Effects Of Aquaporin-4 Knockout On The Promotion Of Neurogenesis By Fluoxetine

Aquaporins (AQPs) are a family of integral membrane pore proteinsmediating transmembrane water movement in most cells. In the centralnervous system (CNS), AQP4 is the most abundant isoform expressed inastrocyte membranes involved in the regulation of brain volumehomeostasis, and has the relationship with physiopathology of cerebraldisorders including stroke, tumor, infection, hydrocephalus, epilepsy, andtraumatic brain injury. Astrocytes play a crucial role in maintaining aproper environment for neuronal activity. This includes: removal,metabolism, and release of neurotransmitters; maintenance ofextracellular K⁺ and H⁺ levels; secretion of neurotrophic factor,promoting neurogeneis; providing energy substrates for their demandingneighbors the neurons. Inhibition of aquaporin-4 expression inastrocytes by RNAi determines alteration in cell morphology, growth,and water transport and induces changes in ischemia-related genes.Astrocyte monolayers can form neurospheres that give rise both toneurons and glia, which confirms that astrocytes globally possess NSCsattributes. NSCs, the family member of astrocytes, have a high expressionof AQP4 too. So, it is therefore reasonable to suppose that AQP4 mayparticipate in the modulation of physiological function of astrocytes andNSCs.In the adult mammalian brain, new neurons are continuously generated from a proliferating population of NSCs and becomeincorporated into the existing neuronal circuitry via a process termedadult neurogenesis. Neurogenesis principally occurs in SVZ and DGareas of the rodent, primates, humans and nonhumans brain throughoutadulthood, and also reside in cortex, hypothalamus, substantia nigra,spinal cord. After injuries, NSCs are activated to make new nerve cellsand take part in the repair of brain tissue. Many studies have revealedthat dysregulation of adult neurognesis may contribute to thepathogenesis of neurodegenetative disorders and psychiatric disorders.Manipulating adult neurogenesis has been regarded as a potential strategyfor cell replacement therapy for functional recovery in degenerativediseases. Chronic mild stress (CMS) is often used as an animal modelof depression in rodents, and has a potent inhibition on dentate cellproliferation. Chronic fluoxetine treatment was reported to increase therate of neurogenesis in adult brain. However, the cellular and molecularmechanisms underlying adult neural stem cell proliferation,differentiation, and migration are largely unknown. Thus, seeking theendogenous target to regulate neurogenesis may offer prospective clinicaltherapeutic benefit for CNS diseases.Antidepressant drug fluoxetine could influence the level of5-hydroxytryptamine (5-HT), modulate astrocytic function, and promotehippocampal neurogenesis. AQP4 is mostly localized in astrocytes andneural stem cells, and has a influence on the astrocytic function. So weinvestigated the role of AQP4 on fluoxetine's effect on hippocampalneurogenesis in vivo and vitro, and some possible mechanisms by usingAQP4 knockout mice.AIM: To investigate the influence of AQP4 deletion onhippocampus neurogenesis by chonic fluoxetine administration both invivo and in vitro, and further the underlying mechanisms. METHODS: 1) Mice of both genotypes (wild-type and AQP4knockout mice) were randomly assigned to either saline or fluoxetinetreatment groups. Mice received a daily i.p. injection of eitherfluoxetine (10 mg·kg^-1) or physiological saline for 28 days. Behavioraltests were conducted during the last several days of drug treatment. Instress, AQP4^+/+ and AQP4^-/- mice were randomly assigned to saline,CMS/saline, CMS/fluoxetine treatment groups and subjected to an8-weeks period of mild, unpredictable stressors. Fluoxetine (10 mg·kg^-1)or saline was administrated i.p. once a day for 28 days, starting at 4weeks after the beginning of the CMS. Behavioral tests were conductedbefore and during the last several days of drug treatment. 2) BrdUImmunohistochemistry was taken for the analysis of neural stem cell(NSC) proliferation and survival in the SGZ of hippocampus. Theneuronal or glial phenotype of BrdU⁺ cells was determined by doubleimmunofluorescence labeling. 3) The cell proliferation rate of NSCsafter fluoxetine treatment was analyzed by [³H] thymidine incorporationin vitro. 4) Western blotting was taken for the analysis ofp-CREB/p-ERK/p-CaMKIV/AQP4 expression.RESULTS: 1) Fluoxetine-treated AQP4^-/- mice showed nodifference on latency to feed compare to saline-treated AQP4^-/- mice,whereas fluoxetine resulted in a significant decrease in latency to feed inAQP4^+/+ mice. CMS induced significant behavioral changes at 4 weeksin both AQP4^+/+ and AQP4^-/- mice, as evidenced by increased immobilitytime on the TST paradigm and reductions in sucrose preference. Theseeffects were reversed by fluoxetine treatment in AQP4^+/+ mice, but not inAQP4^-/- mice. 2) In normal and CMS state, chronic fluoxetineadministration significantly increased the number of BrdU-positive cellsin the dentate gyrus of AQP4^+/+ mice, but had no effect on AQP4^-/- mice.No significant difference was detected between saline-treated AQP4^+/+and AQP4^-/- mice. AQP4 knockout did not affect the survival rate of BrdU-positive cells and differentiation of cells into neurons or glia in thehippocampus. 3) [³H]TdR incorporation assay showed that the cellproliferation was significantly increased in 0.1μM and 1μMfluoxetine-treated AQP4^+/+ NSCs by 31% and 21%. No proliferationpromotion was found in any concentration of fluoxetine in AQP4^-/- NSCs.4) In normal and CMS state, fluoxetine administrtion led to asignificantly increase of the phosphorylation of CREB in thehippocampus of AQP4^+/+ mice, but not in AQP4^-/- mice. Fluoxetineinduced a significant increase of the phosphorylation of CaMKIV inAQP4^+/+ mice, but had no effect on AQP4^-/- mice. Fluoxetine led to anincrease of total ERK expression, and with no significant influence onphosphorylation of ERK in both AQP4^+/+ and AQP4^-/- mice. In normalstate, fluoxetine has no significant influence on the expression of AQP4in hippocampus. In stress state, the expression of AQP4 inhippocampus was significantly increased, After chronic fluoxetinetreatment, the AQP4 expression was reversed to the levels of normalstate.CONCLUSION: AQP4 knockout results in the inhibition to thefluoxetine's effects on hippocampus neurogenesis in vivo and in vitro,The dysfunction maybe associated with the disorganized phosphorylationof CaMKIV and CREB in hippocampus of AQP4^-/- mice.The major contributions of the present study lie in:The first to report AQP4 take part in the modulation of neurogenesis,which accumulates theoretical and experimental evidence for furtherresearch on the great potential of AQP4 in neurobiology, and provide anew strategy for the treatment of CNS disease.