Expression And Significance Of Minichromosome Maintenance Protein 5 In Brain

Research background:Minichromosome maintenance proteins 5(MCM5)is a member of minichromosome maintenance proteins family(MCMs),which mainly includes MCM1-10.Among them,MCM2-7 is found in eukaryotes and archaea,and the sequences of MCM2-7 proteins are obviously similar to each other,and they interact together to form MCM complex hexamer,which plays a role in gene replication in eukaryotes.MCM5 is mainly involved in the unwinding of DNA replication initiation and the extension of replication fork,but the content of MCM5 in cells far exceeds the amount needed for its replication process.Later,it was found that MCM5 is also involved in RNA transcription process.At present,the research of MCM5 mainly focuses on the pathogenesis of tumors,and the abnormal expression of MCM5 is closely related to the diagnosis,treatment and prognosis of many tumors.MCM5 deletion leads to nervous system and embryonic development disorders.In recent years,it has been reported that MCM5 is age-dependent and plays a key role in cell aging.However,the location of MCM5 in the brain is still unclear,so this project will explore the distribution of MCM5 in the brain of mice and provide morphological reference for the study of the mechanism of MCM5 in the central nervous system.Research purposes:To explore the expression location and cell types of MCM5 in the brain of mouse,and to explore the species expression differences of MCM5 in the brains of mice and Tibetan Emirates monkeys,so as to lay a foundation for further study on the role of MCM5 in brain development and diseases.Materials and Methods:1.C57/BL6 male rats were selected for heart perfusion,brain harvesting,postfixation and dehydration,and brain coronal frozen sections.The brain sections were labeled with monoclonal antibodies from rabbit origin of MCM5 for immunofluorescence staining,and the brain sections were scanned by fluorescence microscope to observe the expression and localization of MCM5 in mouse brains.The fixed part of the brain region of Tibetan Emirates monkey was selected for paraffin embedding section,and the expression of MCM5 in some brain regions of Tibetan Emirates monkey was observed by fluorescence microscope.2.The mouse brain tissue was embedded in paraffin and sliced,and the monoclonal antibody MCM5 from rabbit was used to react with mouse anti-neuronal nuclear antigen antibody(Neu N,neuron marker),anti-oligodendrocyte transcription factor 2 antibody(OLIG2,oligodendrocyte marker),ionized calcium linker 1 antibody(Iba1,microglia marker),anti-glial fibrillary acidic protein antibody(GFAP,Astrocyte marker),tyrosine hydrolase antibody(TH,dopaminergic neuron marker),anticholinesterase antibody(CHAT,cholinergic neuron marker),vesicular glutamate transporter-1 antibody(VGLUT1,glutamatergic neuron marker)and glutamate decarboxylase antibody(GAD67,γ-aminobutyric acid neuron marker)were co-immunofluorescent stained,and the expression of MCM5 in different types of neuron and glial cells in mice was observed by fluorescence microscope..3.C57/BL6 wild-type 3-month-old adult male mice and 18-month-old male mice were selected for heart perfusion,brain harvesting,post-fixation,dehydration and paraffin section,and the paraffin sections of adult mice and old mice were labeled with monoclonal antibodies of MCM5,respectively,to observe the expression of MCM5 at different ages.4.C57/BL6 wild-type 3-month-old adult male mice and 18-month-old male mice were selected to extract proteins from five brain regions: cerebral cortex,striatum,hippocampus,thalamus and midbrain.The protein concentration was measured by BCA method,and the protein content of MCM5 in these five brain regions was detected and compared by western blot,and the statistical analysis was made.Result:1.MCM5 positive signal is widely distributed in mouse brain,in the synaptic sphere/peribulbar cell layer(GI),outer plexiform layer(EPI),anterior olfactory region(AO),ventral orbital Cortex(VO),lateral orbital cortex(LO),medial orbital cortex(MO),secondary motor cortex(M2),insular cortex(AI)and Pyriform cortex(Pir)and its 1-3layers,piriform nucleus(En),primary sensory cortex(S1),secondary sensory cortex(S2),primary motor cortex(M1),cingulate cortex 29 C area(A29C),primary visual cortex(V1),lateral secondary visual cortex(V2L),secondary auditory cortex(Au D)and Primary auditory cortex(Au1),caudate nucleus(CPu),globus pallidus(VP)of striatum of basal nucleus of brain,nucleus accumbens(Acb),nucleus septa(MS),nucleus diagonal band(VDB),Pyramidal layer(py),molecular layer(Lmol)and dentate gyrus(DG)of hippocampus,and other areas more apparent positive signal.No MCM5 positive signal was observed in granular cell layer(Gro),corpus callosum(cc),corpus callosum forceps(fmi)and external capsule(ec)of olfactory brain.Subsequently,we selectively observed GI and AO of olfactory brain,M2,LO,En of cerebral cortex,Acb of septal area,CPu and VP of basal nucleus,DG and Py of hippocampus,and found that MCM5 was punctate positive in the nucleus and perinuclear cytoplasm.2.MCM5 positive marker is widely distributed in the diencephalon in mice,in paraventricular nucleus of thalamus(PVA),central medial nucleus(CM),dorsal nucleus(AD),before the thalamic reticular nucleus(Rt),lateral dorsal nucleus(LDDM),ventrolateral(VL),ventral lateral nucleus(VPL),after the ventromedial nucleus(VM),the medial nucleus(VPM)after abdominal,thalamus nucleus of thalamus(Sub)and after the dorsal lateral geniculate body nuclear(DLG),thalamus pulp lines(sm),habenula nucleus(Hb),lateral hypothalamus(LH),the hypothalamus(AHA),the bottom of the side the subthalamic nucleus of thalamus(STh),and other brain regions observed strong positive marker.Then we observed the lateral dorsal nucleus of thalamus(LDDM),ventral lateral nucleus(VL),central medial nucleus(CM),on the side before pulp lines(sm)of the thalamus,hypothalamus(AHA),the subthalamic nucleus of thalamus(STh)and after dorsal lateral geniculate body nuclear(DLG),it was found that MCM5 was punctate positive in nucleus and perinuclear cytoplasm.3.Also found widely distributed in the mice brain stem MCM5 positive signal,the obvious positive signals in the brain stem in the area of the midbrain pronucleus(APTD),the top belly pronucleus(APTV),the ventral tegmental area(VTAR),substantia nigra(SN),periaqueductal nuclear group(DK)and the periaqueductal gray(PAG),red nucleus(RPC),foot(IP),tegmental nucleus(Tg),the inferior colliculus cortex(CIC),pons,pons nucleus(Pn),pons reticular nucleus(Pn C),pons reticular nucleus(Rt Tg)is built,the main branch of trigeminal nerve(Pr5VL),vestibular nuclei(VE),facial nerve nucleus(7N),and other areas.In the midbrain periaqueductal nuclear group(DK),red nucleus(RPC),foot(IP),tegmental nucleus(Tg)and pontine reticular nucleus(Rt Tg)is built,vestibular nuclei(VE),facial nerve nucleus(7N)region of the expression of MCM5 in nucleus and perinuclear cytoplasm was punctate,and in substantia nigra(SN),MCM5 was diffuse and strongly positive in cytoplasm.4.The positive signals of MCM5 are also widely distributed in the cerebellum of mice,and the strong positive signals are found in the vermis(cb),the cerebellar intermedial nucleus(Int A),the lateral cerebellar nucleus(Lat)and the medial cerebellar nucleus(med).We observed these four parts of the cerebellum and found that MCM5 was punctate positive around the nucleus.5.MCM5 positive signals were found in the cerebral cortex,hippocampus,striatum,substantia nigra and thalamus of Tibetan Emirates monkeys.6.MCM5 and Neu N are co-labeled in the nuclei and cytoplasm of cerebral cortex and thalamus neurons,and co-labeled in the cytoplasm of hippocampus and substantia nigra neurons.MCM5 and TH were diffusely positive in the cytoplasm of dopaminergic neurons in substantia nigra.MCM5 and CHAT are co-labeled in the cytoplasm of cholinergic neurons in cerebral cortex and hippocampus,and MCM5 is positive in the nucleus of cholinergic neurons labeled by CHAT in striatum and diagonal band.MCM5 and VGLUT1 are co-labeled in the cytoplasm of glutamatergic neurons in hippocampus,striatum and thalamus,but expressed in the nucleus and cytoplasm of glutamatergic neurons in cerebral cortex.MCM5 has positive signals in the cytoplasm and nucleus of γ-aminobutyric acid neurons in cerebral cortex,striatum and hippocampus.In the posterior hypothalamic nucleus,MCM5 and GAD67 labeled γ-aminobutyric acid neurons are colabeled in the cytoplasm.MCM5 and OLIG2 are co-labeled in the cytoplasm of oligodendrocytes in the cerebral cortex,substantia nigra of midbrain,facial nucleus(7N)and thalamus.MCM5 and Iba1 are co-labeled in the cytoplasm of microglia in cerebral cortex,hippocampus,striatum and substantia nigra of mice.MCM5 and GFAP are not co-labeled in astrocytes of cerebral cortex,hippocampus,striatum and substantia nigra of midbrain.7.We found that the positive signal of MCM5 existed in the cerebral cortex,striatum,hippocampus,thalamus and substantia nigra of normal adult and aged mice by immunofluorescence staining,but the positive signal of MCM5 in aged mice was obviously stronger than that in adult mice.protein western blot once again proved that the expression of MCM5 in aged mice was higher than that in adult mice.Conclusion:1.MCM5 is widely distributed in the brain of mice,and it is diffusely positive in some areas of olfactory bulb,cerebral cortex,basal nucleus of brain,septal area,hippocampus,thalamus,hypothalamus,posterior thalamus,midbrain and cerebellum,especially in the dense part of substantia nigra,and strongly positive in the granular cell layer,corpus callosum and outer capsule of olfactory bulb.This shows that MCM5 may have a wide range of functions in the brain,especially in the substantia nigra.MCM5 was expressed in the cerebral cortex,hippocampus,striatum,substantia nigra and thalamus of Tibetan Emirates monkey,which indicated that the expression of MCM5 was conservative between the two species.2.MCM5 exists in neurons,microglia,oligodendrocytes,cholinergic neurons,glutamatergic neurons,GABA neurons and dopaminergic neurons,but in the same type of nerve cells in different brain regions,MCM5 positive signals may exist only in the nucleus or cytoplasm,or both.The expression of MCM5 in the cytoplasm of dopaminergic neurons is relatively specific and strongly positive,which indicates that MCM5 may have a more specific role in dopaminergic neurons.MCM5 was negative in astrocytes.3.MCM5 was expressed in the cerebral cortex,striatum,hippocampus,thalamus and substantia nigra of normal adult and aged mice,but the expression of MCM5 in aged mice was significantly higher than that in adult mice.protein western blot once again verified this conclusion,indicating that its expression had age differences.