The Internalization And Lysosomal Degradation Of AQP4in Cytotoxic Brain Edema And Its Mechanism

BackgroundBrain edema can produce dysfunction of brain cells and elevateintracranial pressure that leads to impaired vascular perfusion, herniation,even death of patients. The abnormality of water transport in the brain isinvolved in the occurrence and development of brain edema. Aquaporin-4(AQP4) is a membrane-bound water channel. It is concentrated in thebasement membrane of ependymal cells and the astrocytic endfoot apposedto capillaries and the pia mater. The polarized pattern of AQP4distribution isan important structural basis for water regulation and transportation betweenglial cells and cerebrospinal fluid or vessels.Brain edema is classified into two major categories: cytotoxic andvasogenic edema. Cytotoxic edema is characterized by accumulation ofintracellular water without disruption of the blood-brain barrier (BBB). Incontrast, the characteristic of vasogenic edema is the breakdown of BBBwhich causes plasma proteins and electrolytes accompanied by water leaksfrom capillaries into the interstitium. AQP4has different roles in the pathophysiology of cytotoxic and vasogenic brain edema. AQP4facilitatesedema fluid formation in cytotoxic edema but increases the rate of edemafluid elimination in vasogenic brain edema. Therefore, inhibition of AQP4isexpected to protect the brain in cytotoxic edema, whereas activation ofAQP4will be required to facilitate the clearance of vasogenic brain edema.The alteration of AQP4expression level and the loss of AQP4polarized localization have been observed in the brain with edema inducedby kinds of pathological conditions，such as ischemia，epilepsy and glioma.However, the mechanisms underlying these changes remain unclear.It is known that the endocytotic internalization of membrane-boundproteins (such as AQP4) can alter the subcellular localization of theseproteins; moreover, this internalization may or may not be accompanied bysubsequent lysosomal sorting and degradation. Whether AQP4isinternalized in the ischemic brain remains an open question.If the phenomenon of AQP4internalization and lysosomal degradationis verified to be existed in the development of brain edema, it will provideus a new strategy for treatment of brain edema. Promoting theinternalization and lysosomal degradation of AQP4will reduce the watertransported into cells via AQP4, and be beneficial for reducing thecytotoxic brain edema；while inhibiting the internalization and lysosomaldegradation of AQP4will keep the polarized distribution of AQP4, and bebeneficial for the clearance of the excess water in the intercellular space, thus reduce the vasogenic edema.ObjectiveThe present study was conducted to investigate whether AQP4in ratbrains is internalized and/or lysosomal sorting in cerebral ischemia andbacterial meningitis models and to explore the mechanisms of AQP4internalization and lysosomal sorting.MethodsThis experiment consists of two parts. Part1: The experiment forobserving AQP4internalization and lysosomal sorting in the ischemic brainedema models and exploring the mechanisms of AQP4internalization andlysosomal sorting; Part2: The experiment for observing AQP4internalization and lysosomal sorting in rat brains with bacterial meningitis.Part1(1) The experiment for observing AQP4internalization and lysosomalsorting in the ischemic brain edema models:The rat cerebral ischemia model was produced by middle cerebralartery occlusion (MCAO). The tissue infarction of ischemic brain wasshowed by2,3,5-triphenyl-tetrazolium chloride (TTC) staining. The brainultrastructures were observed by transmission electron microscopy (TEM).Double immunofluorescence labeling was applied to detect the co-expression of AQP4with the marker of early endosome (early endosomeantigen-1, EEA1) and the marker of lysosome (lysosomal associatedmembrane protein-1, LAMP1), respectively. The ratio of AQP4coexpressedwith EEA1/LAMP1to total AQP4was measured by Image analysissoftware Image-Pro Plus6.0.(2) The experiment for exploring the mechanisms of AQP4internalization and lysosomal sorting in the ischemic brain edema models：The protein kinase C (PKC) activator phorbol12-myristate13-acetate(PMA) was given intravenously. The alteration of brain water content, theastrocytic swelling and AQP4expression were measured by wet and dryweight methods, TEM, and Western blot, respectively. Doubleimmunofluorescence labeling was applied to detect the co-expression ofAQP4with EEA1and AQP4with LAMP1, respectively. The ratio of AQP4coexpressed with EEA1/LAMP1to total AQP4was measured by Imageanalysis software Image-Pro Plus6.0.Part2The experiment for observing AQP4internalization and lysosomalsorting in the meningitic brain edema models：Group B hemolytic Streptococcus was injected into the cerebellarmedulla pool of rat brains to establish bacterial meningitis model. The brainwater content, integrity of blood-brain barrier (BBB) and expression ofAQP4, α-dystroglycan (α-DG) and β-dystroglycan (β-DG) were measured by wet and dry weight methods, Evans blue (EB) staining, TEM andWestern blot, respectively. Double immunofluorescence labeling wasapplied to detect the co-expression of AQP4with the marker of earlyendosome EEA1, AQP4with the marker of late endosomemannose-6-phosphate receptor (MPR) and AQP4with the marker oflysosome LAMP1, respectively. The ratio of AQP4coexpressed withEEA1/MPR/LAMP1to total AQP4was measured by Image analysissoftware Image-Pro Plus6.0.ResultsPart1(1) The experiment for observing AQP4internalization and lysosomalsorting in the ischemic brain edema models:①No appreciable TTC staining defects appeared in the normal or1hMCAO rat brains; tissue infarction was presented in the3h,6h,12h,24hMCAO rat brains;②Electron microscopy of3h post-ischemia brainrevealed the astrocyte swelling, no disruption of basement membranes ortight junction opening;③At1h,3h,6h,12h,24h post-ischemia, theco-expression of AQP4with EEA1was observed. The ratio of AQP4withEEA1in ischemic brain increased compared with in the control group (P＜0.05);④At3h,6h,12h,24h post-ischemia, the co-expression of AQP4with LAMP1was observed. The ratio of AQP4with LAMP1in ischemic brain increased compared with in control group (P＜0.05).(2) The experiment for exploring the mechanisms of AQP4internalization and lysosomal sorting in the ischemic brain edema models:①The brain water content in the PMA treated animals decreasedsignificantly compared with those in vehicle groups at1h and3hpost-ischemia (P＜0.05); PMA had no obvious effect on brain water contentafter6h post-ischemia (P＞0.05)；②Electron microscopy revealed thatPMA infusion decreased the average foot processes at3h post-ischemicbrain compared with vehicle infusion (P＜0.05)；③PMA infusiondownregulated AQP4expression in the brain cortex at3h post-ischemiacompared with vehicle infusion (P＜0.05)；④PMA infusion did notincrease the ratio of co-expressed AQP4(with EEA1/LAMP1) to totalAQP4compared with those in vehicle infusion group (P＞0.05).Part2The experiment for observing AQP4internalization and lysosomalsorting in the meningitic brain edema models:①Compared with the control group, the brain water content in themodel group increased (P＜0.05)；②Compared with the control group, EBcontent in the model group have not changed significantly (P＞0.05);③Electron microscopy of model group revealed the astrocyte swelling, nodisruption of basement membranes or tight junction opening;④Comparedwith the control group, AQP4expression in the model group increased (P＜ 0.05)；⑤Compared with the control group, α-DG and β-DG expression inthe model group increased (P＜0.05)；⑥The co-expression of AQP4withEEA1, AQP4with MPR, and AQP4with LAMP1were observed inmeningitic rat brains. The ratio of AQP4with EEA1/MPR/LAMP1in modelgroup increased compared with in control group (P＜0.05).Conclusions1. The internalization and lysosomal sorting of AQP4occurs in in theischemic rat brains.2. PKC activator PMA could decrease brain edema accompanied bydownregulating AQP4expression in the early stage poste-ischemia.However, the evidence to prove PKC activator PMA can promote theinternalization or lysosomal degradation of AQP4has not been founded.3. AQP4in rat brains is up-regulated in the bacterial meningitis model.Meanwhile, the internalization and lysosomal sorting of AQP4occurs in thismodel.