Targeted Knockdown of Genes in the Choroid Plexus

Summary

Here, we describe a method to selectively alter gene expressions in the choroid plexus while avoiding any impact in other brain areas.

Abstract

The choroid plexus (ChP) serves as a critical gateway for immune cell infiltration into the central nervous system (CNS) under both physiological and pathological conditions. Recent research has shown that regulating ChP activity may offer protection against CNS disorders. However, studying the biological function of the ChP without affecting other brain regions is challenging due to its delicate structure. This study presents a novel method for gene knockdown in ChP tissue using adeno-associated viruses (AAVs) or cyclization recombination enzyme (Cre) recombinase protein consisting of TAT sequence (CRE-TAT). The results demonstrate that after injecting AAV or CRE-TAT into the lateral ventricle, the fluorescence was exclusively concentrated in the ChP. Using this approach, the study successfully knocked down the adenosine A_2A receptor (A_2AR) in the ChP using RNA interference (RNAi) or Cre/locus of X-overP1 (Cre/LoxP) systems, and showed that this knockdown could alleviate the pathology of experimental autoimmune encephalomyelitis (EAE). This technique may have important implications for future research on the ChP's role in CNS disorders.

Introduction

The choroid plexus (ChP) was often thought to help maintain brain functional homeostasis by secreting cerebrospinal fluid (CSF) and brain-derived neurotrophic factor (BDNF)^1,2. Increasing research over the last three decades has revealed that the ChP represents a distinct pathway for immune cell infiltration into the central nervous system (CNS).

The tight junctions (TJs) of the ChP, composed of a monolayer ChP epithelium, maintain immunological homeostasis by preventing macromolecules and immune cells from entering the brain³. However, under certain pathological conditions, the ChP tissue detects and responds to danger-associated molecular patterns (DAMPs) in the CSF and blood, leading to abnormal immune infiltration and brain dysfunction^4,5. Despite its critical role, the ChP's small size and unique location in the brain make it difficult to study its function without affecting other brain regions. Therefore, manipulating gene expression specifically in the ChP is an ideal approach to understanding its function.

Initially, cyclization recombination enzyme (Cre) transgenic lines, which express Cre under the control of promoters specific to genes expressed in the ChP, were commonly used to delete target genes by breeding with floxed candidate genes^6,7,8. For example, the transcription factor Forkhead box J1 (FoxJ1) is exclusively expressed in the ChP epithelium of the prenatal mouse brain⁷. Thus, the FoxJ1-Cre line was often used to delete genes located in the ChP^6,9. However, the success of this strategy relies heavily on the specificity of the promoter. It was gradually discovered that the FoxJ1 expression pattern was not distinctive enough, as FoxJ1 was also present in ciliated epithelial cells in other parts of the brain and peripheral system⁷. To overcome this limitation, intra-cerebroventricular (ICV) injection of Cre recombinase was performed to deliver recombinase into the ventricles of floxed transgenic lines. This strategy showed high specificity, as evidenced by the presence of tdTomato fluorescence solely in the ChP tissue^10,11. However, this method is still limited by the availability of floxed transgenic mouse lines. To address this issue, researchers have employed ICV injection of adeno-associated virus (AAV) to achieve ChP-specific knockdown or the overexpression of target genes^12,13. A comprehensive evaluation of different AAV serotypes for ChP infection revealed that AAV2/5 and AAV2/8 exhibit strong infection abilities in the ChP, while not infecting other brain regions. However, AAV2/8 was found to infect the ependyma surrounding ventricles, whereas the AAV2/5 group showed no infection¹⁴. This method has the advantage of overcoming the limitations of acquiring floxed transgenic animals.

This article describes a step-by-step protocol for gene knockdown in the ChP using two methods: ICV of AAV2/5 carrying shRNA of the adenosine A_2A receptor (A_2AR) and Cre recombinase protein consisting of TAT sequence (CRE-TAT) recombinase to achieve ChP-specific knockdown of A_2AR. The study findings suggest that knocking down A_2AR in the ChP can alleviate experimental autoimmune encephalomyelitis (EAE). This detailed protocol provides useful guidance for ChP function studies and the specific knockdown of genes in the ChP.

Protocol

All animal procedures described in this study were conducted in accordance with the guidelines outlined in the NIH Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee at Wenzhou Medical University.

1. Animals

1. Purchase male C57BL/6 mice aged 8-12 weeks and weighing 20-22 g.
2. Obtain the transgenic Rosa-LSL (Lox-StoP-Lox)-tdTomato (Ai9) mouse line, and male A_2AR^flox/flox mice.
3. Randomly assign the mice to two groups and house in cages, with a maximum of five mice per cage, under a standard 12 h light/12 h dark cycle for 1 week.
4. Provide the mice with sufficient food and water and maintain them at a constant temperature at 25 °C.

2. ChP-specific knockdown of A_2ARs with AAV2/5-shRNA

1. Weigh each mouse and write down the values.
2. Anesthetize the mice intraperitoneally with 1% pentobarbital sodium at a dosage of 6-8 mL/kg equivalent to 60-80 mg/kg pentobarbital, and place the mouse on a heating pad to keep it warm.
   NOTE: It is crucial to administer the correct dosage of pentobarbital sodium according to the mouse's weight to ensure successful surgery. The anesthesia should not be too deep, as it may result in mortality, but should not be too shallow, as the mouse may wake up during the procedure, potentially affecting the effectiveness of the virus injection. Check for the proper anesthesia dose by toe pinch and ensuring that mice do not respond.
3. Use a specialized mouse shaver to trim the top hair of the anesthetized mice.
4. Apply eye ointment on both eyes to prevent from drying, and fix the mice onto a stereotaxic apparatus to immobilize the brain. Cover the animal with a sterile drape.
5. Thoroughly sterilize the skin of the head and neck of the mice with three rounds of iodophor disinfectant and 75% alcohol to reduce postoperative infection. Then apply 1% lidocaine topically to the scalp of mice and make a small incision to fully expose the skull under a microscope.
   NOTE: Use sterile instruments throughout the procedure.
6. Prepare two 10 µL syringes: one with purchased AAV2/5-A_2AR-shRNA (titer: 6.27 × 10⁹ vg/µL) and the other with purchased AAV2/5-Scramble (titer: 6.21 × 10⁹ vg/µL).
   NOTE: When using the syringe, the front end needs to be connected to a glass capillary, and a range of 10 µL can be obtained by controlling the length of the glass capillary.
7. Use the microscope to locate the bregma and lambda and set the coordinate point (AP: -0.58; ML: ±1.10; DL: -2.20) on the 10 µL syringe (Figure 1). The coordinate point is based on the mouse brain stereotaxic atlas¹⁵.
8. Drill a small hole in the skull at the adjusted coordinate point.
   NOTE: Under the microscope, drilling is performed using a sterile microdrill against the surface of the skull. After drilling, by removing the meninges covering the brain and exposing the underlying brain parenchyma, injury to the blood vessels is prevented. This step prevents the tip of the glass capillary from being broken off by the meninges. The diameter of the drilled hole should be sufficient to allow the needle tip to enter the brain tissue.
9. Administer 2 µL of AAV2/5-A_2AR-shRNA virus into the brain ventricle via lateral injection at a steady rate of 100 nL/min. Keep the needle in place for 10 min before withdrawing. Note that the virus was administered with unilateral injection.
10. Seal the injured skin using medical biofibrin glue promptly to prevent virus loss, which can be rinsed out with CSF after removing the needle if the glue is not applied quickly. Also, do not use cotton buds to wipe the CSF, as it may also cause virus loss.
11. To facilitate recovery, place the mouse on a heating pad to maintain the body temperature at 37.0 ± 0.5 °C, and apply 1% lidocaine topically to the mouse's scalp. Allow the mice to recuperate for 2 weeks before inducing the EAE model, as this period is necessary for AAV2/5-A_2AR-shRNA virus expression and subsequent A_2AR knockdown.

3. ChP-specific knockdown of A_2AR with a Cre/locus of X-overP1 (Cre/LoxP) system

NOTE: The following procedures can be achieved using the method described previously. Refer to steps 2.1-2.11 for detailed injection methods.

1. Inject 2 µL of CRE-TAT recombinase into each lateral ventricle of a Rosa-LSL (Lox-StoP-Lox)-tdTomato mouse as the experimental group and 2 µL of sterile phosphate-buffered saline (PBS) as the control group. Using the same protocol as above (section 2), inject 2 µL of CRE-TAT recombinase into each of the lateral ventricles of A_2AR^flox/flox mice along with 2 µL of sterile PBS as a control group.
2. Prepare frozen tissue sections and perform nuclear staining 2 weeks after the CRE-TAT recombinase injection. See steps 4.1-4.3 for more information.

4. Transcardial perfusion in mice

1. Administer 60-80 mg/kg pentobarbital sodium to deeply anesthetize the mice. Confirm the plane of anesthesia by a toe pinch and perform transcardial perfusion using 40 mL of sterile PBS solution followed by 20 mL of 4% paraformaldehyde (PFA).
   NOTE: Successful perfusion is indicated by a white color in the liver, while the presence of enlarged lungs or PBS outflow from the mouth during perfusion suggests a failure of the procedure.
2. Quickly extract the mouse brain, ensuring minimal protein degradation.
3. Submerge the mouse brain in 4% PFA/PBS overnight for post-fixation, followed by replacement with 30% sucrose PB solution for 72 h.
   ​NOTE: It is critical to avoid excessive dehydration of the brain tissue, so the brain should not be left in the sucrose PB solution for too long.

5. Frozen tissue sectioning and staining

1. Embed the previously dehydrated mouse brain in optimal cutting temperature (OCT) glue, freeze the embedded brain, and use a sliding microtome to cut coronal sections with a thickness of 20 µm.
2. Place the brain sections on the glass slide. Once the brain sections are dry, store them in a -20 °C refrigerator for subsequent experiments.
3. Place each glass slide into a frame case and submerge the frame into a container filled with PBS to thoroughly rinse the slide. Gently rinse the brain slides with PBS solution three times, for 10 min each time.
   NOTE: Caution should be exercised to prevent the brain sections from falling off the glass slide.
4. Stain the brain slides with 4′,6-diamidino-2-phenylindole (DAPI) solution for 10 min.
5. Rinse the brain slides with PBS solution for 5 min.
6. Apply one drop of antifade mounting medium to the brain sections.
7. Place a coverslip on the brain sections, seal the coverslip with nail polish, and analyze the sections using a conventional fluorescence microscope.

6. EAE induction

NOTE: Perform EAE induction after 2 weeks of the shRNA or CRE-TAT recombinase injection¹¹.

1. Create an aqueous solution by mixing 2.5 mg myelin oligodendrocyte glycoprotein (MOG_35-55) with 2 mL of PBS. Prepare a complete Freunds adjuvant (CFA) oil solution by mixing M. tuberculosis (H37Ra) with incomplete Freunds adjuvant (IFA).
2. Mix the aqueous and oil solutions in a 1:1 ratio. Use a tee pipe to whip the mixture into an oil-in-water state.
   NOTE: The tee pipe is also called a three-way pipe. It is a plastic tube that can control the direction of flowing solution to fully mix the MOG_35-55 and CFA.
3. Use a high-speed homogenizer to make the MOG antigen emulsion for the EAE model under ice bath conditions.
4. Anesthetize the mice intraperitoneally with 1% pentobarbital sodium at a dosage of 6-8 mL/kg equivalent to 60-80 mg/kg pentobarbital.
5. Subcutaneously inject the MOG antigen emulsion into four different points (neck, back, left and right hips) at a volume of 10 mL/kg for a total of four injections each.
   NOTE: It is important to carefully select the injection site, as different sites may have varying effects on the morbidity and mortality of mice. Additionally, repeated MOG injections can lead to immune tolerance, so the research team chose a single injection method to prevent this potential issue.
6. Immediately inject 500 ng/mL of pertussis toxin (PT) intraperitoneally at a dosage of 5 mg/kg after MOG injection.
   NOTE: PT increases the permeability of the blood-brain barrier (BBB) and facilitates the infiltration of T cells into the brain.
7. After 48 h, intraperitoneally inject the PT solution at the same volume.

7. Neurological deficit score

1. Evaluate and grade the mice daily using a rating scale¹⁶ ranging from 0 to 15 to assess the incidence and severity of EAE according to the following neurological deficits:
   Tail: 0 indicates no signs, 1 represents a half-paralyzed tail, and 2 indicates a fully paralyzed tail.
   Limbs: 0 indicates no signs, 1 represents a weak or altered gait, 2 indicates paresis, and 3 represents a fully paralyzed limb.
2. Assign a quadriplegic animal having complete paralysis a score of 12, and assign mortality a score of 15.

8. Hematoxylin-eosin (H&E) staining

1. Take the dehydrated mouse brain and embed it in molten paraffin. Allow the paraffin block to cool and solidify for later use.
   NOTE: The paraffin block should be completely dry and cool to avoid tissue damage.
2. Cut the mouse brain paraffin block into 5 µm thick slides. Place the paraffin mouse brain section on a glass slide and dry it in a 60 °C oven for 3 h.
3. Submerge the slides sequentially in xylene solution I for 10 min, xylene solution II for 10 min, 100% alcohol I for 3 min, 100% alcohol II for 3 min, 95% alcohol for 3 min, 90% alcohol for 3 min, 80% alcohol for 3 min, 70% alcohol for 3 min, and distilled water for 1 min.

9. Quantitative polymerase chain reaction (qPCR) analysis

1. After perfusing the mice with PBS, remove the ChP from the ventricles and extract RNA with Trizol. Synthesize the cDNA using the first Strand cDNA Synthesis Kit.
2. Carry out qPCR analysis using Ex Taq SYBR-green premix and a real-time PCR system. Use the following A_2AR primers: forward - GCCATCCCATTCGCCATCA; reverse - GCAATAGCCAAGAGGCTGAAGA.

Results

ChP-specific A_2AR knockdown by ICV injection of AAV2/5-shRNA or CRE-TAT
The role of A_2AR in the ChP as a powerful regulator of neural information in EAE pathogenesis remains unclear. Knocking down ChP-specific A_2AR expression could shed light on the A_2AR regulatory effects on the central immune system in EAE and other nervous system inflammations. This study used ICV injection of CRE-TAT to decrease A_2AR expression in the ChP of A_2AR...

Discussion

The research presented two distinct approaches for the targeted knockdown of ChP genes. The first approach involved the ICV injection of CRE-TAT, which contains Cre recombinase, into A_2AR^flox/flox mice. The second approach entailed ICV injection of AAV2/5 carrying shRNA of A_2AR. By utilizing these two strategies, the work achieved the selective knockdown of A_2AR within the ChP and was able to demonstrate the protective effects of inhibiting A_2AR signaling in the ChP ...

Materials

Name	Company	Catalog Number	Comments
A2ARflox/flox mice	State Key Laboratory of Ophthalmology, Optometry and Visual Science, Wenzhou Medical University
AAV2/5-A2AR-ShRNA virus	Shanghai Heyuan Biotechnology Co. LTD	pt-4828
antifade mounting medium	Beyotime Biotechnology	0100-01
borosilicate glass capillary	Beijing Meiyaxian Technology Co. Ltd	B100-50-10
brain stereotaxic apparatus	RWD, Shenzhen	69100
C57BL/6 mice	Beijing Vital Charles River Laboratory Animal Technology Company
CRE-TAT recombinase	Millipore	SCR508
DAPI	Absin	B25A031
frozen slicing machine	Leica	CM1950
H37Ra	Becton Dickinson and company	231141
Hamilton syringe	Hamilton, American	P/N: 86259
Incomplete Freunds adjuvant	Sigma	F5506
Laser confocal microscope	Zeiss	LSM900
MOG35-55	Suzhou Qiangyao Biotechnology Co., LTD	4010006243
OCT glue	Epredia	6502p
paraformaldehyde	Chengdu Kelong Chemical Reagent Company	30525-89-4
pentobarbital sodium	Boyun Biotech	PC13003
Pipette gun	Eppendorf	N45014F
PrimeScript 1st Strand cDNA Synthesis Kit	Takara	6110A
Real- Time PCR System	BioRad	CFX96
Rosa-LSL (Lox-StoP-Lox)-tdTomato mice	Jackson Laboratory
sucrose	Sangon Biotech	A502792-0500
super high speed homogenizer	IKA	3737025
Trizol	Invitrogen	15596026
xylene solution	Chengdu Kelong Chemical Reagent Company	1330-20-7