Jul 08, 2024

Public workspaceGenetic expression suppressor screen

DOI
dx.doi.org/10.17504/protocols.io.ewov19z67lr2/v1
  • 1Department of Biochemistry, Weill Cornell Medicine, New York, NY 10065, USA;
  • 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, Maryland 20815, USA
Alexandros C Kokotos
Open access
DOI: dx.doi.org/10.17504/protocols.io.ewov19z67lr2/v1
Protocol Citation: Alexandros C Kokotos, Tim Ryan 2024. Genetic expression suppressor screen. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov19z67lr2/v1
Manuscript citation:
Phosphoglycerate kinase is a central leverage point in Parkinson’s Disease driven neuronal metabolic deficits
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 08, 2024
Last Modified: July 08, 2024
Protocol Integer ID: 103021
Keywords: ASAPCRN, Live-cell imaging, neurons, low-throughput screening, glycolytic enzyme
Funders Acknowledgement:
ASAP
Grant ID: 000580
Abstract
This protocol describes a low-throughput genetic screen to identify glycolytic enzymes that improve neuronal function under hypometabolic conditions.
Materials
Media Supplies
  • 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; Alomone Labs, Cat. No. C-141)
  • Calcium Chloride (Millipore-Sigma, Cat. No. C3881)
  • DL-2-Amino-5-phosphonovaleric acid (APV; Millipore-Sigma A5282)
  • Glucose (Millipore-Sigma, Cat. No. G7021)
  • HEPES (Millipore-Sigma, Cat. No. H3375)
  • Magnesium Chloride (Millipore-Sigma, Cat. No. M0250)
  • Potassium Chloride (Millipore-Sigma, Cat. No. P9333)
  • Sodium Chloride (Millipore-Sigma, Cat. No. S5761)

Stock solutions
APV (25mM Stock)
  1. Add 20L ddH2O to whole bottle of APV
  2. Sonicate at ~55C for 30 minutes.
  3. Make 1mL aliquots and store at -20C.

CNQX (10mM Stock)
  1. Add 10 mL of ultrapure H2O to the whole bottle of CNQX.
  2. Vortex until dissolved.
  3. Make 1 mL aliquots and store at -20 oC.
Media solutions
0.1mM glucose tyrodes (119mM NaCl, 2.5mM KCl, 0.1mM D-Glucose, 54.9mM HEPES, 2mM CaCl2, 2mM MgCl2, 50uM APV, 10uM CNQX)

NH4Cl tyrodes (119mM NaCl, 2.5mM KCl, 5mM HEPES, 50mM NH4Cl, 2mM CaCl2, 2mM MgCl2, 50uM APV, 10uM CNQX)
Equipment
  • 40x, 1.3 N.A Objective (like Zeiss-Fluar)
  • 488nm Laser (like Coherent-OBIS 488nm LX Laser)
  • CO2 Incubator (like VWR Symphony)
  • EMCCD Camera (like Andor iXon Ultra 897)
  • Epifluorescence Microscope (like Zeiss ObserverA1)
  • Filter cube set for GFP excitation and emission (like Chroma filters)
  • Laser speckle reducer (like Optotune LSR 3005)
  • Laser/stage/stimulation-Master board controller (like a custom-designed Arduino board)
  • Light transmission microscope (like Olympus CK40)
  • Mercury arc lamp (like Zeiss HBO 100)
  • Perfusion system (like Automate Scientific Valvelink 8.2)
  • Microscope XYZ Stage (like Applied Scientific Instrumentation stage)
  • Stimulus isolator (like World precision Instruments A385)
  • Temperature controller for Objective (like Minco)

Plasmids
  • vGLUT1-pHluorin
  • mTagBFP2-N1
  • hSyn HK1 (Addgene plasmid #220915; http://n2t.net/addgene:220915; RRID:Addgene_220915)
  • hSyn AldoA (Addgene plasmid #220916; http://n2t.net/addgene:220916; RRID:Addgene_220916)
  • hSyn AldoC (Addgene plasmid #220917; http://n2t.net/addgene:220917; RRID:Addgene_220917)
  • hSyn GAPDH (Addgene plasmid #220918; http://n2t.net/addgene:220918; RRID:Addgene_220918)
  • hSyn PGK1 (Addgene plasmid #220919; http://n2t.net/addgene:220919; RRID:Addgene_220919)
  • hSyn PKM1 (Addgene plasmid #220920; http://n2t.net/addgene:220920; RRID:Addgene_220920)
Neuronal culture and transfection
Neuronal culture and transfection
Use cultured rodent primary hippocampal neurons, transfected with vGlutI-pH, mTagBFP2 and a construct expressing a single glycolytic enzyme, as explained in dx.doi.org/10.17504/protocols.io.ewov1qxr2gr2/v1.

Live-cell imaging
Live-cell imaging
25m
Remove the cells from the incubator and bathe them in Tyrodes containing 0.1 mM glucose.
Mount the coverslips with the cells on the imaging chamber and image as explained in dx.doi.org/10.17504/protocols.io.q26g7pn4qgwz/v1.
Locate cells in an unbiased manner using the mTagBPF2 channel and center on their axon, judging by their morphology.
After a total of Duration00:05:00 that the cells have been in the 0.1 mM glucose tyrode buffer, start the live-cell imaging experiment in the vGlutI-pH channel.

5m
Stimulate the cells with 100 APs delivered at 10 Hz every Duration00:01:00 , for a total of 10 rounds of activity.

1m
At the end of the experiment, perfuse cells with Tyrodes containing 50 mM NH4Cl.
Analyze the collected data accordingly.
Protocol references