Felodipine induces autophagy in mouse brains with pharmacokinetics amenable to repurposing

Felodipine induces autophagy in mouse brains with pharmacokinetics amenable to repurposing

Publication date: Apr 19, 2019

After incubation in HBSS with 0. 25% trypsin (Gibco) for 20 min at 37 ^0C, dissociated neurons were resuspended in HBSS and seeded on poly-D-lysine-coated 6-multiwell or 12-multiwell plates, or MatTek glass bottom culture dishes (P35G-1. 0-14-C).

Cells were cultured in maintenance media (Neurobasal-A medium (#12349015, Thermo Fisher Scientific) supplemented with 2 mM GlutaMAX, 200 mM B27 supplement and 1% Penicillin-Streptomycin) at 37 ^0C in a humidified incubator with 5% CO .

Synthetic RNA oligonucleotides (target site: 5′-GTGGTGCATGGTGTGGCAAC-3′, 225 pmol crRNA/tracrRNA, IDT) were annealed by heating to 95 ^0C for 2 min in duplex buffer (IDT) then cooled slowly, followed by addition of 122 pmol recombinant eSpCas9_1. 1 protein (in 10 mM Tris-HCl, pH 7. 4, 300 mM NaCl, 0. 1 mM EDTA, 1 mM DTT), incubation at room temperature for 20 min, and addition of 500 pmol of a 100 nt ssDNA oligonucleotide (IDT Ultramer) as a homology-directed repair template to introduce the desired base change.

After recovery, plating at single cell density and colony picking into 96 well plates, 288 clones were screened for heterozygous and homozygous mutations by high throughput sequencing of amplicons spanning the target site using an Illumina MiSeq instrument using primers A53TintF and A53TintR Final cell lines were expanded and further validated by a second round of sequencing.

Human iPSCs were cultured in TeSR-E8 medium (Stem Cell Technologies) on Vitronectin (Thermo Fisher Scientific) coated plates.

Cells were grown for 11 days in knockout serum replacement media (KSR) supplemented with LDN-193189 (100 nM, Stemgent), SB431542 (10 uM, R&D Systems), SAG (100 nM, Enzo Life Sciences), Purmorphamine (2 uM, Stemgent), FGF8a (100 ng/ml, R&D Systems) and CHIR99021 (3 uM, Stemgent) and 2 mM L-glutamine (Thermo Fisher Scientific).

Media was changed to NB medium on day 12 containing Neurobasal medium, B27 (1cD7) and 2 mM L-glutamine (Thermo Fisher Scientific) supplemented with CHIR99021 (3 uM, Stemgent) (until day 14) and with BDNF (20 ng/ml, Peprotech), GDNF (20 ng/ml, Peprotech), Ascorbic Acid (200 uM, Sigma-Aldrich), TGFβ3, 1 ng/ml, Thermo Fisher Scientific), dibutyryl cAMP (500 uM; Sigma-Aldrich), and DAPT (10 uM, Stemgent).

At day 21 cells were dissociated with StemPro Accutase (Thermo Fisher Scientific) and replated at 300,000 cells/cm2 in dishes pre-coated with Matrigel in final differentiation medium (NB with BDNF, GDNF, TGFb3, DAPT, dbcAMP and Ascorbic Acid).

Maintenance of zebrafish stocks and transgenic lines: Zebrafish were bred and maintained under standard conditions at 28. 5 +/- 0. 5 ^0C on a 14 h light: 10 h dark cycle.

Embryos were collected at 4-5 h post-fertilisation in embryo medium (EM) (5 mM NaCl, 0. 17 mM KCl, 0. 33 mMCaCl , 0. 33 mM Mg SO , 5 mM HEPES) and thereafter kept in a temperature controlled incubator at 28 ^0C and staged according to established criteria29.

Hu α-syn (A53T) mice, also called SNCA(A53T)G2-3 line, or PrPsynA53T Tg mice from the Jackson Laboratory (Bar Harbour, ME, USA) express a 436 bp human α-synuclein cDNA bearing the familial Parkinson’s disease-linked A53T missense mutation driven by the mouse prion protein promoter21.

The primer sequences for mRFP-GFP-LC3 mice genotyping are: Minipigs: Wild-type female Bama pigs, weighing 12-33 kg (Guangzhou Institutes of Biomedic

Concepts Keywords
3M Genotyping
ACC Genomics
Actin Antibodies
Allele Vehicle control
Amino Acid Huntingtin
Amino Acids Studies protocol
Amplicons Pharmacokinetics
Annealed Digital camera
Antibodies Bioluminescence
Antibody Green fluorescent protein
Apochromat Transgene
Ascorbic Acid Zebrafish
Assay Autophagosome
Autophagy Autophagy
Backcrossed Neuron
BDNF Brain
CAMP Genetic engineering
Carl Zeiss Branches of biology
Cas9 PFA
CDNA Episodic seizures dystonia
Central Nervous System Behaviour tremor
Cerebral Cortices MS
Chinese Familial Parkinsons disease
Codon Posture tremor
Colony Studies protocol
Confocal Microscopy Oil immersion lens
Corning 5ml fresh media
Cortical Ethanol castor oil
CRISPR Individual experimental
Culture Medium Oligonucleotides target site
DMSO Software slides
Dopaminergic
DTT
Duplex
E8
EDTA
EGFP
Embryo
Exon
Fertilisation
Fish
GDNF
Genetic
Gestation
GFP
Glutamine
Guangzhou
HCl
Heterozygous
High Throughput Sequencing
Histological Analysis
Home Office
Homozygous
Huntingtin
IDT
Illumina
Infection
Jackson Laboratory
KCl
Knockout
Lens
Lentiviral
Lysed
Lysine
Meninges
Mice
Missense Mutation
Mutagenesis
Mutant
Mutation
N Terminal
NaCl
Neurodegenerative
Neuron
Neurons
Oligonucleotide
Oligonucleotides
Outcrossed
Pharmacokinetics
Primers
Prion
Rabbit
Recombinant
Rho
Ribonucleoprotein
Rod Photoreceptors
SAG
Sequencing
Serum
Sigma Aldrich
Single Density
Standard Conditions
Temperature
Thermo Fisher Scientific
Transgenic
Trypsin
Vesicles
Western Blotting
Wild Type
Wildtype
Zebrafish
Zen

Semantics

Type Source Name
gene UNIPROT BDH1
drug DRUGBANK L-Lysine
drug DRUGBANK Gelatin
gene UNIPROT PTPN5
gene UNIPROT WASHC1
drug DRUGBANK Biotin
drug DRUGBANK Hydrogen peroxide
gene UNIPROT MBOAT4
drug DRUGBANK Sodium Citrate
drug DRUGBANK Formaldehyde
drug DRUGBANK Sucrose
drug DRUGBANK Abacavir
gene UNIPROT ABCB6
gene UNIPROT NOVA2
disease MESH multiple
gene UNIPROT GOPC
gene UNIPROT DEPP1
gene UNIPROT PIGS
disease DOID dystonia
disease MESH seizures
disease MESH posture
disease MESH tremor
gene UNIPROT RNF40
gene UNIPROT ACTBL2
gene UNIPROT SERPINA3
gene UNIPROT ACTG1
gene UNIPROT POTEM
gene UNIPROT FHL5
gene UNIPROT TNFSF14
gene UNIPROT SLC35G1
drug DRUGBANK Potassium Chloride
gene UNIPROT RHOD
gene UNIPROT RHO
gene UNIPROT ATG7
gene UNIPROT SH3PXD2A
disease MESH stop codon
gene UNIPROT ADA2
gene UNIPROT LGALS4
gene UNIPROT MAPT
gene UNIPROT KNTC1
drug DRUGBANK Water
disease MESH neurodegenerative disease
disease DOID neurodegenerative disease
gene UNIPROT LAT2
gene UNIPROT BFAR
gene UNIPROT ADRB2
gene UNIPROT NR1H4
drug DRUGBANK Amino acids
gene UNIPROT FYN
gene UNIPROT SYNM
gene UNIPROT SNCA
disease MESH familial
gene UNIPROT ACTG2
gene UNIPROT ACOT7
drug DRUGBANK Dimethyl sulfoxide
gene UNIPROT GDNF
gene UNIPROT BDNF
drug DRUGBANK L-Glutamine
gene UNIPROT KIT
gene UNIPROT RNPC3
drug DRUGBANK Tromethamine
drug DRUGBANK Edetic Acid
gene UNIPROT KSR1
gene UNIPROT RNF7
gene UNIPROT SAG
gene UNIPROT DMBT1
drug DRUGBANK Medical air
disease MESH death
gene UNIPROT CASP1
gene UNIPROT CES2
gene UNIPROT EFNA5
gene UNIPROT GAPDH
gene UNIPROT PRDM6
gene UNIPROT TGFB3
pathway BSID Homology Directed Repair
gene UNIPROT SCARA5
gene UNIPROT ACACA
gene UNIPROT DYRK3
gene UNIPROT BMS1
gene UNIPROT IK
gene UNIPROT TRIM27
drug DRUGBANK Rifampicin
disease MESH infection
gene UNIPROT MRAP
drug DRUGBANK D-Lysine
gene UNIPROT SLC7A5
gene UNIPROT MAP1LC3A
gene UNIPROT ANKH
drug DRUGBANK Trypsin
drug DRUGBANK Diaminopropanol tetraacetic acid
gene UNIPROT CHAMP1
gene UNIPROT CAMP
drug DRUGBANK Cyclic Adenosine Monophosphate
drug DRUGBANK Ascorbic acid
drug DRUGBANK Felodipine
disease MESH separated
drug DRUGBANK Isoflurane
gene UNIPROT KCNK3
gene UNIPROT SET
gene UNIPROT CD36
gene UNIPROT FAT1
drug DRUGBANK Palmitic Acid
gene UNIPROT SDS
gene UNIPROT SBDS
drug DRUGBANK Sodium lauryl sulfate
gene UNIPROT NR4A3
drug DRUGBANK Carbon dioxide
gene UNIPROT ARID1B
gene UNIPROT ARID3A
disease MESH optic nerve head
gene UNIPROT WIPF2
gene UNIPROT GRIP1
disease MESH weight loss
drug DRUGBANK Polyethylene glycol
gene UNIPROT AKR1C1
drug DRUGBANK Castor oil
drug DRUGBANK Ethanol
gene UNIPROT TSPO
gene UNIPROT CHRM3
drug DRUGBANK Dihydrotachysterol
drug DRUGBANK Verapamil
drug DRUGBANK Propylthiouracil

Original Article

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