Visualizing the DNA Damage Response in Purkinje Cells Using Cerebellar Organotypic Cultures

Summary

Cerebellar Purkinje cells (PCs) are particularly sensitive to deficiencies in the DNA damage response. A protocol is presented for the visual evaluation of the dynamics of PC response to DNA damage, which involves staining protein-bound poly(ADP-ribose) chains within cerebellar organotypic cultures.

Abstract

Cerebellar Purkinje cells (PCs) exhibit a unique interplay of high metabolic rates, specific chromatin architecture, and extensive transcriptional activity, making them particularly vulnerable to DNA damage. This necessitates an efficient DNA damage response (DDR) to prevent cerebellar degeneration, often initiated by PC dysfunction or loss. A notable example is the genome instability syndrome, ataxia-telangiectasia (A-T), marked by progressive PC depletion and cerebellar deterioration. Investigating DDR mechanisms in PCs is vital for elucidating the pathways leading to their degeneration in such disorders. However, the complexity of isolating and cultivating PCs in vitro has long hindered research efforts. Murine cerebellar organotypic (slice) cultures offer a feasible alternative, closely mimicking the in vivo tissue environment. Yet, this model is constrained to DDR indicators amenable to microscopic imaging. We have refined the organotypic culture protocol, demonstrating that fluorescent imaging of protein-bound poly(ADP-ribose) (PAR) chains, a rapid and early DDR indicator, effectively reveals DDR dynamics in PCs within these cultures, in response to genotoxic stress.

Introduction

The integrity of cellular DNA is constantly under threat from DNA damaging agents, predominantly metabolic by-products like reactive oxygen species, which inflict tens of thousands of DNA lesions per cell daily¹. The persistent upkeep of genome stability is essential for cellular homeostasis^2,3. The cornerstone of this maintenance is the DNA damage response (DDR) - an intricate, layered signaling network that initiates specific DNA repair pathways while carefully adjusting many other cellular processes^4,5. Deficits in the DDR are commonly manifested as 'genome instability syndromes,' marked by chromosomal instability, progressive tissue deterioration, impaired growth or development, a predisposition to cancer, and heightened sensitivity to particular DNA-damaging agents^6,7,8,9. Notably, neurodegeneration, which often includes cerebellar atrophy, is a distinct feature of many genome instability syndromes^7,10,11,12.

The autosomal recessive disorder, ataxia-telangiectasia (A-T), is a well-documented example of a genome instability disorder^13,14,15. This condition arises from null mutations in the ATM (A-T, mutated) gene, responsible for coding the pivotal protein kinase, ATM, which is known primarily as a DDR mobilizer in response to DNA double-strand breaks (DSBs)^16,17. A-T manifests as a multisystem disorder, predominantly characterized by progressive cerebellar degeneration, leading to acute motor impairments, immunodeficiency, gonadal atrophy, cancer predisposition, and extreme sensitivity to ionizing radiation. Cultured cells from individuals with A-T show chromosomal instability and increased sensitivity to genotoxic agents, especially those causing DSBs^15,18,19. Importantly, ATM also plays a role in repairing other DNA lesions, underscoring its broad significance in maintaining genome stability^20,21,22.

Despite thorough research into ATM's numerous roles, the specific mechanism leading to cerebellar degeneration in A-T remains a topic of active debate, with various models proposed to elucidate this process^{23,24,25,26,27,28,29,30}. Our model²⁸ suggests that cerebellar degeneration in A-T patients begins with the dysfunction and eventual loss of Purkinje cells (PCs). Considering ATM's critical role in preserving genome stability in the face of ongoing DNA damage, PCs are particularly vulnerable to the absence of ATM. We attribute this vulnerability to the combination of their high metabolic activity, distinctive chromatin structure, and extensive transcriptional activity. Ultimately, it is suggested that the loss of PC function, and hence, their degeneration, is due to the stochastic, functional inactivation of genes, a consequence of producing defective transcripts²⁸.

The study of PC biology in the laboratory is impeded by challenges in cultivating isolated PCs, as these cells rely heavily on their natural milieu and neighboring cells for survival and function, rendering them incompatible with dissociated culture growth. Nonetheless, PCs can remain viable for extended periods in tissue slice cultures. Cerebellar organotypic cultures, which are tissue slices typically derived from rodent cerebella, maintain the tissue's structural organization and support various experimental manipulations analogous to those possible with cultured cells. Therefore, these cultures allow for cerebellar studies within a controlled setting^{31,32,33,34,35,36,37,38,39,40,41,42,43}. Specifically, in the context of A-T, murine cerebellar organotypic cultures have proven to be instrumental in exploring the DDR in Atm-deficient PCs^40,41,42,43. While Atm-deficient mice display only a subtle cerebellar phenotype^44,45,46,47, presumably due to differences between human and mouse cerebellar physiology, the assumption is that ATM's roles are largely conserved across these species. This notion is supported by our observations that the deficient response to DNA DSBs in Atm^-/- murine PCs aligns with that observed in other murine and human ATM/Atm-deficient cell types^40,41,42,43.

A limitation in analyzing PC responses to various stimuli or stresses within organotypic cultures is the necessity to rely on microscopic imaging for readouts. The DDR is usually studied using bulk biochemical readouts, although common immunofluorescent markers are utilized as well, such as following the dynamics of formation and resolution of nuclear foci of phosphorylated histone H2AX (γH2AX) and the 53BP1 protein, which are considered indicators of DSBs^48,49. A broader measure is the fluorescent imaging of poly(ADP-ribose) (PAR) chain formation on proteins, a rapid and robust early DNA damage response, particularly to strand breaks^7,50. We modified the protocol by Komulainen et al.⁵¹ for PAR staining in cerebellar organotypic cultures. We observed a pronounced PAR response in the sizeable nuclei of PCs. Presented here is our refined protocol for establishing murine cerebellar organotypic cultures and for visualizing the PAR response under genotoxic stress.

Protocol

See Table of Materials for details of materials, equipment, and antibodies. Animal procedures were employed under the ethical guidelines of Tel Aviv University's Ethics Committee after approval. The procedure is carried out on 10-day old mouse pups, regardless of their sex. If needed, genotyping is performed on the previous day using a tail biopsy and standard methods. Solutions are sterile and stored at 4 °C unless indicated otherwise; see Table 1.

1. Preparation of cultures

NOTE: Conduct the procedure within a sterile environment and disinfect the work surface in advance using 70% ethanol.

1. Add 1 mL of BME to each well of 6-well plates. Using sterile forceps, place a single cell culture insert into each well and incubate the plates in a 37 °C/5% CO₂ incubator for a minimum of 2 h prior to the dissection procedure.
2. Set up a dissection area within a biological hood, containing the tissue chopper, 30 mm culture plates, a binocular microscope, and a light source.
3. Immerse the surgical tools in 70% ethanol throughout the process. Use a urinary cup filled with 70% ethanol for immersion, and a separate urinary cup with 1x PBS to wash off the ethanol before use.
4. For each dissection (i.e., for each cerebellum), prepare two 30 mm culture plates filled with cold dissection medium and leave them on ice.
5. Spray the animal with 70% ethanol.
6. Using sharp scissors, swiftly decapitate the animal. To expose the brain, make a small incision with scissors away from the cerebellum. Using tweezers with rounded ends, gently peel off the skull. Remove the skull in small pieces to avoid damaging the cerebellar tissue.
7. Immediately transfer the brain into cold dissection medium or directly remove the cerebellum from within the skull. To do this, expose the cerebellum and separate the cerebellar cortex from the brainstem by disconnecting the three pairs of peduncles, using a fine curved iris spatula. To identify the peduncles, view from rostral to caudal: the paired superior cerebellar peduncles connect the midbrain to each side of the cerebellum; the paired middle cerebellar peduncles connect the pons to each side of the cerebellum; and the paired inferior cerebellar peduncles connect to the medulla to each side of to the cerebellum. Once the cerebellum is visible, separate it from the cerebrum using the same spatula: slide the spatula between the cerebellum, the superior colliculus, and the brainstem.
   NOTE: Do not make a special effort to remove the meninges as they readily detach after the cerebellum is sliced.
8. Set the tissue chopper to a slice thickness of 350 µm.
9. Prior to positioning the cerebellum on the chopper's base, remove excess dissecting buffer from the tissue. This will improve the tissue's adhesion to the chopper's base.
10. Position the cerebellum on the cutting platform in a vertical orientation and carry out parasagittal slicing at a slice thickness of 350 µm. Gently separate the slices using a fine iris spatula and put them into cold dissection medium.
11. Transfer the sliced tissue back to a fresh cold dissection buffer. Use a 1,000 µL tip to delicately guide the tissue slices into the dish containing cold dissection buffer.
12. Under the binocular use two curved iris spatulas to carefully separate the tissue slices from each other. For cultures, use slices derived from the cerebellar vermis situated in the medial cortico-nuclear zone of the organ.
13. Transfer each tissue slice onto a culture insert using a wide spatula and move each insert carrying a tissue slice into a well in the 6-well plates containing the pre-warmed medium. Leave the plates in the incubator.
14. Replace the medium every other day: aspirate the used medium using a sterile glass pipette, and with a 5 mL serological pipette, add 1 mL of fresh MBE with the pipette's tip leaning against the well's wall. Make sure not to direct the medium flow onto the tissue.
    NOTE: The tissue slice will exhibit an inflammation-like appearance for the first 5-7 days in culture and will be ready for experiments as soon as this phenomenon disappears and up to 2 weeks after culture establishment.

2. Treatment with DNA damaging agents, fixation, staining, and microscopic imaging

1. Add DNA-damaging chemicals directly to the culture medium at appropriate final concentrations, for predetermined durations. After the exposure period, remove the chemicals by replacing the medium with fresh culture medium.
2. If the DDR readout involves PAR staining, add an inhibitor of poly(ADP-ribose) glycohydrolase (PARG) to the culture medium at a final concentration of 10 µM, 30 min prior to fixation.
   NOTE: This treatment is critical as the PAR modification is short-lived and continuously degraded by PARG.
3. Immediately prior to fixation, aspirate the medium and replace it with 1 mL of 0.1 M phosphate buffer at room temperature.
   NOTE: Cold buffer might cause detachment of the tissue slices from the insert.
4. Immediately fix the slices by replacing the buffer with precooled (-20 °C) acetone:methanol mix (1:1) such that the tissue slice is submerged in this mix and leave for 20 min at -20 °C.
   NOTE: It is possible to stop at this stage and proceed with immunostaining later. To do so, wash the tissues 3x with 0.1 M phosphate buffer precooled to 4 °C, making sure the tissue is submerged in the buffer. Tissues can be stored now in 0.1M phosphate buffer for up to 2 weeks at 4 °C.
5. Transfer the tissue slice into a well in a 48-well plate.
6. Use a scalpel and a cutting board to remove the margins of the insert, cutting around the tissue slice. Place each slice in a well of a 48-well plate. Add 100 µL of 0.1 M phosphate buffer.
7. For permeabilization, aspirate the buffer, add to the well 100 µL of 0.1 M phosphate buffer containing 1% Triton X-100, and leave for 5 min at room temperature.
8. Aspirate the solution and add 100 µL of blocking solution (see Table 1) per well. Shake gently at room temperature for 2 h.
9. During the blocking step, dilute the primary antibody or anti-PAR reagent (1:800) in the blocking solution.
10. Remove the blocking solution (no need to wash) and add 100 µL of primary antibody mix to each well. Apply gentle shaking for 2 h at room temperature.
11. Dilute the secondary antibodies in 0.1 M phosphate buffer containing 0.2% Triton X-100 (1:500) while minimizing their exposure to light.
12. Aspirate the primary antibody solution and wash the slices 3 x 5 min with 0.1 M phosphate buffer containing 0.2% Triton X-100, at room temperature, with gentle shaking.
13. Replace the wash solution with 100 µL of secondary antibody solution (1:500 dilution). Continue gentle shaking for 2 h at room temperature in the dark (cover the plates with aluminum foil).
14. To prepare a DAPI solution, dilute the stock solution (2 mg/mL) to 1 µg/mL in 1x PBS.
15. Twenty minutes before the end of the 2 h incubation with the secondary antibody, add 20 µL of the final DAPI solution to each well and continue shaking for 20 min. Aspirate the liquid and wash with the wash solution for 3 x 5 min with gentle shaking.
16. For mounting, place the tissue on a microscope glass slide with the tissue facing up, add mounting medium, and cover with a cover glass. Place the slides on a flat surface and let them dry overnight in the dark, at room temperature (avoid using mounting medium containing DAPI to prevent blurriness).
17. Store the slides at 4 °C in a lightproof box.
18. Capture microscopic images using a confocal microscope and appropriate filters.

Results

Figure 1 illustrates the general appearance of the cerebellar organotypic cultures. The top row in Figure 1A shows the cerebellar foliation, which is maintained in culture, while the bottom row shows the PCs stained for Calbindin D-28k (green) and the neuronal nuclei stained for NeuN (red). In Figure 1B, the astrocytes in the PCs (red) are stained for GFAP (green), an intermediate filament type III protein. Figure 2 and Figure 3 show the PAR staining observed after treating these cultures with two DNA-damaging agents, both of which are strong oxidants: potassium bromate (a final concentration of 10 mM for 12 h) and paraquat. The PAR response was marked in the treated PCs in the cerebellar folium compared to the control.

Figure 1: Overview of murine cerebellar organotypic cultures. (A) The top row illustrates the cerebellar foliation, which is maintained in culture. The bottom row focuses on the PC layer. PCs are highlighted by Calbindin D-28k (green). NeuN staining (red) identifies neuronal nuclei across the layers. (B) A closer look at PCs (red). GFAP (green), an intermediate filament type III protein, which highlights astrocytes. Scale bars = 968.8 µm (A, top row), 42.1 µm (A, second row), 72.7 µm (B, top row), 18.2 µm (B, second row). Abbreviations: PC = Purkinje cells; GFAP = glial fibrillary acidic protein; DAPI = 4',6-diamidino-2-phenylindole. Please click here to view a larger version of this figure.

Figure 2: PAR staining in a cerebellar slice treated with potassium bromate. The cerebellar slice was treated with potassium bromate (KBrO₃)-a potent oxidizing agent, at a final concentration of 10 mM for 12 h. Scale bars = 775 µm. Abbreviations: PAR = poly(ADP-ribose); DAPI = 4',6-diamidino-2-phenylindole. Please click here to view a larger version of this figure.

Figure 3: PAR response in cerebellar slices exposed to paraquat, a strong producer of superoxide anions. (A) A general view of a cerebellar folium. Note the robust PAR response particularly in PCs compare to the control. (B) A closer examination shows the PAR response in PCs and other cell types. Scale bars = 193.8 µm (A), 18.2 µm (B). Abbreviations: PAR = poly(ADP-ribose); DAPI = 4',6-diamidino-2-phenylindole. Please click here to view a larger version of this figure.

Solutions and buffers
Solution	Ingredients
Dissection medium	Hank’s balanced salt solution (HBSS) containing 0.5% D-glucose. Add 0.5 mL of 50% D-glucose solution to 50 mL of HBSS without Mg2+ and Ca2+.
BME culture medium	Mix 100 mL of Basal Medium Eagle (BME) containing phenol red but not L-glutamine or HEPES,  with 50 mL of HBSS with phenol red, 50 mL of heat-inactivated horse serum, 2 mL of 50% D-glucose, and 1 mL of 200 mM L-glutamine. Mix and filter through a 0.22 μm filter in a sterile environment. Store at 4 °C
Blocking solution for immunostaining	in 0.1 M Phosphate buffer: add 0.5% Triton X-100, 10% Goat serum
Secondary antibody dilution buffer	0.1 M phosphate buffer, 0.2%Triton X-100
Wash buffer	0.1 M phosphate buffer, 0.2%Triton X-100
Fixative solution	Actenoe:methanol (1:1 v:v)
Permeabilization solution	0.1 M phosphate buffer, 1%Triton X-100
DAPI solution	Stock solution (2 mg/mL) diluted to 1 µg/mL in 1x PBS
Phosphate Buffer	Prepare two soulutions A and B (see below). Mix solutions according to the ratios specified below to make 0.2 M phosphate buffer. Then dilute the 0.2M solution  with DDW (1:1) to make 0.1 M phosphate buffer (working solution).
solution A (for 0.2 M)	12 g of Sodium Phosphate Monobasic Anhydrous (Na2HPO4 FW 141.96)  500 mL of DDW
Solution B (for 0.2 M)	14.2 g of Sodium phosphate diabasic anhydrous (Na2HPO4 FW 141.96) 500 mL of DDW
Mixed solutions to make 0.2 M	Prepare 1 L: 230 mL of solution A and 770 mL of solution B
0.1 M phosphate buffer	Dilute the mixed solution with DDW (1:1)

Table 1: Solutions and their composition.

Discussion

General comments
The major advantage of the organotypic culture system is that it facilitates studies using the cerebellar cortex tissue, preserving its structural organization for several weeks in the culture dish setup. This system is useful for conducting in-depth morphological analyses of Purkinje cells (PCs), including detailed examinations of dendritic spines and ultrastructural features^{52,53,54,55,56,57,58,59}. Our observations indicate that the DNA damage response (DDR) is notably active in PCs within these cultures. However, the major limitations of this system include the limited duration of its use and its reliance on microscopic imaging to monitor cellular processes, highlighting the ongoing need to develop or adapt suitable imaging techniques.

Another word of caution pertains to the cerebellar portion that is dissected for preparing the tissue slices. We have observed that different cerebellar lobes can yield cultures with markedly different internal organization and overall quality. Therefore, we prefer to establish cultures from slices obtained via midsagittal sections of the cerebellar vermis. In these slices, the folia of the cerebellar cortex are conveniently visible.

It is advisable to utilize tissue slices obtained from the cerebellar vermis, which exhibit the characteristic foliation of the cerebellar cortex. These slices maintain their organizational structure in culture for several weeks. Furthermore, it is recommended to commence experimental procedures with the cultures approximately 7-12 days post establishment. A typical experiment designed to assess the DNA damage response in these cultures involves initiating damage by irradiation or by the addition of DNA-damaging chemicals to the culture medium. Subsequently, tissue fixation and staining are performed at various time intervals. To assess recovery from chemical-induced damage, a period of incubation in fresh medium is included. It is advisable to initiate long-term experiments at the earliest opportunity, ideally around 7 days following culture establishment.

The cerebellar organotypic cultures are maintained in a medium devoid of antibiotics; thus, maintaining a sterile environment during their establishment is imperative. The age of the pups from which the cerebella are harvested is critical, with a recommended range of 9-12 days. This age range is optimal as the cerebellar cortex is sufficiently developed for sectioning. Notably, in older animals, the tissues tend to adhere to the blade. It is crucial to keep the cerebellum immersed in a cold dissection solution until dissected.

During the initial 5-7 days in culture, the tissue slices may exhibit signs reminiscent of inflammation, characterized by swelling and milky discoloration. These attributes can potentially affect the measurement of physiological parameters and the effectiveness of immunostaining techniques.

It is highly recommended not to slice the cerebellum thinner than 350 µm to avoid inconsistency in thickness during slicing. Keep in mind that a 350 µm-slice tissue gets much thinner after 7 days in culture to around 90 µm (based on Z stack measurement).

Here, we introduce PAR staining^7,50 to the toolkit used to investigate the DDR in PCs. This readout is rapid, sensitive, and captures the dynamic nature of the DDR, proving especially useful in our analysis of the DDR in wild-type and Atm-deficient PCs. The protocols outlined here should be particularly beneficial for researchers studying genomic instability syndromes that affect the cerebellum.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagent and Equipment
150 x 30 cm Petri dish	Corning Inc.	3261
35 x 10 mm Petri dish	Corning Inc.	3294
Acetone	Invitrogen	25030081
Acetone	Sigma-Aldrich	270725-1L
Adhesive microscope slides	Marienfeld	48187	76 x 26 x 1 mm
Blades for chopper	TED PELLA	Model TC752-1	Style Razor Blades
BME	GIBCO	41010-026 500ml.	No L-Glut
Cell culture inserts	Millipore	PICM0RG50
D-Glucose	Biological Indo.	02-015-1A
HBSS w/o phenol red	Sigma-Aldrich	H-1138-500L	No Ca+,No Mg+
HBSS with phenol red	Sartorius	02-015-1A
Horse serum	GIBCO	26050088	heat inactivated
Iris forceps	CellPath	GZX-0100-00A	130mm blunt serrated
Iris spatula	F.S.T	10092-12
L-Glutamine (200 mM)	Gibco	41010026
Methanol	Sigma-Aldrich	322412-1L
Methanol	Sigma-Aldrich	G5767
Microscope	Olympus
Mounting Medium without	GRIGENE. Ltd, Israel	K239320A
Paraquat	Sigma-Aldrich	856177-250MG	Paraquat dichloride (Methyl viologen)
PARG- Poly(ADP-Ribose) Glycohydrolase inhibitor	Tocris Bioscience, United Kingdom	PDD 00017273	light sensitive
Phosphate buffer	Biological Indo.	02-018-1A
Scissors, skin-tissue
Six-well plates	Corning incorporated	CLS3516
Spare Chopping Discs	TED PELLA	Model 10180-01
Tissue chopper	McIlwain	Model TC752	10180-220
Tweezers, pointed
Urinary cups
Antibodies
Alexa fluor 488 donkey anti Rabbit IgG	Invitrogen	A21206	1:500; secondary ab; Secondary dilution buffer;  Incubation for 2 h at room temperature
Anti glial fibrillary acidic protien	Milliphore	MAB3402	1:1500; primary ab; blocking; Host:mouse
Anti-calbindin-D-28K	Swant	CB300	1:2000; primary ab; blocking; host: rabbit
Anti-calbindin-D-28K	Swant	CB-38a	1:2000; primary ab; blocking
Anti-pan-ADP-ribose reagent	Milliphore	MABE1016	1:800; primary ab; blocking; incubation for 2 h at room temperature
DAPI (4',6-Diamidine-2'-phenylindole dihydrochloride)	Cell signaling	4084	1:1000; secondary dilution buffer; incubation for 20 min at room temperature