We gratefully acknowledge the support of the National Natural Science Foundation of China (Grant No. 31800903, awarded to W. Zheng) and the Wenzhou Science and Technology Project (no. Y2020426, awarded to Y. Y. Weng) for this work.

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
<ol>
	<li>Purchase male C57BL/6 mice aged 8-12 weeks and weighing 20-22 g.</li>
	<li>Obtain the transgenic Rosa-LSL (Lox-StoP-Lox)-tdTomato (Ai9) mouse line, and male A2ARflox/flox mice.</li>
	<li>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.</li>
	<li>Provide the mice with sufficient food and water and maintain them at a constant temperature at 25 &#176;C.</li>
</ol>
2. ChP-specific knockdown of A2ARs with AAV2/5-shRNA
<ol>
	<li>Weigh each mouse and write down the values.</li>
	<li>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&#39;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.</li>
	<li>Use a specialized mouse shaver to trim the top hair of the anesthetized mice.</li>
	<li>Apply&#160;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.</li>
	<li>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.</li>
	<li>Prepare two 10 &#181;L syringes: one with purchased AAV2/5-A2AR-shRNA (titer: 6.27 &#215; 109 vg/&#181;L) and the other with purchased AAV2/5-Scramble (titer: 6.21 &#215; 109 vg/&#181;L). 
	NOTE: When using the syringe, the front end needs to be connected to a glass capillary, and a range of 10 &#181;L can be obtained by controlling the length of the glass capillary.</li>
	<li>Use the microscope to locate the bregma and lambda and set the coordinate point (AP: -0.58; ML: &#177;1.10; DL: -2.20) on the 10 &#181;L syringe (Figure 1). The coordinate point is based on the mouse brain stereotaxic atlas15.</li>
	<li>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.</li>
	<li>Administer 2 &#181;L of AAV2/5-A2AR-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.</li>
	<li>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.</li>
	<li>To facilitate recovery, place the mouse on a heating pad to maintain the body temperature at&#160;37.0 &#177; 0.5 &#176;C, and apply 1% lidocaine topically to the mouse&#39;s scalp. Allow the mice to recuperate for 2 weeks before inducing the EAE model, as this period is necessary for AAV2/5-A2AR-shRNA virus expression and subsequent A2AR knockdown.</li>
</ol>
3. ChP-specific knockdown of A2AR 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.
<ol>
	<li>Inject 2 &#181;L of CRE-TAT recombinase into each lateral ventricle of a Rosa-LSL (Lox-StoP-Lox)-tdTomato mouse as the experimental group and 2 &#181;L of sterile phosphate-buffered saline (PBS) as the control group. Using the same protocol as above (section 2), inject 2 &#181;L of CRE-TAT recombinase into each of the lateral ventricles of A2ARflox/flox mice along with 2 &#181;L of sterile PBS as a control group.</li>
	<li>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.</li>
</ol>
4. Transcardial perfusion in mice
<ol>
	<li>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.</li>
	<li>Quickly extract the mouse brain, ensuring minimal protein degradation.</li>
	<li>Submerge the mouse brain in 4% PFA/PBS overnight for post-fixation, followed by replacement with 30% sucrose PB solution for 72 h. 
	&#8203;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.</li>
</ol>
5. Frozen tissue sectioning and staining
<ol>
	<li>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 &#181;m.</li>
	<li>Place the brain sections on the glass slide. Once the brain sections are dry, store them in a -20 &#176;C refrigerator for subsequent experiments.</li>
	<li>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.</li>
	<li>Stain the brain slides with 4&#8242;,6-diamidino-2-phenylindole (DAPI) solution for 10 min.</li>
	<li>Rinse the brain slides with PBS solution for 5 min.</li>
	<li>Apply one drop of antifade mounting medium to the brain sections.</li>
	<li>Place a coverslip on the brain sections, seal the coverslip with nail polish, and analyze the sections using a conventional fluorescence microscope.</li>
</ol>
6. EAE induction
NOTE: Perform EAE induction after 2 weeks of the shRNA or CRE-TAT recombinase injection11.
<ol>
	<li>Create an aqueous solution by mixing 2.5 mg myelin oligodendrocyte glycoprotein (MOG35-55) with 2 mL of PBS. Prepare a complete Freunds adjuvant (CFA) oil solution by mixing M. tuberculosis (H37Ra) with incomplete Freunds adjuvant (IFA).</li>
	<li>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 MOG35-55 and CFA.</li>
	<li>Use a high-speed homogenizer to make the MOG antigen emulsion for the EAE model under ice bath conditions.</li>
	<li>Anesthetize the mice intraperitoneally with 1% pentobarbital sodium at a dosage of 6-8 mL/kg equivalent to 60-80 mg/kg pentobarbital.</li>
	<li>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.</li>
	<li>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.</li>
	<li>After 48 h, intraperitoneally inject the PT solution at the same volume.</li>
</ol>
7. Neurological deficit score
<ol>
	<li>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.</li>
	<li>Assign a quadriplegic animal having complete paralysis&#160;a score of 12, and assign mortality a score of 15.</li>
</ol>
8. Hematoxylin-eosin (H&#38;E) staining
<ol>
	<li>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.</li>
	<li>Cut the mouse brain paraffin block into 5 &#181;m thick slides. Place the paraffin mouse brain section on a glass slide and dry it in a 60 &#176;C oven for 3 h.</li>
	<li>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.</li>
</ol>
9. Quantitative polymerase chain reaction (qPCR) analysis
<ol>
	<li>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.</li>
	<li>Carry out qPCR analysis using Ex Taq SYBR-green premix and a real-time PCR system. Use the following A2AR primers: forward - GCCATCCCATTCGCCATCA; reverse - GCAATAGCCAAGAGGCTGAAGA.</li>
</ol>

Selective Gene Silencing in the Choroid Plexus

<ol>
	<li>Damkier, H. H., Brown, P. D., Praetorius, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebrospinal+fluid+secretion+by+the+choroid+plexus.">Cerebrospinal fluid secretion by the choroid plexus.</a> Physiological Reviews. 93 (4), 1847-1892 (2013).</li><li>Lun, M. P., Monuki, E. S., Lehtinen, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+functions+of+the+choroid+plexus-cerebrospinal+fluid+system.">Development and functions of the choroid plexus-cerebrospinal fluid system.</a> Nature Reviews: Neuroscience. 16 (8), 445-457 (2015).</li><li>Wolburg, H., Paulus, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choroid+plexus:+biology+and+pathology.">Choroid plexus: biology and pathology.</a> Acta Neuropathologica. 119 (1), 75-88 (2010).</li><li>Solar, P., Zamani, A., Kubickova, L., Dubovy, P., Joukal, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choroid+plexus+and+the+blood-cerebrospinal+fluid+barrier+in+disease.">Choroid plexus and the blood-cerebrospinal fluid barrier in disease.</a> Fluids Barriers CNS. 17 (1), 35 (2020).</li><li>Marques, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+choroid+plexus+in+health+and+in+disease:+dialogues+into+and+out+of+the+brain.">The choroid plexus in health and in disease: dialogues into and out of the brain.</a> Neurobiology of Disease. 107, 32-40 (2017).</li><li>Myung, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+choroid+plexus+is+an+important+circadian+clock+component.">The choroid plexus is an important circadian clock component.</a> Nature Communications. 9 (1), 1062 (2018).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transgenic+FOXJ1-Cre+system+for+gene+inactivation+in+ciliated+epithelial+cells.">A transgenic FOXJ1-Cre system for gene inactivation in ciliated epithelial cells.</a> American Journal of Respiratory Cell and Molecular Biology. 36 (5), 515-519 (2007).</li><li>Johansson, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcription+factor+Otx2+regulates+choroid+plexus+development+and+function.">The transcription factor Otx2 regulates choroid plexus development and function.</a> Development. 140 (5), 1055-1066 (2013).</li><li>Xu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choroid+plexus+NKCC1+mediates+cerebrospinal+fluid+clearance+during+mouse+early+postnatal+development.">Choroid plexus NKCC1 mediates cerebrospinal fluid clearance during mouse early postnatal development.</a> Nature Communications. 12 (1), 447 (2021).</li><li>Spatazza, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choroid-plexus-derived+Otx2+homeoprotein+constrains+adult+cortical+plasticity.">Choroid-plexus-derived Otx2 homeoprotein constrains adult cortical plasticity.</a> Cell Reports. 3 (6), 1815-1823 (2013).</li><li>Zheng, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choroid+plexus-selective+inactivation+of+adenosine+A2A+receptors+protects+against+T+cell+infiltration+and+experimental+autoimmune+encephalomyelitis.">Choroid plexus-selective inactivation of adenosine A2A receptors protects against T cell infiltration and experimental autoimmune encephalomyelitis.</a> Journal of Neuroinflammation. 19 (1), 52 (2022).</li><li>Steffensen, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cotransporter-mediated+water+transport+underlying+cerebrospinal+fluid+formation.">Cotransporter-mediated water transport underlying cerebrospinal fluid formation.</a> Nature Communications. 9 (1), 2167 (2018).</li><li>Zhu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Klotho+controls+the+brain-immune+system+interface+in+the+choroid+plexus.">Klotho controls the brain-immune system interface in the choroid plexus.</a> Proceedings of the National Academy of Sciences. 115 (48), E11388-E11396 (2018).</li><li>Chen, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+serotypes+of+adeno-associated+virus+vector-+and+lentivirus-mediated+tropism+in+choroid+plexus+by+intracerebroventricular+delivery.">Different serotypes of adeno-associated virus vector- and lentivirus-mediated tropism in choroid plexus by intracerebroventricular delivery.</a> Human Gene Therapy. 31 (7-8), 440-447 (2020).</li><li>Konsman, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+brain+in+stereotaxic+coordinates.">The mouse brain in stereotaxic coordinates.</a> Psychoneuroendocrinology. 6 (28), 827-828 (2003).</li><li>Weaver, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+elevated+matrix+metalloproteinase+(MMP)+in+an+animal+model+of+multiple+sclerosis+is+protective+by+affecting+Th1/Th2+polarization.">An elevated matrix metalloproteinase (MMP) in an animal model of multiple sclerosis is protective by affecting Th1/Th2 polarization.</a> FASEB J. 19 (12), 1668-1670 (2005).</li><li>Kertser, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corticosteroid+signaling+at+the+brain-immune+interface+impedes+coping+with+severe+psychological+stress.">Corticosteroid signaling at the brain-immune interface impedes coping with severe psychological stress.</a> Science Advances. 5 (5), 4111 (2019).</li><li>Kaiser, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MEIS-WNT5A+axis+regulates+development+of+fourth+ventricle+choroid+plexus.">MEIS-WNT5A axis regulates development of fourth ventricle choroid plexus.</a> Development. 148 (10), (2021).</li><li>Compston, A., Coles, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+sclerosis.">Multiple sclerosis.</a> Lancet. 372 (9648), 1502-1517 (2008).</li><li>Reboldi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C-C+chemokine+receptor+6-regulated+entry+of+TH-17+cells+into+the+CNS+through+the+choroid+plexus+is+required+for+the+initiation+of+EAE.">C-C chemokine receptor 6-regulated entry of TH-17 cells into the CNS through the choroid plexus is required for the initiation of EAE.</a> Nature Immunology. 10 (5), 514-523 (2009).</li><li>Jovanova-Nesic, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choroid+plexus+connexin+43+expression+and+gap+junction+flexibility+are+associated+with+clinical+features+of+acute+EAE.">Choroid plexus connexin 43 expression and gap junction flexibility are associated with clinical features of acute EAE.</a> Annals of the New York Academy of Sciences. 1173, 75-82 (2009).</li><li>Jovanova-Nesic, K., Jovicic, S., Sovilj, M., Spector, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+brain+stimulation+upregulates+adhesion+and+prevents+Eae:+MMP-2,+ICAM-1,+and+VCAM-1+in+the+choroid+plexus+as+a+target.">Magnetic brain stimulation upregulates adhesion and prevents Eae: MMP-2, ICAM-1, and VCAM-1 in the choroid plexus as a target.</a> International Journal of Neuroscience. 119 (9), 1399-1418 (2009).</li><li>Mills, J. H., Alabanza, L. M., Mahamed, D. A., Bynoe, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+adenosine+signaling+induces+CX3CL1+expression+in+the+brain+to+promote+experimental+autoimmune+encephalomyelitis.">Extracellular adenosine signaling induces CX3CL1 expression in the brain to promote experimental autoimmune encephalomyelitis.</a> Journal of Neuroinflammation. 9, 193 (2012).</li></ol>

The authors state that they have no competing financial interests or other disclosures to declare.

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 A2ARflox/flox mice. The second approach entailed ICV injection of AAV2/5 carrying shRNA of A2AR. By utilizing these two strategies, the work achieved the selective knockdown of A2AR within the ChP and was able to demonstrate the protective effects of inhibiting A2AR signaling in the ChP ...

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 dysfunction4,5. Despite its critical role, the ChP&#39;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 genes6,7,8. 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 ChP6,9. 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 tissue10,11. 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 genes12,13. 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 A2A receptor (A2AR) and Cre recombinase protein consisting of TAT sequence (CRE-TAT) recombinase to achieve ChP-specific knockdown of A2AR. The study findings suggest that knocking down A2AR 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>A2ARflox/flox mice</td>
			<td>State Key Laboratory of Ophthalmology, Optometry and Visual Science, Wenzhou Medical University</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AAV2/5-A2AR-ShRNA virus</td>
			<td>Shanghai Heyuan Biotechnology Co. LTD</td>
			<td>pt-4828</td>
			<td></td>
		</tr>
		<tr>
			<td>antifade mounting medium</td>
			<td>Beyotime Biotechnology</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>borosilicate glass capillary</td>
			<td>Beijing Meiyaxian Technology Co. Ltd</td>
			<td>B100-50-10</td>
			<td></td>
		</tr>
		<tr>
			<td>brain stereotaxic apparatus</td>
			<td>RWD, Shenzhen</td>
			<td>69100</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 mice</td>
			<td>Beijing Vital Charles River Laboratory Animal Technology Company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CRE-TAT recombinase</td>
			<td>Millipore</td>
			<td>SCR508</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Absin</td>
			<td>B25A031</td>
			<td></td>
		</tr>
		<tr>
			<td>frozen slicing machine</td>
			<td>Leica</td>
			<td>CM1950</td>
			<td></td>
		</tr>
		<tr>
			<td>H37Ra</td>
			<td>Becton Dickinson and company</td>
			<td>231141</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe</td>
			<td>Hamilton, American</td>
			<td>P/N:&#160;86259</td>
			<td></td>
		</tr>
		<tr>
			<td>Incomplete Freunds adjuvant</td>
			<td>Sigma</td>
			<td>F5506</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser confocal microscope</td>
			<td>Zeiss</td>
			<td>LSM900</td>
			<td></td>
		</tr>
		<tr>
			<td>MOG35-55</td>
			<td>Suzhou Qiangyao Biotechnology Co., LTD</td>
			<td>4010006243</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT glue</td>
			<td>Epredia</td>
			<td>6502p</td>
			<td></td>
		</tr>
		<tr>
			<td>paraformaldehyde</td>
			<td>Chengdu Kelong Chemical Reagent Company</td>
			<td>30525-89-4</td>
			<td></td>
		</tr>
		<tr>
			<td>pentobarbital sodium</td>
			<td>Boyun Biotech</td>
			<td>PC13003</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette gun</td>
			<td>Eppendorf</td>
			<td>N45014F</td>
			<td></td>
		</tr>
		<tr>
			<td>PrimeScript 1st Strand cDNA Synthesis Kit</td>
			<td>Takara</td>
			<td>&#160;6110A</td>
			<td></td>
		</tr>
		<tr>
			<td>Real- Time PCR System</td>
			<td>BioRad</td>
			<td>CFX96</td>
			<td></td>
		</tr>
		<tr>
			<td>Rosa-LSL (Lox-StoP-Lox)-tdTomato mice</td>
			<td>Jackson Laboratory</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sucrose</td>
			<td>Sangon Biotech</td>
			<td>A502792-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>super high speed homogenizer</td>
			<td>IKA</td>
			<td>3737025</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Invitrogen</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr>
			<td>xylene solution</td>
			<td>Chengdu Kelong Chemical Reagent Company</td>
			<td>1330-20-7</td>
			<td></td>
		</tr>
	</tbody>
</table>

targeted knockdown of genes in the choroid plexus

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 A2A receptor (A2AR) 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.

Author Spotlight: Insights and Innovations in Gene Expression Manipulation Techniques for Choroid Plexus Research 

Targeted Knockdown of Genes in the Choroid Plexus

The objective of this research is to investigate the techniques for manipulating gene expression in the choroid plexus. Our study has introduced the two methods that allow for technique exclusively in the choroid plexus without any discernible impact on other regions. Our findings can potentially enhance future function studies of the choroid plexus in neuroscience.Our research has reviewed that inhibiting T-cell infiltration across the choroid plexus through selective lockdown of adenosine to a receptor is sufficient to protect mice from EAE pathology. This finding supports the notion that detecting gateway activity in the choroid plexus could present a promising new strategy for treating multiple sclerosis. This study presents two alternative methods for gene expression manipulation in the choroid plexus.ICV injection of Cre recombinase into those transgenic mice or ICV injection of AAV2/5, or it's targeted SHI in the WT mice. These methods overcome issues with promoting specificity and transgenetic mouse breeding.

ChP-specific A2AR knockdown by ICV injection of AAV2/5-shRNA or CRE-TAT 
The role of A2AR in the ChP as a powerful regulator of neural information in EAE pathogenesis remains unclear. Knocking down ChP-specific A2AR expression could shed light on the A2AR regulatory effects on the central immune system in EAE and other nervous system inflammations. This study used ICV injection of CRE-TAT to decrease A2AR expression in the ChP of A2AR...

Watch this Scientific Journal Video about Insights and Innovations in Gene Expression Manipulation Techniques for Choroid Plexus Research at JoVE.com

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

The choroid plexus (ChP) serves as a critical gateway for immune cell infiltration into the central nervous system (CNS) under both physiological and ...

targeted-knockdown-of-genes-in-the-choroid-plexus

Research

JoVE Journal

Neuroscience

ChP Specific Knockdown of A2ARs with AAV2/5-shRNA and Cre/LoxP System

ChP Specific Knockdown of A2ARs with AAV2/5-shRNA and Cre/LoxP System  

To begin, place the anesthetized mice on the surgical platform. After trimming the hair, apply eye ointment down both eyes to prevent them from drying. Then fix the mice onto a stereotaxic apparatus for immobilization.Make a small incision under a microscope to fully expose the skull. Prepare two 10 microliter syringes, one containing AAV2/5-adenosine 2A receptor shRNA, and the other containing AAV2/5 scramble. Then use the microscope to locate the bregma and lambda and set the coordinate point on the 10 microliter syringe based on the mouse brain stereotaxic atlas.Drill a small hole in the skull at the established coordinate point. Inject two microliters of AAV2/5-Adenosine 2A receptor shRNA virus into the brain ventricle via lateral injection at a steady rate of 100 nanoliters per minute. Keep the needle in place for 10 minutes before withdrawing.Seal the injured skin promptly using medical bio fibrin glue to prevent virus loss. Place the mouse on a heating pad, maintaining its body temperature approximately at 37 degrees Celsius to facilitate recovery. Inject two microliters of CRE-TAT Recombinase into each lateral ventricle of Arosa L-D-L-T-D tomato mouse for the experimental group, and two microliters of sterile PBS for the control group.Two weeks after the CRE-TAT Recombinase injection, prepare frozen tissue sections, perform nuclear staining and prepare the slide for imaging Knocking down adenosine 2A receptor in the choroid plexus in homozygous adenosine 2A receptor fluxed mouse, using CRE-TAT, led to decreased EAE clinical scores. Similarly, knocking down choroid plexus in adenosine 2A receptor wild-type mice, using AAV2/5 shRNA decreased EAE clinical scores. The QPCR analysis showed that the mRNA levels of adenosine 2A receptor decreased in the AAV2/5 shRNA and CRE-TAT groups, compared to each control.Silencing adenosine 2A receptor, specifically in the choroid plexus, reduced immune cell infiltration into the spinal cord.

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

Selective Gene Silencing in the Choroid Plexus

Summary