Effects Of Aquaporin-4 On Neurotransmissions And Its Relationships With Parkinson's Disease

Aquaporins (AQPs) are integral membrane proteins that serve aschannels in the transfer of water, and in some cases, small solutes acrossthe membrane. In the central nervous system, aquaporin-4 (AQP4) isthe most abundant isoform and is localized in ependymal cells lining theventricles and astrocyte membranes, where the most abundant expressionis in the perivascular glial processes. This expression pattern suggestsAQP4 involvement in the movement of water between blood and brain,and between brain and cerebrospinal fluid compartments. AQP4 hasgained much attraction due to its involvement in the physiopathology ofcerebral disorders including stroke, tumor, infection, hydrocephalus,epilepsy, and traumatic brain injury. Using RNA interferencetechnology, knockdown of AQP4 in astrocyte primary cultures reducedmembrane water permeability, impaired cell growth, and altered cellmorphology. Recently, more studies provided compelling evidence forinvolvement of AQP4 in functions of astrocytes, such as astroglialmigration, potassium buffering, and neural signal transduction.Therefore, AQP4 may serve as a modulator of astroglial functionincluding regulating neurotransmission in mammalian brain.Parkinson's disease (PD) is a common and disabling idiopathicneurodegenerative disorder characterized by tremor, muscle stiffness,paucity of voluntary movements, and postural instability. Astrocyticreaction, which is found in PD patients as well as in experimental modelsof PD, suggests that this feature is involved in the pathogenesis of the disease. The reactive astrocytes may provide permissive and energysubstratum in supporting neuronal survival, axonal outgrowth andsynaptic reorganization through release of surface recognition molecules,neurotrophic factors and cytokines. Further, astrocytes play a veryimportant function in the differentiation, survival, pharmacologicalproperties, and resistance to injury of DA neurons, and are potentialmanipulation targets in novel cell replacement therapy of PD. Becauseof these, we hypothesized that AQP4 may participate in progression andmortality of PD.The aims of the present studies are: 1) to investigate whether AQP4participates in the regulation of neurotransmission; 2) to investigate therelationships between AQP4 and PD employing classical PD animalmodels. The results revealed here will be beneficial to knowing theneurobiology of AQP4 and its mechanisms involved in PD.Partâ… Effects of AQP4 deficiency on neurotransmission1. Effects of AQP4 deficiency on basal levels of neurotransmittersAIM: To investigate whether AQP4 participates in the regulation ofbasal neurotransmission.METHODS: 1) Transgenic knockout mice deficient in AQP4 weregenerated by targeted gene disruption. Eight-week-old wildtype (WT)and AQP4 knockout (KO) mice were used for the experiments. Themice were killed by cervical dislocation. The medial prefrontal cortex(mPFC), striatum, hippocampus, and hypothalamus were rapidlydissected out free on ice and were then ultrasonicated. Homogenateswere centrifuged, and the supernatant was filtered and stored before assay.2) The levels of amino acids and monoamines in each sample weremeasured by high-performance liquid chromatography.RESULTS: 1) The significant alterations of aspartate and glutaminecontent in brain regions (mPFC, striatum, hippocampus) were found inboth genders of KO mice, whereas an increased levels of glutamate was only detected in female and an decreased levels of glycine only in male;2) in KO male mice, alterations of the dopaminergic and serotonergicsystem were detected in the mPFC and striatum, whereas only theserotonergic system was increased in the hypothalamus; and 3) in KOfemale mice, no difference in dopamine metabolism was detected in allregions, whereas an increase in the metabolism of serotonin wasmeasured in the mPFC and hypothalamus.CONCLUSION: AQP4 participates in the regulation ofneurotransmission. The lack of AQP4 expression in mice is paralleledby sex- and region-specific abnormalities in the metabolisms of basalamino acids and monoamines.2. Effects of AQP4 deficiency on K~+-stimulated releases of striatalneurotransmittersAIM: To investigate whether AQP4 participates in the regulation ofK~+-stimulated releases of striatal neurotransmitter.METHODS: 1) Eight-week-old WT and AQP4 KO mice were used forthe experiments. The mice were placed in a stereotaxic apparatus. Theconcentric microdialysis probe was implanted into the right ventralstriatum. The whole assembly was secured with the dental cement.For the first set of groups (probes without guides cemented directly to theskull), neurotransmitter release was measured in the striatum of mice at24 h after surgery. In the second set of groups, neurotransmitter releasewas measured in the striatum of mice at 7d after surgery. On day of theexperiment, the probes were perfused with an artificial cerebrospinal fluid(aCSF) for 100 min. After collection of five high-K+ samples for 100min, the mice were continually injected by aCSF for 160 min tonormalize the levels of neurotransmitters. Each collected sample wasstored for analysis of amino acids and monoamines levels. 2) The levelsof amino acids and monoamines in dialysate samples were measured byhigh-performance liquid chromatography.RESULTS: When measured at 24 h after surgery, neurotransmitter releases under the basal or W-stimulated condition in the striatum ofAQP4 KO mice were similar to that of WT mice, except for taurine.However, when measured at 7 d after surgery, the basal extracellularlevels of DA, DOPAC, and HVA were significantly increased in KO mice,followed by reduced (DA) or no response (DOPAC, HVA, and 5-HIAA)to depolarizing stimuli. In addition, there were higher responses ofaspartate, glutamate, taurine and GABA to high K~+ stimulus in KO mice.CONCLUSION: AQP4 participates in the regulation of W-stimulatedreleases of striatal neurotransmitters. These findings provide in vivoevidence for that AQP4 plays different roles in neurotransmissions ofamino acids and monoamines under baseline and K~+ perfusion conditions.Partâ…¡Effects of AQP4 deficiency on the neurotoxicity of MPTP inmiceAIM: To investigate the hypothesis that abnormal AQP4 expression isinvolved in the pathophysiology of PD.METHODS: AQP4 KO and WT mice were injected intraperitoneallyfour times with MPTP-HC1 (20 mg/kg) in saline at 2 h intervals, the totaldose per mouse being 80 mg/kg, and were killed at 7 days after the lastinjection. Alocomotor activity test and a pole test were performed underbaseline conditions and at 2, 7 days after the final injection of MPTP orsaline. The total numbers of TH-positive neurons in the SNc andGFAP-positive cells densities in the SNc and striatum were obtainedstereologically using the optical fractionator method. Total RNA wasextracted from selected brain regions after MPTP and used for RT-PCRanalysis of AQP4 and GAPDH. HPLC with electrochemicaldetectionwas used to measure striatal levels of DA, DOPAC and HVA.The UV detector was set to 295 nm for MPP~+ detection. Thedithiobinitrobenzoic acid method was used to test GSH level.Concentration of GDNF protein in striatal tissue was determined byELISA. The levels of IL-6 and CNTF mRNA in striatal and midbraintissue were determined by realtime-PCR. Astroglial activity and proliferation were determined respectively by MTT and [~3H]-TdR.RESULTS: AQP4 deficiency increased the sensitivity ofdopaminergic neurons to MPTP neurotoxicity. The AQP4 KO micecontained lower levels of DA, DOPAC and HVA in the striatum, and aless number of TH-positive dopaminergic neurons in the SNc, comparedto WT mice after MPTP injections. Furthermore, this study providedadditional evidence: (1) the treatment with MPTP resulted in worseperformances in the pole test in KO mice, (2) astroglial proliferationinduced by MPTP was inhibited by AQP4 deficiency, (3) KO micedecreased the synthesis of astrocytic GDNF protein after MPTPtreatments, and (4) AQP4 deficiency resulted in the impairment of theregulation of IL-6 mRNA, and (5) AQP4 deficiency increased thesensitivity of astrocytes to MPP~+ toxicity.CONCLUSION: AQP4 is important in the MPTP neurotoxic processand indicates that the therapeutic strategy targeted to astrocyticmodulation with AQP4 may offer a great potential for the development ofnew treatment for PD.In summary, we firstly explored that AQP4 participates in theregulation of neurotransmission under baseline and K~+ perfusionconditions and is important in the MPTP neurotoxic process. The resultsof the present study suggest: 1) AQP4 might be involved in the regulationof astroglial function and plays different roles in neurotransmissions ofamino acids and monoamines. 2) The therapeutic strategy targeted toastrocytic modulation with AQP4 may offer a great potential for thedevelopment of new treatment for PD.