TITLE: Testing ER stress induction in Cultured Induced Neurons via measuring ATF4 protein level or XBP-1 mRNA splicing
AUTHORS: Melissa Hoyer, Harper JW

[DESCRIPTION]
The endoplasmic reticulum (ER) has a vast proteomic landscape to preform many diverse functions including protein and
lipid synthesis, calcium ion flux, and inter-organelle communication. The ER proteome is remodeled in part through membrane-embedded receptors linking ER to degradative autophagy machinery (selective ER-phagy). A refined tubular ER network is formed in neurons within highly polarized dendrites and axons. Autophagy-deficient neurons in vivo display axonal ER accumulation within synaptic ER boutons, and the ER-phagy receptor FAM134B has been genetically linked with human sensory and autonomic neuropathy. However, mechanisms and receptor selectivity underlying ER remodeling by autophagy in neurons is limited. Here, we combine a genetically tractable induced neuron (iNeuron) system for monitoring extensive ER remodeling during differentiation. To test if any of our knockout iNeurons lead to induction of ER stress programs, we either left iNeurons with different genotypes untreated or treated them with tunicamycin as a positive control for ER stress induction. We then compared ATF4 protein induction and XBP-1 splicing levels in untreated versus tunicamycin treated iNeurons. 




[STEPS]
SECTION: Genetically modify Ngn2-inducible embryonic stem (ES) cell H9 line using Cas9
1.  Genetic editing of Ngn2-inducible ES cells is done using the following protocol
“Electroporation of Cas9 protein into human pluripotent stem cells” (dx.doi.org/10.17504/protocols.io.br87m9zn)
SECTION: Differentiate Stable Cell ES H9 line to induced neurons (iN)
2. Differentiation to induced neurons (iN) is done by following the protocol “Neural
differentiation of AAVS1-TREG3-NGN2 pluripotent stem cells (dx.doi.org/10.17504/protocols.io.br9em93e)
SECTION: iN Treatment
3. iN Treatment with or without tunicamycin
SECTION: iN Treatment
3.1. At day 12, iN with each genotype are left untreated or treated with tunicamycin (variable concentrations and times can be used but we found that optimal ER stress response was 1.0
microgram/ml) tunicamycin, 6h treatment). 
SECTION: iN Treatment
3.2. After treatment, all iN are scrapped off the dishes, pelleted and washed three
times with 1X Phosphate Buffered Saline (PBS)
SECTION: iN Treatment
3.3. Determine Number of cells. Pellet cells into two tubes-one for subsequent western blot analysis and the other for mRNA analysis (each pellet should have >1x106 cells)

SECTION: iN Treatment
3.4. Snap freeze pelleted cells in liquid nitrogen. Pellets can be stored at -80C for a few days before use.

SECTION: ATF4 protein level detection
4. ATF4 protein level detection
SECTION: ATF4 protein level detection
4.1. Thaw
cell pellets and resuspend them in Urea lysis buffer (8M Urea, 75mM NaCl, 150mM
Tris pH 7.4, 1X protease inhibitors, 1X phosphatase inhibitors). Approximately
75 µL per sample. Sonicate pellets 2x for 5 sec each.
SECTION: ATF4 protein level detection
4.2. Clarify the lysate: Spin down 4C for 20,000xg 10min, keep supernatant

SECTION: ATF4 protein level detection
4.3. Measure protein concentration using a BCA assay (follow kit directions).



SECTION: ATF4 protein level detection
4.4. Create samples with equal concentrations of protein, add 4x Laemmli Sample Buffer and 2-Mercaptoethanol to 1X in each sample. Heat samples at 65C for 10min



SECTION: ATF4 protein level detection
4.5. With equal amounts of each sample perform a western blot and probe for ATF-4 (B-3) 1:1000
with Goat anti-Mouse IgG HRP conjugate 1:3000 and hFAB‱ Rhodamine Anti-Tubulin 1:10,000.
Image blot on BioRad ChemiDoc imager and quantify levels with Image Lab BioRad Software 6.1



SECTION: XBP-1 splice isoform detection
5. XBP-1 splice isoform detection



SECTION: XBP-1 splice isoform detection
5.1. Thaw cell pellets and resuspend them in freshly prepared RLT buffer (350 µL per sample for 1x106 cells cells) from the RNAeasy Qiagen kit 

SECTION: XBP-1 splice isoform detection
5.2. Add Dnase1 digestion buffer to cells and subsequently lyse
via passage through a Qiashredder column 

SECTION: XBP-1 splice isoform detection
5.3. Add One volume of 70% ethanol to the lysate and transfer
the lysate-EtOH solution to a RNAeasy spin column

SECTION: XBP-1 splice isoform detection
5.4. Perform the following spins: 
1) on column DNAseI (Thermo EN0521) digestion, 
2) buffer washes, and 
3) RNA elution via the RNAeasy Qiagen kit directions

SECTION: XBP-1 splice isoform detection
5.5. Measure final extracted RNA concentration for each condition using a NanoDrop



SECTION: XBP-1 splice isoform detection
5.6. Perform reverse transcription reactions for each condition (using the same amount of starting µg of RNA, 0.5 µg, in each reaction) with Superscript III reverse transcriptase master mix using oligo dT20 primers and dNTPs to create complementary DNA (cDNA).  Incubate at 50 or 55˚C for 50 minutes, then quench the reaction by heating to 85˚C for 5 minutes. Freeze
these samples as cDNA at -20 until ready to use. 
SECTION: XBP-1 splice isoform detection
5.7. With the cDNA, perform PCR reactions to amplify cDNA from GAPDH mRNA (forward
5′GGATGATGTTCTGGAGAGCC3′, reverse 5′CATCACCATCTTCCAGGAGC3′) ; or to amplify
cDNA from unspliced or spliced XBP1 mRNA (forward 5′CCTTGTAGTTGAGAACCAGG 3′, reverse 5′GGGGCTTGGTATATATGTGG 3′) (as performed in van
Schadewijk et al 2012, Yoshida et al 2001).

SECTION: XBP-1 splice isoform detection
5.8. Perform gel electrophoresis on PCR products using a 2.5 percent agarose gel, run for 30min at 100V. Image gel on BioRad ChemiDoc imager and quantify levels with Image Lab BioRad Software 6.1 GAPDH levels should be even in all samples. The size difference between the spliced and the unspliced XBP1 is 26 nucleotides.