Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Summary

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

Abstract

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

Introduction

Cerebellar granule neurons (CGNs) are well characterized in culture and have served as an effective model to study neuronal death and development ^1,2,3,4,5,6. The early expression of N-methyl-D-aspartate (NMDA) receptors in CGN cultures in vitro makes them an attractive model to study NMDA-induced signalling. Activation of these receptors with NMDA in conjunction with membrane depolarization is used to model physiological neuronal activity stimulation, and has allowed for research into the mechanisms of synaptic plasticity ^7,8. On the contrary, over-stimulation of these receptors by NMDA ligand can be used to model excitotoxicity, a major mechanism of neuronal loss in acute brain damage and neurodegenerative diseases ⁹. One mechanism for the induction of excitotoxicity is through ATP starvation with reduced oxygen, as seen with acute neuronal injury. This results in membrane depolarization and elevated levels of glutamate release at the synapse. The subsequent overstimulation of the NMDA receptor by elevated glutamate results in excessive Ca²⁺ influx via these receptors, which in turn activates several pathways including Ca²⁺-activated proteases, phospholipases, and endonucleases, resulting in the uncontrolled degradation of critical cellular components and cell death. Additionally, high intracellular Ca²⁺ leads to the generation of oxygen free radicals and mitochondrial damage ^10,11.

While the majority of neuronal loss following NMDA-induced neuronal excitotoxicity is due to calcium influx and is Bax/Bak independent, other mechanisms of cell death cannot be excluded from this model. The appearance of both necrotic and apoptotic like cell death due to excitotoxicity is partially due to the generation of reactive oxygen species (ROS) and DNA damage caused by high intracellular Ca²⁺ levels ¹². DNA damage results in neuronal death through apoptotic mechanisms, being correlated with hallmarks of apoptotic cell death, such as the appearance of chromatin masses and apoptotic bodies. Induction of apoptosis is mediated through the release of cytochrome c from the mitochondria, and has been shown to be dependent on Bax/Bak oligomerization ¹³. Bax/Bak oligomerization promotes pore formation in the outer mitochondrial membrane, resulting in cytochrome c release and the activation of pro-apoptotic regulators as seen with mild ischemic injury ¹⁴.

Generation of ROS is a significant issue in the brain due to the low endogenous levels of antioxidants, coupled with the large oxygen requirement for neuronal functioning ¹⁵. When exposed to an ischemic event, nitric oxide synthase is upregulated , producing nitric oxide and increasing reactive oxygen species ¹⁴. The increased concentration of oxygen radicals can result in DNA damage and indirectly cause energy starvation. High levels of DNA double-stranded breaks are remedied by the activation of poly ADP-ribose polymerase-1 (PARP-1), an eukaryotic chromatin-bound protein responsible for catalyzing the transfer of ADP-ribose units from NAD⁺, a process integral to DNA repair ¹⁶. However, with excessive damage due to oxidative stress, PARP-1 activation can cause energy starvation due to the increased drain on NAD⁺, a necessary substrate for ATP production through oxidative phosphorylation. Ultimately, oxidative stress will trigger apoptosis in a Bax/Bak dependant manner leading to mitochondrial cytochrome c release, and has been shown to induce mitochondrial remodelling in CGNs ¹⁷.

Finally, changes in concentration of potassium chloride (KCl) in CGN cultures can be used to model low potassium/depolarization mediated apoptosis ^18,19,20. When exposed to low levels of K⁺, CGNs undergo distinct physiological changes, resulting in reductions of both mitochondrial respiration and glycolysis, attributed to decreased cellular demand ²¹, as well as reduction in levels of nuclear factor-κB (NFκB) which regulates activities including inflammation and synaptic transmission ²². This model is of particular interest for the study of cell death during neuronal development. The low K⁺ environment more closely resembles physiological conditions, and causes hallmarks of cell death seen during neuronal development ²³.

In summary, CGNs provide a longstanding model to investigate the underlying molecular mechanisms of neuronal death and degeneration. The following protocol will allow isolation and culturing of CGNs, expression or repression of a particular genetic pathway using viruses and the induction of neuronal death via different mechanisms representing neuronal injury and degeneration.

Protocol

This protocol is based on modifications of procedures that have been described previously ^{18,24,25,26,27}. This protocol is approved by the Animal Care Committee at McGill University.

1. Experimental Preparation

NOTE: The following stock solutions can be prepared and maintained until use.

1. Dissection Solution
   1. Dissolve 3.62 g of sodium chloride (NaCl), 0.2 g of potassium chloride (KCl), 0.069 g of sodium phosphate (NaH₂PO₄ ∙ H₂O), and 1.306 g of D-(+)-Glucose in 500 mL of distilled H₂O. Then add 12.5 mL of HEPES Buffer, 450 µL of Phenol red solution, and 20 mL of BSA fraction V to the solution. Adjust to pH 7.4 using 1 N sodium hydroxide (NaOH).
   2. Sterilize with a 0.22 µm filter and store at 4 °C. This solution will remain stable for up to one week to 10 days.
2. Trypsin
   1. Prepare a stock solution at a concentration of 25 g/L of trypsin in 0.9% sodium chloride. Store the solution at -20 °C.
3. Trypsin Inhibitor
   1. Prepare a stock with a concentration of 10 mg/mL by dissolving 1 g of chicken egg white trypsin inhibitor in 100 mL of 1 mM HCl. Store this solution at -20 °C.
4. DNase
   1. Dissolve 100 mg of DNase 1 in 10 mL of sterile water to generate a 10 mg/mL stock. Store the solution at -20 °C.
5. MgSO₄
   1. Add 6.02 g of magnesium sulfate (MgSO₄) to 50 mL of distilled water (H₂O) to generate a 1 M stock. Store the solution at 4 °C.
6. CaCl ₂
   1. Dissolve 0.735 g of calcium chloride (CaCl₂∙ 2H₂O) in 50 mL of distilled H₂O to a stock concentration of 100 mM. Store solution at 4 °C.
7. KCl
   1. Dissolve 3.7275 g of KCl in 50 mL of distilled H₂O to a stock concentration of 1 M. Store solution at 4 °C.
8. D-(+)-Glucose
   1. Dissolve 1.802 g of D-(+)-glucose in 50 mL of distilled H₂O to a stock concentration of 100 mM. Store solution at 4 °C.
9. Cell Culture Media
   1. Prepare cell culture media as follows: 5 mL of heat inactivated dialyzed Fetal Bovine Serum, 500 µL of 200 mM L-Glutamine, 100 µL of 50 mg/mL Gentamycin and 1 mL of 1.0 M KCL should be added to 45 mL of filter sterilized Eagle's minimal essential media (E-MEM) that is supplemented with 1.125 g/L D-Glucose to generate 50 mL of cerebellar granule neuron culture media. Store media at 4 °C.
10. Cytosine Beta-D- Arabino Furanoside
    1. Dissolve 48 mg of cytosine beta-D-arabino furanoside in 10 mL of growth media to a stock concentration of 20 mM. Store solution at -20 °C.
11. Poly-D-Lysine
    1. To generate a 2 mg/mL poly D-lysine stock, add 10 mL of sterile H₂O to 20 mg of poly D-lysine. Stock poly D-lysine should be stored at -80 °C in 100 µL aliquots.
12. NOTE: The following solutions should be prepared prior to dissection and maintained at 4 °C until use.
13. MgSO₄ Supplemented Dissection Solution
    1. Add 300 µL of stock MgSO₄ to 250 mL of dissection solution.
14. Trypsin Dissection Solution
    1. Add 100 µL of stock trypsin and 12 µL of stock MgSO₄ to 10 mL of dissection solution.
15. Trypsin Inhibitor Solution 1
    1. Add 164 µL of stock trypsin inhibitor, 250 µL of stock DNase 1 and 12 µL of stock MgSO₄ to 10 mL of dissection solution.
16. Trypsin Inhibitor Solution 2
    1. Add 1040 µL of stock trypsin inhibitor, 750 µL of stock DNase 1 and 12 µL of stock MgSO4 to 10 mL of dissection solution.
17. CaCl ₂ supplemented dissection solution
    1. Add 10 µL of stock CaCl₂ and 25 µL of stock MgSO₄ to 10 mL of dissection solution.
18. Poly D-Lysine Coated Culture Dishes
    1. To coat culture dishes, dilute 100 µL of stock poly D-lysine in 40 mL of sterile H₂O. For 35 mm Nunc culture dishes, add 2 mL of diluted poly D-Lysine solution. For 4 well plates, add 300 µL of diluted poly D-Lysine to each well.
    2. Let the poly D-lysine sit for 1 h before aspirating and washing each well with 4 mL or 600 µL (for 35 mm culture dishes or 4 well plates, respectively) of sterile H₂O. Then aspirate the H₂O and let the dishes air dry for 1 h.

2. Brain Extraction and Isolation of Cerebellum

1. Decapitate a 6-7 day old mouse pup using decapitation scissors. Perform the dissection of the brain in a sterile tissue culture hood.
2. To remove the brain, grasp the head using a pair of forceps and cut through the skin toward the anterior of the head using microdissection scissors as demonstrated in Figure 1. Then cut through the skull bone using a new pair of scissors to minimize risk of contamination. Remove the skull covering the brain using forceps and tease out the brain using forceps or a spatula.
   1. As needed, cut through the optic nerve to facilitate getting the brain out as a whole.
3. Place the brain in the dissection solution which is supplemented with MgSO₄. Keep the solution and the brain on ice.
4. Following brain removal, under a dissection microscope, dissect out the cerebellum from the brain while still in MgSO₄ supplemented dissection solution, and remove the meninges using fine forceps. Turn the cerebellum to its ventral side and insure removal of the choroid plexus.
5. Pool the cerebella into a 35-mm dish containing ~1 mL of MgSO₄ supplemented dissection solution.
6. Chop the tissue into small pieces and transfer into a 50 mL tube containing 30 mL of MgSO₄ buffered dissection solution.

3. Mouse Cerebellar Granule Neuron Isolation and Culturing

1. Centrifuge the 50 mL tube containing the chopped cerebral tissue for 5 min at 644 x g and 4 °C.
2. Remove the supernatant and add 10 mL of trypsin dissection solution. Then shake the tube at high speed for 15 min at 37 °C.
3. Add 10 mL of trypsin inhibitor solution 1 to the tube and rock gently for 2 min.
4. Centrifuge the tube for 5 min at 644 x g and 4 °C.
5. Remove the supernatant and add 2 mL of trypsin inhibitor solution 2, and then transfer to a 15 mL tube.
6. Triturate the tissue in the 15 mL tube until the solution becomes murky. Then let settle for 5 min.
7. Remove the clear supernatant and transfer the supernatant to a new tube containing 1 mL of CaCl₂ supplemented dissection solution.
8. Add another mL of trypsin inhibitor solution 2 to the bottom of the tube containing the pellet from step 3.6. Triturate again and let settle for 5 min. Remove the supernatant and add it to the tube containing the supernatant from step 3.7 (That is a repeat of steps 3.6-3.7). Repeat this process until most of the tissue is mechanically dissociated.
9. Add 0.3 mL of CaCl₂ supplemented dissection solution to the supernatant collection for every mL of supernatant.
10. Mix the contents of the tube and then centrifuge for 5 min at 644 x g.
11. Remove the supernatant and add 10 mL of fresh media to the pellet and mix.
12. Cells may then be counted and diluted to a concentration of 1.5 x 10⁶ cells/mL. Please note that in general 10 million cells are expected from each dissected brain, dependent on trypsinization time and efficiency of dissection.Plate cells on previously made poly D-lysine plates. For 4-well plates, plate 0.5 mL, giving 7.5 x 10⁵ cells per well. For 35-mm dishes, plate 4 mL, giving 6 x 10⁶ cells per plate.
13. After 24 h, add cytosine-β-arabino furanoside (AraC) to the plates to reduce glial contamination. For each mL of media 0.5 µL of 20 mM AraC is required. Addition of AraC does not require a media change. If cells are to be maintained for 7-8 days, repeat this treatment on day 3.
14. Maintain cultures in a 5% CO₂ incubator at 37 °C and feed with 100 µL of 100 mM glucose added to the culture for every 2 mL of media every 2 days past 5 days. A complete media change is not required.

4. Lentivirus Packaging, Purification and Titration

NOTE: The protocol for the packaging, concentration, purification and titration of lentivirus has previously been described in detail without the use of a kit ²⁸, including alternative methods for titration, such as flow cytometry ²⁹. Here we briefly present the protocol used in our lab for the production of lentivirus to study neuronal injury using speedy virus purification solution and a qPCR lentiviral titration kit.

1. Plate Hek293T cells in 10 cm culture dishes with Dulbecco's modified Eagle's minimal essential medium (DMEM) supplemented with 10% FBS. For obtaining a high viral titer, grow at least 5 plates of Hek293T cells for each transfection. Make sure on the day of transfection cells are 70% confluent. Change media three hours prior to transfection.
2. For each transfection, prepare two tubes. In tube 1 add 1.5 mL MEM-reduced serum media and 41 µL of transfection reagent, mix well. In tube 2, add 1.5 mL of MEM-reduced serum media and 6 µg of transfer plasmid, 6 µg of pCMV-dR8.2 vector (packaging plasmid), 3 µg pCMV-VSV-G vector (envelope plasmid), and 35 µL of p3000 enhancer reagent (supplied with lipofectamine). Mix well, and combine tubes 1 and 2, vortex and incubate for 15 min.
3. Remove 50% of the media from Hek293T plates and add DNA-lipid complexes to cells. Incubate for 6 h at 37 °C, 5% CO₂. Remove media and replace with 5 mL of fresh DMEM, incubate overnight.
4. At 24 h post-transfection, collect the first batch of viral supernatant from each plate. Keep the viral media at 4 °C and add 5 mL of fresh media into each plate of Hek293T cells. Incubate overnight.
5. 48 h post-transfection, collect the second batch of viral supernatant, combine it with the first batch and centrifuge combined viral supernatant for 10 min at 600 x g.
6. Filter the supernatant through a filter with 45 µm pore size.
7. Purify the virus using a speedy virus purification solution. For every 45 mL of viral supernatant, add 5 mL of lentiviral binding solution, mix, and centrifuge for 10 min at >5,000 x g and 4 °C.
8. Remove supernatant carefully so as not to disturb the pellet.
9. Add 20-40 µL of medium and re-suspend the pellet. Aliquot and store at -80 °C.
10. To obtain the lentiviral titer, a qPCR lentiviral titration kit is used. Dilute 2 µL of purified virus in 500 µL of PBS. Add 2 µL of the diluted virus to 18 µL of virus lysis buffer (provided in the kit).
11. Set up three different RT-q-PCR reactions in triplicates and label them as, viral lysate, positive control 1 (STD1), and positive control 2 (STD2). For each reaction add, 12.5 µL of 2x qPCR MasterMix, 2.5 µL of either viral lysate or STD1 (provided in the kit) or STD2 (provided in the kit), and 10 µL of reagent-mix (provided in the kit) in 0.2 mL PCR tubes.
12. Set the PCR program as follows: 20 min at 42 °C for reverse transcription, 10 min at 95 °C for enzyme activation, 40 cycles of 15 s at 95 °C for denaturation and 1 min at 60 °C for annealing/extension.
13. Calculate the viral titer using this formula:
    Titer of viral lysate = 5 x 10⁷/2^{3(Ctx-Ct1)/(Ct2-Ct1)}.
    Ctx = average Ct value of unknown sample, Ct1 = average value of STD1, Ct2 = average value of STD2.
    NOTE: CGNs can be best transduced with lentiviruses or infected with adenoviruses depending on the mode of injury being studied. Addition of lentiviruses at an MOI of 2-3 to cell culture solution at the time of plating is used to study NMDA induced signalling at day 7 of the culture. This results in more than 80% transduction and is appropriate to pursue with biochemical analyses of these neurons (Figure 2). Addition of adenoviruses at day 5 of culture (at MOI 50) and studying NMDA induced signalling at day 7-8 is used for other techniques such as imaging. This treatment results in less than 10% infection of neurons, making it ideal for imaging and co-localization of proteins. Adenoviruses can be used at the time of plating to study DNA damage induced cell death by addition of camptothecin at day 2-3 or oxidative stress by addition of hydrogen peroxide. The presence of fluorescence proteins (GFP, RFP, CFP or YFP) tagged to the gene of interest can be used to estimate percent transduction and potential toxicity. With the recommended MOI, we see minimal toxicity and maximum transduction (Figure 3).

5. Modeling Neuronal Injury

1. To induce NMDA induced neuronal excitotoxicity, following 7 days in vitro, treat CGNs with 100 µM NMDA and 10 µM glycine for 1 h. Replace all the medium with conditioned medium from parallel cultures with no treatment. This concentration results in 50% cell death at 24 h post treatment (Figure 3C). Mitochondrial fragmentation is evident at 6-8 h post treatment (See Jahani-Asl et al.^26,27).
2. To induce ROS-induced cell death (oxidative stress), treat neurons with 75-100 µM hydrogen peroxide (H₂O₂) and switch to conditioned media from parallel cultures following 5 min exposure to H₂O₂. Note, due to the instability of H₂O₂, optimize the concentration to a level that induces 50-70% cell death in culture following 24 h of treatment.
3. To induce DNA damage induced cell death, treat CGNs with 10 µM camptothecin. This concentration induces more than 50% cell death in 24 h. Mitochondrial fragmentation and early apoptotic signalling occurs 2-3 h after addition of camptothecin at this concentration (See Jahani-Asl et al.¹⁸)
4. To induce low K+ induced neuronal apoptosis in CGNs, change media containing 25 mM K⁺ to low potassium media with 5 mM K⁺ following 7 days in vitro.

Results

With careful dissection, the intact brain should be removed with minimal damage as seen in Figure 1A-B. Effort should be taken to minimize damage to the brain during removal, particularly damage to the cerebellum. Damaging the cerebellum makes for more difficult identification and complete removal of the meninges, and increases the likelihood of contamination of the neuronal culture. Once the meninges have been removed, the cerebellum can be ...

Discussion

Here we provide a simple method for the culturing of primary mouse cerebellar granule neurons (CGNs), loss and gain of function studies, and modeling different mechanisms of cell death. Several factors affect the reproducibility of the results using this procedure which require close monitoring. These include the purity of the culture including the elimination of glial cells in the culture, the confluence of the culture, and maintaining healthy cells. Introducing variability in these factors can bias the results and chal...

Materials

Name	Company	Catalog Number	Comments
qPCR lentivitral titration kit	ABM	#LV900
speedy virus purification solution	ABM	#LV999
pCMV-dR8.2	Addgene	#8455
pCMV-VS.VG	Addgene	#8454
Distilled water	Gibco	#15230162
200 mM L-Glutamine	Gibco	#25030081
35 mm Nunc culture dishes	Gibco	#174913
PowerUP SYBR green master mix	life technologies	#A25742
BSA V Solution	Sigma Aldrich	#A-8412
CaCl2 • 2H2O	Sigma Aldrich	#C-7902
Camptothecin	Sigma Aldrich	#C-9911
Chicken Egg White Trypsin Inhibitor	Sigma Aldrich	#10109878001
Cytosine beta-D-Arabino Furanoside	Sigma Aldrich	#C-1768
D-(+)-Glucose	Sigma Aldrich	#G-7528
DNase1	Sigma Aldrich	#11284932001
Eagle-minimal essential medium	Sigma Aldrich	#M-2279
Glycine	Sigma Aldrich	#G-5417
Heat inactivated dialyzed Fetal Bovine Serum	Sigma Aldrich	#F-0392
Hepes Buffer	Sigma Aldrich	#H-0887
Hydrogen peroxide	Sigma Aldrich	#216763
50 mg/mL Gentamycin	Sigma Aldrich	#G-1397
MgSO4	Sigma Aldrich	#M-2643
N-Methyl-D-aspartic acid	Sigma Aldrich	#M-3262
Phenol Red Solution	Sigma Aldrich	#P-0290
Trypsin	Sigma Aldrich	#T-4549
Lipofectamine 3000	Thermo Fisher Scientific	L3000-008
p3000 enhancer reagent	Thermo Fisher Scientific	L3000-008
Opti-MEM I Reduced Serum Medium	Thermo Fisher Scientific	31985070
KCl	VWR	#CABDH9258
NaCl	VWR	#CABDH9286
NaH2PO4H2O	VWR	#CABDH9298
Poly D-lysine	VWR	#89134-858
DMEM	Wisent	#319-005-CL
FBS	Wisent	#080-450