The Expression Of ZNT7 In The Mouse Nervous System And The Mechanism Of Zinc Deficiency Induced Learning And Memory Impairments

PrefaceZinc,an essential trace element for mammals,is abundant in the nervous system and has been suggested to be involved in many biological functions.In the brain,about 85%zinc ions are tightly bound to macromolecules or serve as a cofactor for a large number of enzymes.A small fraction of zinc ions exist,however,in synaptic vesicles of the zinc-enriched(ZEN)neurons and are free or weakly bound.Such weakly bound zinc ions can be detected by zinc-staining techniques such as zinquin and 6-methoxy 8-para toluene sulfonamide quinoline(TSQ).Zinc ions are also histochemically detectable with Timm staining or autometallography(AMG).Zinc cannot travel across biological membranes by passive diffusion.Specific membrane transporters and channels involved in its transfer and metabolism.Zinc transporter(ZNT)is one of the important protein family involved in zinc metabolism in the brain.Until now,eight members of the ZNT family(ZNT1-8)have been characterized.Most of the ZNT family members are predicted to have 6 transmembrane domains and a large histidine-rich intracellular loop between domainsⅣandⅤ,which is supposed to function as a potential zinc-binding site.ZNT family members are responsible for the extrusion of zinc outside the cytoplasm to the extracellular space or intracellular organelles.ZNT7 is a 42 kDa membrane protein that is highly expressed in the mouse nervous system including brain and spinal cord.Many studies have demonstrated that ZNT7 resides in the Golgi apparatus,suggesting that ZNT7 may be responsible for transporting cytoplasmic zinc ions into the Golgi cisterns.In this research,we focused on studying the expression and distribution of ZNT7 in cerebellum and sympathetic ganglion of CD-1 mice.Hippocampus is a critical area for converting short-term memories into long-term memories and also plays a pivotal role in encoding,consolidation and retrieval of associations responsible for episodic memory.Within the DG and CA3 areas,large amounts of chelatable zinc ions exist in the presynaptic vesicles in the giant boutons of mossy fibers.During synaptic activities,these zinc ions may be released to serve as a neuromodulator to modulate different types of receptors.Recent studies have shown that zinc concentration in the hippocampus is significantly decreased by dietary zinc deficiency.However,the underlying mechanism of impairing hippocampus-dependent learning and memory under zinc deficiency is poorly understood.The role of neurogenesis in hippocampus is associated with neuronal plasticity.The hippocampal DG is among the few regions in the brain in which production of new neurons continues in the adulthood.It is well known that the neuronal progenitor cells in the subgranular zone(SGZ)can differentiate into neurons and glial cells.Some of the newborn neurons migrate to the granular layer and extend mossy fiber axons,indicating that they are integrated into the functional circuitry of the hippocampus.However,the mechanism of impairing hippocampus-dependent learning and memory under zinc deficiency through affecting neurogenesis and increasing apoptosis is not fully understood.Long-term potentiation(LTP)in the hippocampus is considered to be important for elucidating the molecular mechanism of learning and memory.Several lines of evidence have shown that certain protein kinases are involved in LTP formation.It has been confirmed that synaptically released zinc may play an essential role in the induction of LTP in mossy fibers input into CA3 neurons.In the present study,we examined the expression levels of proteins in CaM-CaMKⅡ-CREB pathways of zinc deficiency mouse hippocampus,in order to analyze the involvement of zinc deficiency on impaired learning and memory.In this study,we used morphology technology and molecular technology to analyze the expression and distribution of zinc ions and ZNT7 in the CD-1 mice nervous system.To test that zinc deficiency may impair learning and memory,we examined the possible mechanisms,by analyzing the proliferation of newborn neurons, apoptosis of neurons and the expression of proteins in CaM-CaMKⅡ-CREB pathways under zinc deficiency.MethodsAdult male CD-1 mice were used for study the expression of ZNT7 in nervous system;CD-1 mice(3-week-old)which were fed with zinc-deficient diet(zinc:0.85 mg/kg,Egg white-based AIN-76A,Research Diets Company,USA)were used as the zinc-deficient animal model,control group were fed with zinc-adequate diets(zinc:30 mg/kg).The expression of free zinc ions in CD-1 mouse nervous system were evaluated by AMG.The distribution of ZNT7 in nervous system was detected by immunostainning, Western blot,double immunofluorescence and confocal laser scanning microscopy. The changes of zinc ions expression levels in zinc deficient hippocampus were studied by TSQ and AMG analyses.Immunostainning,Western blot,double immunofluorescence and confocal laser scanning microscopy were used to detect the hippocampus neurogenesis in zinc deficient hippocampus.Western blot and TUNEL were used to reveal the mechanism of cell apoptosis in zinc deficient mouse.The expressions of LTP-related proteins in zinc deficient mouse hippocampus were detected by Western blot.Results1.Expression of ZNT7 protein in the mouse nervous systemTo assess the ZNT7 expression in the nervous system,we used Western blot to semi-quantify ZNT7 content.The specificity of the rabbit anti-ZNT7 antibody used for immunohistochemistry was confirmed.ZNT7 immunoreactivity(IR)was detectable in the nervous system including hippocampus,cerebral cortex,cerebellum and superior cervical ganglion(SCG),spinal cord and brain stem.The bands of ZNT7 immunoreactive protein were strong in the hippocampus and cerebral cortex and cerebellum.In comparison with the other nervous tissues,the ZNT7 levels were clearly lower in the mouse SCG,spinal cord and brain stem.2.Abundant expression of ZNT7 in CD-1 mice cerebellum and SCGZNT7-IR was predominately present in the somas and primary dendrites of most,if not all,Purkinje cells.ZNT7-IR was also present in the Bergmann glial cell bodies and processes.At high magnification,it was shown that the positive radial processes of Bergmann glia extended into the molecular layer.A few ZNT7-positive neuronal somas were seen in the molecular cell layer.In the granule cell layer,ZNT7 immunoreactivity displayed as puncta or semilunar structures.TGN38 was used as a marker for the Golgi apparatus.Double immunofluorescence staining demonstrated a colocalization of ZNT7 and TGN38 in the somas of Purkinje cells.Structures positive to both ZNT7 and TGN38 staining were also distributed in the granule cell layer.These double-stained puncta or semilunar structures were the Golgi apparatus in the cytoplasm of granule cells.ZNT7-IR was present in almost all of the SCG neurons of different sizes.The percentage of ZNT7-positive cells was 94.53±4.28%.At a higher magnification,the ZNT7 positive products were located predominantly in the perinuclear regions of the neurons like flower petals around the nucleus.3.Distribution of zinc ions in CD-1 mouse cerebellum and superior cervical ganglionThe selenite AMG was performed to analyze the distribution of chelatable zinc ions in the mouse cerebellum.In general,fine AMG grains were present throughout the cerebellar cortex.A subpopulation of SCG cells expressed varying degrees of AMG-positive staining.The staining intensity of the ZEN neurons was unrelated to the size or location of the neuronal cells.The percentage of AMG-positive ganglionic cells in the SCG was 89.27±8.11%.4.Dietary zinc deficiency causes poor growth and low hippocampal zinc loading in developing mouseLow dietary zinc status may cause poor growth in animals.We therefore monitored the body weight gain of zinc-deficient mice and controls.Zinc-deficient mice grew slower than their littermates.Meanwhile,the body weight of zinc-deficient mice(17.51±3.98 g)was significantly lower than that of control mice(31.97±3.66 g) over a period of 5 weeks.Furthermore,zinc deficiency also caused a lower weight of different brain regions,including the forebrain,the cerebellum and the hippocampus.To evaluate the chelatable zinc accumulation in hippocampal mossy fibers,we employed both TSQ fluorescence and AMG on hippocampal sections.The intensity of fluorescence in the CA1 and CA3 area of zinc deficient mice was weaker than that of control mice.The similar results were also detected in the AMG staining of the hippocampus.The density of AMG staining was decreased obviously in the CA1 and CA3 regions of the zinc deficiency group.5.Effect zinc deficient on neurogenesis in mouse hippocampus To investigate whether hippocampal neurogenesis is impaired by zinc deficiency, the distribution and expression of doublecortin(DCX),a marker for newborn neurons, were analyzed by immunohistochemistry and Western blot.Immunostaining revealed that DCX~+ cells were predominately located in the SGZ.In zinc deficient mice,the processes of DCX~+cell were shortened and their branches were reduced.Cell counting analysis demonstrated that the number of total DCX~+ cells in zinc deficient group was obviously less than that in the control group(p＜0.01).Western blot results showed that the densitometry of DCX immunoreactive bands in the zinc deficient mice were substantially lower than that in control mice(p＜0.01).We further analyzed the hippocampal neurogenesis by measuring 5-bromodeoxyuridine(BrdU)-IR in the hippocampus.Consistent with DCX immunostaining,BrdU~+ nuclei,which represent proliferating cells,were also located in the SGZ.The average number of BrdU~+ nuclei in the SGZ in zinc deficient mice was less than that in controls(p＜0.05).Confocal microscopic results showed that the number of double-labeled BrdU~+/DCX~+ neurons were lower in zinc deficiency group than that in control group(p＜0.05).6.Zinc deficiency results in increase in hippocampal neuronal death through Fas/FasL and AIF pathwaysThe peroxidase TUNEL staining was performed on hippocampal sections from both zinc deficient and control mice.Quantitative results indicated that the number of TUNEL-positive cells in zinc deficient hippocampus was significantly increased in the subareas(CA1,CA3 and DG)of the hippocampus(p＜0.01).We next examined whether Fas/Fas ligand(FasL),Bax,caspase-3 and apoptosis inducing factor(AIF)are involved in zinc deficiency-induced hippocampal cell apoptosis with Western blot immunoassays.Following t-test analyses,statistically obvious increases in expression levels of Fas and FasL were detected in zinc deficient hippocampus(p＜0.05).The cleaved(active)caspase-3 was also increased evidently, but not the procaspase-3(inactive),in zinc deficiency mice.On the other hand,zinc deficiency enhanced the expression of AIF,a caspase-independent apoptotic molecule (p＜0.05).However,the expression level of Bax in zinc deficient hippocampus remained unchanged compared to control group(p＞0.05).7.Zinc deficiency changes basal levels of key signaling molecules involved in learning and memory in mouse hippocampusTo analyze the changes of LTP-related molecules in dietary zinc deficient mouse hippocampus,we focused on detecting CaM-CaMKⅡ-CREB signaling proteins with Western blot analysis.In hippocampal homogenates,our Western blot results demonstrated that Calmodulin(CaM)protein level in zinc deficient group was decreased about 16.53±5.09%compared with control group(p＜0.01).Zinc deficiency reduced the basal level of p-CaMKⅡto 83.47±3.91%,compared with control group(p＜0.01).In contrast,there was no significant change in total CaMKⅡprotein(p＞0.05).Furthermore,zinc deficiency markedly reduced the basal level of p-CREB(p＜0.01),the active form of CREB,but the level of total CREB remained unchanged(p＞0.05).In zinc deficient mice,hippocampal neurogranin/RC3(Ng)protein level was decreased to 66.11±15.17%,compared with control mice(p＜0.05).However, compared with control group,no significant changes in calcineurin(CaN)levels were detected in the zinc deficient mice(p＞0.05).In addition,there were no obvious differences in p-ERK1/2 and total ERK1/2 levels between zinc deficient hippocampus and control's(p＞0.05).Conclusion1.Zinc ions and ZNT7 protein are abundantly expressed in the CD-1 mice nervous system.Localization of ZNT7 in the Golgi complex indicates that ZNT7 might be involved in zinc-containing protein packaging.2.Dietary zinc deficiency causes poor growth and low hippocampal zinc loading in developing mouse.3.Hippocampal neurogenesis is reduced,and the apoptotic cells are abnormally increased after zinc deficient treatment.The altered neurogenisis and increase in cell death in zinc deficient hippocampus are involved in the alternation of learning and memory.4.Zinc deficiency associates with inactivation of the CaM-CaMKⅡ-CREB signaling pathway in the hippocampus.