treatment of cerebellar organotypic cultures with dna damaging agents for fluorescent imaging of par chains to assess genotoxic stress response

Cerebellar Purkinje Cells: DNA Damage Response in Organotypic Cultures

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 homeostasis2,3. 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 processes4,5. Deficits in the DDR are commonly manifested as &#39;genome instability syndromes,&#39; marked by chromosomal instability, progressive tissue deterioration, impaired growth or development, a predisposition to cancer, and heightened sensitivity to particular DNA-damaging agents6,7,8,9. Notably, neurodegeneration, which often includes cerebellar atrophy, is a distinct feature of many genome instability syndromes7,10,11,12.
The autosomal recessive disorder, ataxia-telangiectasia (A-T), is a well-documented example of a genome instability disorder13,14,15. 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 DSBs15,18,19. Importantly, ATM also plays a role in repairing other DNA lesions, underscoring its broad significance in maintaining genome stability20,21,22.
Despite thorough research into ATM&#39;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 process23,24,25,26,27,28,29,30. Our model28 suggests that cerebellar degeneration in A-T patients begins with the dysfunction and eventual loss of Purkinje cells (PCs). Considering ATM&#39;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&#39;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 setting31,32,33,34,35,36,37,38,39,40,41,42,43. Specifically, in the context of A-T, murine cerebellar organotypic cultures have proven to be instrumental in exploring the DDR in Atm-deficient PCs40,41,42,43. While Atm-deficient mice display only a subtle cerebellar phenotype44,45,46,47, presumably due to differences between human and mouse cerebellar physiology, the assumption is that ATM&#39;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 types40,41,42,43.
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 (&#947;H2AX) and the 53BP1 protein, which are considered indicators of DSBs48,49. 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 breaks7,50. 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.

Author Spotlight: Deciphering the Role of ATM in Ataxia-Telangiectasia and the Associated Cerebellar Degeneration

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

Our work is focused on understanding a human genetic disorder called ataxia-telangiectasia, or A-T for short. It is caused by loss of the protein kinase ATM. ATM has many functions and A-T is characterized by many symptoms, but the major symptom of A-T is progressive cerebellar degeneration and it is currently unclear which of the many functions of the ATM protein is the one whose loss is specifically responsible for this devastating symptom.We're trying to investigate this central problem in A-T research and for this we are utilizing cerebellar organotypic cultures derived from wild-type and ATM-deficient mice. They allow us to focus on the cell type that is probably the first to be lost in the degenerating cerebellum of A-T patients, Purkinje cells. Like many fields in life sciences research in A-T leveraging a variety of advanced techniques and methods.Those methods include animal models, novel types of tissue cultures, induced polyploid stem cells, cutting-edge imaging technologies, induced omic methods and single cell partials imaging. The source of cerebellar deterioration in A-T is deterioration of Purkinje cells. Those cells are notoriously difficult to isolate and cannot survive in cultures without supporting of other cell types.A unique experimental challenge is obtaining mature, functioning human Purkinje cells using induced polyploid stem cell technology. Since growing isolated Purkinje cells is currently impossible, cerebellar organotypic cultures provide valuable opportunity to study those cells in a culture dish within the natural tissue context. Using protein correlation as a readout for DNA damage response is highly informative, particularly concerning Purkinje cells.A major function of the ATM protein kinase is regulating the DNA damage response, which is critical for maintaining genome integrity and cellular homeostasis in the face of the ongoing DNA damage which occurs in every cell. We need to pinpoint the critical DNA lesion whose repair is defective in ATM-deficient Purkinje cells and we hope to do this using the cerebellar organotypic cultures. This should allow us to understand better the physiological and molecular basis of the most important and devastating symptom of A-T, the cerebellar degeneration.

Treatment of Cerebellar Organotypic Cultures with DNA Damaging Agents for Fluorescent Imaging of PAR Chains to Assess Genotoxic Stress Response

To begin, culture the chopped cerebellar tissue retrieved from 10 day-old mouse pup in basal medium eagle. Add DNA damaging chemicals directly to the culture medium at appropriate final concentrations. After the exposure period, replace the medium with a freshly prepared mixed medium containing the DNA damaging chemicals and the poly ADP ribose glycohydrolase inhibitor, and incubate for 30 minutes.After 30 minutes, wash the slices with 0.1 molar phosphate buffer at room temperature and immediately fix them with a pre-cooled acetone and methanol mixture. Incubate the plate for 20 minutes at minus 20 degrees Celsius. Then remove the fixation solution and wash it with 0.4 molar phosphate buffer.Add 100 microliters of 0.1 molar phosphate buffer to each well of a 48 well plate. Then use a scalpel and a cutting board to remove the margins of the insert, cutting around the tissue slice. Place the slice in a well of the 48 well plate.After aspirating the buffer, incubate the insert with 100 microliters of 0.1 molar phosphate buffer containing 1%Triton X100. Aspirate the solution and serially incubate the insert in each well on a shaker with appropriate amounts of blocking solution followed by the primary in the secondary antibodies. Add 20 microliters of the final DAPI solution to each well 20 minutes before the end of the two hour incubation with the secondary antibody and continue shaking for 20 minutes.For mounting, place the tissue on a microscope glass slide with the tissue facing up. Add the mounting medium and cover with a cover glass. Place the slides on a flat surface and let them dry overnight in the dark at four degrees Celsius.The cerebellar foliation was maintained in the culture. Purkinje cells were stained for Calbindin D-28K and neural nuclei stained for NeuN. The astrocytes in the Purkinje cells were stained for GFAP.PAR staining increased in treated Purkinje cells after exposure to potassium bromate and paraquat indicating DNA damage.

treatment-cerebellar-organotypic-cultures-with-dna-damaging-agents

Research

JoVE Journal

Biology

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.

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.

Cerebellar Purkinje cells (PCs) exhibit a unique interplay of high metabolic rates, specific chromatin architecture, and extensive transcriptional ...

Isolation of Cerebellum from Mouse Pups for Obtaining Organotypic Cultures to Study DNA Damage Response

To begin, add one milliliter of basal medium eagle to each well of the six-well plate. And using sterile forceps, place a single cell culture insert into each well. Incubate the plate in a 37 degrees Celsius, 5%carbon dioxide incubator for a minimum of two hours.After setting up the dissection area within a biological hood, wash the surgical tools in 70%ethanol, followed by PBS. Then, position the euthanized animal in the dissection area and make a small incision with scissors away from the cerebellum to expose the brain. Using tweezers with rounded ends, gently peel off the skull and remove the skull in small pieces to avoid damaging the cerebellar tissue.Expose the brain tissue to extract the cerebellum and disconnect the three pairs of peduncles using a fine curved iris spatula to separate the cerebellar cortex from the brain stem. Then, slide the spatula between the cerebellum, the superior colliculus, and the brain stem to retrieve the cerebellum.

Preparation of Cerebellar Organotypic Cultures for Investigating DNA Damage Response in Purkinje Cells

To begin, obtain the cerebellum from a 10-day old mouse pup in cold dissecting buffer. After removing the excess buffer, position the cerebellum on the cutting platform in a vertical orientation. Carry out parasagittal slicing at a slice thickness of 350 micrometers.Gently separate the slices with a fine iris spatula and place them into cold dissection medium. 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 corticonuclear zone of the organ.Using a wide spatula, transfer each tissue slice onto a culture insert. Move each insert carrying a tissue slice into a well in the six-well plate containing the pre-warmed medium. During the incubation, aspirate the used medium with a sterile glass pipette.Using a five milliliter serological pipette, add one milliliter of fresh basal medium eagle with the pipette's tip leaning against the well's wall without disturbing the tissue.