and others. Protein aggregation is thought to occur due to disturbed proteostasis, i.e. the imbalance between the arising and degradation of misfolded proteins. Of note, the same proteins are found aggregated in sporadic forms of these diseases that are mutant in rare variants of familial forms.
Schizophrenia is a chronic progressive brain condition that in many cases goes along with a permanent and irreversible cognitive deficit. In a candidate gene approach, we investigated whether Disrupted-in-schizophrenia 1 (DISC1), a gene cloned in a Scottish family with linkage to chronic mental disease1, 2, could be found as insoluble aggregates in the brain of sporadic cases of schizophrenia3. Using the SMRI CC, we identified in approximately 20 % of cases with CMD but not normal controls or patients with neurodegenerative diseases sarkosyl-insoluble DISC1 immunoreactivity after biochemical fractionation. Subsequent studies in vitro revealed that the aggregation propensity of DISC1 was influenced by disease-associated polymorphism S704C4, and that DISC1 aggresomes generated in vitro were cell-invasive5, similar to what had been shown for Aβ6, tau7-9, α-synuclein10, polyglutamine11, or SOD1 aggregates12. These findings prompted us to propose that at least a subset of cases with CMD, those with aggregated DISC1 might be protein conformational disorders.
Here we describe how we generate DISC1 aggresomes in mammalian cells, purify them on a sucrose gradient and use them for cell-invasiveness studies. Similarly, we describe how we generate an exclusively multimeric C-terminal DISC1 fragment, label and purify it for cell invasiveness studies. Using the recombinant multimers of DISC1 we achieve similar cell invasiveness as for a similarly labeled synthetic α-synuclein fragment. We also show that this fragment is taken up in vivo when stereotactically injected into the brain of recipient animals.
1. Preparation of Aggresome Donor Cells: Transfection of Monomeric Red Fluorescent Protein (mRFP) or Enhanced Green Fluorescent Protein (eGFP)-tagged Human Full Length (FL) DISC1
2. Aggresome Purification from Transfected Donor Cells
The protocol described here is a combination of a method to purify Lewy-Bodies from brain tissue13 and a protocol to isolate large aggregates and aggresomes14. Since already existing methods are based on the usage of detergents, the isolation of detergent-free protein for the application in cell-invasiveness assays is critical.
3. Treatment of Recipient SH-SY5Y Cells with mRFP/eGFP-DISC1 Aggresomes
Yet, uptake/invasion of exogenous protein might not essentially be detected in parallel with recruitment of host cell protein. In our example mRFP-tagged DISC1 aggresomes recruit soluble GFP-tagged DISC1(598-854) protein expressed by the host cell (see Figure 3). Pictures were taken on a Zeiss LSM 510 confocal- or a Zeiss Axiovision Apotome2 Microscope.
4. Generation of Recombinant DISC1(598-785)
In a previous publication we analyzed the ability of various DISC1 protein fragments to self-interact based on the presence of self-association domains. Among all protein fragments tested human DISC1(598-785) displayed the strongest multimerization propensity4 (Figure 4). Therefore, human DISC1(598-785) was cloned into pET15b (Novagen, Madison, Wisconsin) containing an N-terminal 6-histidine tag, expressed in E. coli BL21-(lDE3) Rosetta (Novagen, US), and purified under denaturing conditions in 8 mol/l urea as described4. In short, the protocol is outlined below.
From 1 L bacterial starting culture it is possible to isolate up to 50 mg of recombinant DISC1(598-785) protein.
For experiments with α-synuclein, 500 μg of the recombinant protein (Sigma-Aldrich, USA) was dissolved in PBS at a concentration of 1 mg/ml. To obtain oligomers, refolding was performed overnight at 37 °C as described in a previous publication10.
5. Labeling of Recombinant DISC1(598-785) with DyLight594
To further increase the purity of the labeled protein a His6-tagged based affinity-purification on a Ni-NTA column is performed.
optional concentration step
The labeling and purification (Ni-NTA column) of the recombinant α-synuclein was performed in parallel to DISC1(598-785). The total starting material was 500 μg instead of 1 mg for DISC1(598-785).
6. Treatment of Recipient SH-SY5Y Cells with Labeled Recombinant DISC1 (598-785) Protein
In our example rec. DISC1(598-785)* at least in part recruits soluble GFP-tagged DISC1(598-854) protein expressed by the host cell (see Figure 5A). For recombinant α-synuclein invasion but no recruitment is shown (Figure 5B). Pictures were taken on a Zeiss LSM 510 confocal- or a Zeiss Axiovision Apotome2 Microscope.
The injection of the concentrated (ca. 4 μl of 2.5 μg/μl), labeled DISC1(598-785)* into the mPFC results in the spreading of the protein around the injection site which can be detected even after perfusion of the animal with 4% PFA in PBS pH 7.4 with a rhodamine filterset. In our example DISC1(598-785) is taken up by a distinct number of neurons and can be monitored with Z-stack imaging (Figure 6).
In general, the injection of proteins other then the ones used here do not necessarily lead to cell-invasion. The invasion event is essentially dependent on the nature of the protein, nevertheless a clean purification is a prerequisite for further analysis.
7. Representative Results
Aggresomes consisting of recombinant FL DISC1-eGFP purified from NLF cells invaded recipient SH-SY5Y cells at low efficiency (approximately 0.3% 5) as seen by colocalization with confocal microscopy (Figure 3). As previously reported, DISC1(598-785) expressed and purified from E. coli formed multimers 4 were equally cell-invasive at an efficiency of approximately 20% 5 (Figure 5A) similar to that of α-synuclein that was used in parallel as positive control (Figure 5B). Labeled recombinant DISC1(598-785) expressed and purified from E. coli was also cell-invasive to neurons in vivo when the multimeric protein was stereotactically injected into the medial prefrontal cortex of a rat (Figure 6; Pum, Bader, Huston, Korth, unpublished).
Figure 1. NLF neuroblastoma cells transiently expressing FP-DISC1 (red). Red fluorescent aggresomes can be detected within the cells.
Figure 2. Purified mRFP-tagged DISC1 aggresomes after the purification in a sucrose gradient.
Figure 3. Fluorescent picture of invaded aggresomes. mRFP-tagged flDISC1 aggresomes were purified and incubated with SH-SY5Y neuroblastoma cells overexpressing soluble GFP-tagged DISC1(598-854). An uptake of labeled aggresomes is monitored by Z-stack imaging on a Laser-Scan microscope (Zeiss LSM 510).
Figure 4. SEC-profile of rec. DISC1(598-785) containing the S704 and the C704 single nucleotide polymorphisms (SNP). Both variants show a disruption of ordered oligomerization and the formation of high molecular weight multimers (red arrow). This picture is modified from the publication of Leliveld et al., Biochemistry 2009.
Figure 5. Fluorescent Z-stack picture of invasive DyLight-labeled recombinant DISC1(598-785). SH-SY5Y neuroblastoma cells expressing soluble GFP-DISC1(598-854) were incubated with labeled, recombinant protein at a concentration of 5 μg/ml. (A) Red aggregates shows the invasion of rec. DISC1(598-785) protein and yellow dots indicate a recruitment of soluble GFP-DISC1(598-854) into aggregates. (B) Recombinant α-synuclein (red) invades the cells without recruitment with a frequency of about 20%. Click here to view larger figure.
Figure 6. Immunofluorescent picture of the injected labeled, recombinant DISC1(598-785) protein. Z-stack imaging confirms the presence of rec. DISC1(598-785) protein in rat cortical neurons stained with an antibody against neuronal nuclei (NeuN, green).
In this study we describe the purification of mRFP/eGFP-DISC1 aggresomes from a transfected neuroblastoma cell line, the preparation and labeling of a recombinant DISC1 protein species and their application in cell-invasion experiments of recipient cells in vitro and in vivo.
The purification of native mRFP/eGFP-DISC1 aggresomes was developed from protocols to isolate Lewy body-like structures and larger aggregates13, 14, but modified to avoid the use of detergents and to minimize the risk of false positive cell-invasion that might occur due to detergent-facilitated membrane penetration.
One possible future improvement might be the in to further increase the purity of the aggresomes by sonication to completely separate remaining membranes and cytoskeleton from the aggresomes. By increasing overall purity, the number of total invasion events that recorded for large aggresomes may be increased5. Whether the limited definition of our suggested 3-phase sucrose is sufficient for aggresomes of other protein species has to be tested, in this sense the protocol outlined here should be considered as a starting point for individual optimization. The purified aggresomes proved to be quite robust in our hands, additional washing steps (without detergent) to eliminate traces of sucrose did not significantly reduce the overall yield.
In a previous publication we described that oligomer assembly of DISC1 is dependent on distinct multimerization domains in the C-terminus4 (Figure 4). We chose this fragment for recombinant soluble expression in E. coli and subsequent and Ni-NTA purification. To monitor its multimerization and to compare its cell invasiveness with a described cell-invasive protein like α-synuclein, we labeled DISC1(598-785) with the fluorescent dye DyLight594, and compared its cell-invasiveness with that of equally labeled α-synuclein. The cell-invasiveness of the recombinant DISC1 fragment was dramatically increased compared to that of native, full length DISC1 aggresomes, likely due to its smaller size and much higher purity. Again, purity seems to play a decisive role in cell-invasiveness.
In our example the invasive aggresomes recruited soluble homologue protein of the target cell line that was recombinantly expressed in a soluble, i.e. cell-dispersed form. The expression of a fluorescent protein (e.g. GFP or RFP) in the recipient cell line is an advantage since it allows the colocalization of invasive aggresomes and recombinant proteins within the recipient cell limit via Z-stack imaging.
Cell-invasive DISC1 aggresomes and multimeric fragments expressed and purified from E. coli could be a defining feature of a protein conformational diseases relating to DISC1, DISC1opathies 15.
Low aggresome yield after sucrose gradient purification:
The sucrose gradient introduced in this protocol works well with eGFP/mRFP-DISC1(FL) aggresomes. Aggresomes consisting of other proteins might be of variable dimensions therefore the sucrose concentrations should be optimized by other users.
Insufficient purification of recombinant protein:
Any bacterial contaminants in the purification process of the recombinant protein will also be labeled in later steps of the protocol. To avoid contaminations, run the protein on a SDS-PAGE and confirm that the recombinant protein is at least 95% pure by staining with Coomassie-Blue. To ensure highest quality and yield, the protocol has to be optimized for each individual protein.
Low labeling efficiency of recombinant proteins:
Any contaminations that contain free SH-groups will decrease efficient labeling of the protein. Therefore, extensive dialysis and Ni-NTA based purification is mandatory.
No conflicts of interest declared.
This work was funded by NEURON-ERANET DISCover (BMBF 01EW1003) to C.K. and J. P. H., DFG (Ko 1679/3-1; GRK1033) to C.K. V.B. is supported by a grant of the Forschungskommission of the Medical Faculty of the University of Düsseldorf.
|RPMI 1640||Invitrogen||11875-093||Medium is dependent on the host cell line. The optimal recipient cell line should be determined by the user.|
|DMEM/F-12||Invitrogen||11320-033||Medium is dependent on the recipient cell line. The optimal recipient cell line should be determined by the user.|
|Metafectene||Biontex||T020-1.0||Other transfection reagents might work as well but were not tested with our protocol.|
|Ni-NTA agarose||Qiagen||30210||The experiments were done with Ni_NTA agarose from Qiagen, other suppliers should work as well.|
|DNase I||Roche||04716728001||There is no need for RNase free DNase in the process of aggresomes purification.|
|Opti-MEM||Invitrogen||31985-062||Serum-free medium works as well|
|Penicillin/Streptomycin||Invitrogen||15140122||Supplement for SH-SY5Y and NLF medium|
|Non-essential-amino-acids (NEAA)||Sigma-Aldrich||M7145||Supplement for SH-SY5Y medium|
|Trypan Blue 0.4%||Sigma-Aldrich||T8154||Toxic reagent|
|DyLight 594 Maleimide||Thermo-Fisher Scientific||46608||Reduced cysteine reactive dye to form stable thioether bonds. Also available in other colors.|
|ProLong Gold with DAPI||Invitrogen||P36935||This antifade liquid mountant gave superior results in our hands.|
|Protease Inhibitor cocktail||Roche||11873580001||Dissolve 1 table in 500 μl H2O for 100X solution.|
|Synthetic a-synuclein||Sigma||S7820||Refold as described in text.|
|Table of specific reagents.|
|Precellys 24||Bertin Technologies||03119.200.RD000||We have not tested other mechanical homogenizers other than this. Other detergent free homogenization method might work as well, but have not been tested for this protocol.|
|5301 Concentrator (speedvac)||Eppendorf||5301 000.210||Only necessary if protein has to be concentrated.|
|LSM 510 and Axiovision Apotome2||Zeiss||See manufacturers catalog||Both microscopes harbor the ability to perform Z-stack imaging. This is a prerequisite for solid and serious evaluation of invasion events.|
|Cell culture plastic material||Nunc||10 cm dishes #172958, 24-well plates #142475||The use of different plastic material might influence the interaction of recombinant proteins or aggresomes and the plastic surfaces. We have not tested materials other than the ones described here.|
|Table of specific material and equipment.|