The generation, purification and cell invasion of intracellular, cytoplasmic full length DISC1 protein aggresomes from cell cultures and of a labeled, multimeric recombinant DISC1 protein fragment in E. coli are described. Cell invasiveness is shown for recipient cells in cell culture and for neurons in vivo after stereotactical brain inoculation.
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.
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.
Trouble shooting:
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.
The authors have nothing to disclose.
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.
Reagent name | Company | Catalog number | Comments | ||||||||||||||||||||||||||||||||||||
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. | ||||||||||||||||||||||||||||||||||||
PBS | Invitrogen | 14190-250 | |||||||||||||||||||||||||||||||||||||
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. | ||||||||||||||||||||||||||||||||||||
Sucrose | Sigma-Aldrich | S0389 | |||||||||||||||||||||||||||||||||||||
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. | |||||||||||||||||||||||||||||||||||||||
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