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 pluripotent 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 pluripotent stem cell technology.
Since growing isolated Purkinje cells is currently impossible, cellular organotypic cultures provide valuable opportunity to study those cells in a culture dish within their 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 these 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. 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 degree 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 brainstem.
Then, slide the spatula between the cerebellum, the superior colliculus, and the brainstem to retrieve the cerebellum. 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. 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 X-100.
Aspirate the solution and serially incubate the insert in each well on a shaker with appropriate amounts of blocking solution followed by the primary and 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 neuronal 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.
