All cell culture work was performed in sterile conditions using one way material (3.2). Testing for mycoplasma contamination was performed using the Venor®GeM-OneStep
PCR kit (Minerva Biolabs, Berlin, Germany). The following cell lines were used:
Human SH-SY5Y neuroblastoma cells (Biedler et al., 1973) (ATCC® Number: CRL-
2266™) were kindly provided by Dr. Gary Brooks (Institute of Neuropathology, RWTH Aachen University, Aachen, Germany).
The human cervix carcinoma cell line HeLa (Gey et al., 1952; Scherer et al., 1953) (ATCC® Number: CCL-2™) were obtained from the American type culture collection
(ATCC).
Lenti-X 293T lentiviral packaging cells derived from HEK 293T human kidney cells were obtained from Clontech (Mountain View, CA, USA).
3.6.2 Cultivation, treatment, differentiation and transfection of cell lines
For treatment of cultured SH-SY5Y cells, the following substances were used: Doxycycline (Sigma), all-trans retinoic acid (Sigma), brain derived neurotrophic factor (BDNF) (Peprotech), harmine (Fluka), leucettine L41 (kindly provided by Laurent Meijer, ManRos Therapeutics, Roscoff, France, Tahtouh et al., 2012), LiCl (Sigma), CHIR99021 (Cayman Chemical), staurosporine (Enzo Life Sciences).
SH-SY5Y-tetON-GFP-DYRK1A/K188R cells were cultured in Dulbecco's modified Eagle medium (DMEM) high glucose (PAA) supplemented with 10% FCS (PAA) at 37°C, 5% CO2 and constant humidity. At 75% confluence, cells were washed twice with PBS,
trypsinized and resuspended in fresh medium. Cells were centrifuged for 5 min at 500 g, resuspended in fresh medium and then plated into multiwell plates or dishes according to the experimental requirements. DYRK1A overexpression was induced by treatment with 2 µg/ml doxycycline or as indicated. Neuronal differentiation was induced with 10 µM retinoic acid dissolved in 100% EtOH. For terminal differentiation, the RA containing medium was removed after 3 days and replaced by serum free DMEM high glucose containing 50 ng/ml BDNF for 2 additional days. For long-term differentiation with BDNF, culture dishes were pre-coated with 0.05 mg/ml collagen from rat-tail.
Transfection of 2.0x106 SH-SY5Y cells with 2 µg plasmid DNA was performed using the
AMAXA Nucleofector II and the Cell Line Nucleofector Kit V (Lonza) according to the manual. After nucleofection, 400,000 cells were plated per well into 6-well plates.
constant humidity. For transfection, 100,000 cells were seeded into 6-well plates and transfected with 0.5 µg of a single expression vector using FuGene HD transfection reagent as recommended (Promega). In the case of co-transfection 0.5 µg of each expression vector was transfected. All expression vectors used for transfection are listed in the supplemental material (chapter 7.3, table 19).
Pulse labeling of heterologously expressed HaloTag-Septin4 was performed using the HaloTag Biotin Ligand (Promega) according to the manufacturer’s instructions.
3.6.3 Lentivirus production
4×106 Lenti-X 293T cells were plated in 10 cm culture dishes pre-coated with 0.05 mg/ml
collagen. The next day cells were transfected for lentivirus production with a 2nd
generation packaging system (Zufferey et al., 1997) as stated in table 10.
Table 10: Vector composition for lentivirus production in Lenti-X 293T cells
Vector Amount
FUW-tetON plasmid with gene of interest
or FUW-M2rtTA (see chapter 3.6.4) 3.75 µg pMD2.G VSV G envelope plasmid (Addgene
12259) 1.32 µg
psPAX2 packaging plasmid
(Addgene 12260) 2.43 µg
7.50 µg
For transfection, JetPEI transfection reagent (Polyplus, Illkirch, France) was used as recommended by the manufacturer. The virus containing medium was collected 24 h, 48 h and 72 h after transfection and stored at 4°C. After 72 h, all collected virus containing samples were combined, cleared by centrifugation (5 min, 500 g) and the supernatant filtered using a 0.20 µm syringe filter. Afterwards, the viruses were pelleted for 2 h at 50,000 g. Virus pellets were dissolved in sterile PBS (1 µl PBS/500 µl supernatant), and used for cell transduction. Aliquots were frozen in PCR tubes at -80°C until further usage.
3.6.4 Generation of tet-ON-GFP-DYRK1A SH-SY5Y cells
A second generation lentiviral packaging system was used (3.6.3) to generate SH-SY5Y sublines with controllable overexpression of GFP-DYRK1A (wild type) or GFP-DYRK1A- K188R (KR), which is a DYRK1A point mutant with minimal catalytic activity due to abolished ATP binding (Kentrup et al., 1996). In brief, rat DYRK1A and DYRK1A-K188R cDNA were amplified from pEGFP-DYRK1A (Becker et al., 1998) and integrated into the
insert from Addgene plasmid 20723 with EcoRI) by homologous recombination in E. coli (Zhang et al., 1998) resulting in FUW-tetON-GFP-DYRK1A and FUW-tetON-GFP- DYRK1A-K188R (see section 3.5.5). Co-transduction of the FUW-tetON vector with FUW- M2rtTA (Addgene 20342), which encodes the tetracycline-dependent transactivator, allows for tetracycline-regulated expression of the transgene (Urlinger et al., 2000; Hockemeyer et al., 2008). Co-transduction of SH-SY5Y cells was titrated with different amounts of FUW-tetON-GFP-DYRK1A/KR and FUW-M2rtTA containing lentivirus to achieve maximal transduction efficiency as controlled by doxycycline-induced GFP fluorescence (paragraph 4.1.1). Due to the absence of an antibiotic selection marker in the lentiviral expression vector, cell populations with a GFP-positive ratio >90% were considered as stably transduced. Cells of a parallel transduced well, not treated with doxycycline were expanded, frozen, and used for further experiments. All experiments were performed with cells derived from the same transduction.
3.6.5 Preparation and cultivation of mouse hippocampal neurons
ICR mice were housed, bred, treated and used in experiments according to the corresponding Spanish laws (particularly, 32/2007, Nov 7 and 1201/2005, Oct 10). The Commission of Ethics in Animal Experimentation of the Instituto de Neurociencias and the Bioethics Commission of the CSIC (Spanish Upper Research Council) approved specific experimental procedures.
Hippocampal neurons were isolated from E17.5 mouse embryos and cultured according to Kaech and Banker (2006). In detail, before neuron preparation, 12 mm coverslips (Thermo, Menzel Gläser, Germany) were incubated in 69% HNO3 for at least 48 h on a
shaker. Coverslips were washed 4x for 30 min in ddH2O, dried and sterilized for 4 h at
225°C. Afterwards, coverslips were transferred into 24-well plates and coated with poly-L- lysine (1 mg/ml in borate buffer) over night. The next day, poly-L-lysine was removed and cover slips were washed once with ddH2O. Wells were filled with 250 µl ddH2O until cells
were prepared for plating. E17.5 mouse embryos were removed from the uterus after cervical dislocation of the pregnant dam and transferred to 10 cm dishes on ice containing sterile PBS. The skulls were subsequently separated, washed again in PBS and finally transferred to a new 10 cm dish with fresh PBS on ice. Immediately, the cranium was opened and brains dissected using a binocular loupe, working under a cell culture hood. Brains were collected in sterile PBS on ice. Preparation of hippocampi was started by removing the meninges from single brains and dissecting the telencephalon hemispheres from the brain stem. Hippocampi were removed from the telencephalon, taking care to avoid glia contamination by eliminating all fimbria. Afterwards, hippocampi were cut into small pieces, transferred to cold HBSS and washed twice. Finally, hippocampi were
resuspended in 2 ml HBSS, supplemented with 20 µl DNAse (10 mg/ml) and 200 µl trypsin (2.5%), and incubated at 37°C for 10 min. Meanwhile, water was removed from the coverslips and wells prefilled with 500 µl plating medium. After incubation, the trypsin/DNase solution was removed and hippocampi were washed in 5 ml dissociation medium. Immediately, a second wash was performed by incubating the hippocampi in dissociation medium for 3 min and afterwards adding fresh dissociation medium to a final volume of 1.5 ml, supplemented with 15 µl DNase. Hippocampi were dissociated using a wide Pasteur pipette first and secondly using a narrow Pasteur pipette. After 3 min incubation, the cell containing supernatant was collected and cell viability was determined using trypan blue. If viability was higher than 75%, hippocampal neurons were pelleted for 5 min at 120 g and the cells resuspended in dissociation medium to plate 50,000 cells/well. After 2.5 h, when the neurons attached to the coverslips, plating medium was carefully removed and 500 µl neuronal medium per well were added. The composition of all used media is stated in table 11.
Table 11: Media for preparation and culture of mouse hippocampal neurons
Glial and plating medium Final concentration 50 ml
FCS (Gibco) 10% 5 ml
Pen/Strep (100x) 1% 0.5 ml
DMEM (+Glutamax) (Gibco) 89% 44.5 ml
Neurobasal medium Final concentration 25 ml
Neurobasal medium (Invitrogen) 96% 24 ml
Pen/Strep (100x) (Invitrogen) 1% 0.25 ml
B27 supplement (50x) (Invitrogen) 2% 0.5 ml
Glutamax (100x) (Invitrogen) 1% 0.25 ml
Dissociation medium Final concentration 15 ml
FCS 10% 1.5 ml
DMEM (+Glutamax) 90% 13.5 ml
HBSS/HEPES buffer Final concentration 50 ml
HBSS (Hank´s balanced salt solution) (Invitrogen) 99% 49.5 ml
HEPES [1 M] 0.01 M 0.5 ml
Borate buffer [0.1M] in ddH2O, pH 8.5 MW 400 ml
Boric acid 61.83 g/mol 1.24 g
added when changing the plating medium to neuronal medium. This time point is considered as t0 since no signs of neuronal differentiation were observed before. After
20 h of inhibitor treatment, neurons were fixed with 4% paraformaldehyde. Preparation and culture of mouse hippocampal neurons was planned and performed at the Instituto de Neurociencias (Alicante, Spain) in cooperation with the division of developmental neurogenetics (Dr. F.J. Tejedor).
3.6.6 Mouse embryo culture
Dissection and culture of mouse embryos was carried out comparably as described by (Takahashi et al., 2008). E12 embryos were cultured in the presence or absence of the DYRK1A inhibitors harmine (4 µM), epigallocatechin gallate (EGCG) (10 µM), leucettine L41 (8 µM) (kindly provided by Laurent Meijer, ManRos Therapeutics, Roscoff, France) or without inhibitor for 6 h. EdU (5-ethynyl-2’-deoxyuridine) (Invitrogen/Life Technologies) was added to the culture medium at a final concentration of 20 µM 1 h prior to the end of culture. Brains of the embryos were immediately dissected in cold PBS and fixed in 4% paraformaldehyde. After washing out the fixative, brains were embedded in 4% low gelling temperature agarose and sectioned in frontal orientation with a vibratome. Preparation and culture of mouse embryos was planned and performed at the Instituto de Neurociencias (Alicante, Spain) in cooperation with the division of developmental neurogenetics (Dr. F.J. Tejedor).
3.7 Cell proliferation analysis, flow cytometry and imaging