| Background:Rab GTPase is by far the largest family of the Ras superfamily,they function as multifaceted coordinators in the regulation of intracellular membrane trafficking,which includes vesicle budding,uncoating,motility,fusion and the maintenance of membrane identity.Rab proteins possess four GDP/GTP binding and GTPase domains(switch Ⅰ throughⅣ),and a C-terminal motif for membrane binding.Most known phosphorylation sites in Rab proteins reside in the highly conserved switch Ⅱ motif,the Thr or Ser site of which could be phosphorylated by Parkinson’s disease(PD)associated LRRK2 kinase,and finally contributes to the pathogenesis of PD.Among the dozens of Rab GTPases,Rab 10 appears to be rather distinctive and attracts our great attention.Firstly,Rab 10 is ubiquitously distributed in intracellular membranes and is implicated in a myriad of membrane trafficking processes,such as endoplasmic reticulum dynamics,exocytic transport,axon development,ciliary transport and ciliogenesis,lysosome tubulation and exocytosis,and autophagy.Secondly,the function of Rab 10 varies with cell types and the stages of development involved.Most importantly,Rab 10 and its phosphorylation have been implicated in the pathogenesis of neurodegenerative diseases.Aberrantly enhanced LRRK2 kinase activity and concomitant elevated Rab 10 phosphorylation have been detected in the brains of sporadic and familial PD patients.There are also compelling evidences for the involvement of Rab 10 in the pathogenesis of Alzheimer’s disease(AD).Immunocytochemical analysis reveals that pRab 10-T73 is enriched in the hippocampus specimens of AD cases and highly co-localized with neurofibrillary tangles(NFTs),without any salient changes in total Rab10 level.Moreover,systematic proteomic and phosphoproteomic analysis identifies Rab10 as the substrate with the strongest affinity to LRRK2.Therefore,it is of great importance to uncover the physiological and pathological role of Rab10 and its phosphorylation.Recently,several studies have focused on the effect of Rab10 Thr73 phosphorylation by the PD related LRRK2 mutants.To be specific,studies revealed that Rab10 phosphorylation could strengthen its intrinsic ability to block ciliogenesis and promote deciliation.Furthermore,LRRK2 could be recruited to stressed lysosomes and phosphorylate Rab10,Rab8a and Rab35 therein,facilitating the tubulation and secretion lysosome.A recent study reveals that Rab10 and its phosphorylation have also been linked to mitophagy:Rab10 accumulates on depolarized mitochondria in a PINK1-and PRKN-dependent manner,recruits the autophagy receptor OPTN and facilitates mitophagy,which could be impaired by LRRK2 phosphorylation.However,these studies were mainly carried out by LRRK2 mutant cell models in vitro,and failed to decipher the role of Rab10 Thr73 phosphorylation in vivo under general physiological conditions,notably in the central nervous system,which is also of great importance and remains to be fully elucidated.Aims:1.To analyze the effect of Rab10 Thr73 phosphorylation on mouse behaviors.2.To find out the responsible brain region and cell type affected by Rab10 Thr73 phosphorylation.3.To investigate the effects of Rab10 Thr73 phosphorylation on the morphology and function of neurons in the brain and the underlying molecular mechanisms.Methods:1.Generation of non-phosphorylatable and phosphomimetic Rab10 mutant mice modelsNon-phosphorylatable Rab10 T73V and phosphomimetic Rab10 T73D mutant mice were generated using CRISPR/Cas9-mediated gene targeting.The mutation and the expression of Rab10 were confirmed by DNA sequencing and western blot(WB)analysis.2.Analysis of the behavioral phenotypes of Rab10 mutant mice3-month-old Rab10T73V/T73V mice,Rab10T73D/+mice at the age of 3,5,7,and 9 months,and age-matched littermates were subjected to behavior assessment batteries,including open field test(locomotor activity and anxiety-like behavior),elevated plus maze(anxiety-like behavior),light/dark box(anxiety-like behavior),Y maze(working memory),zero maze(anxiety-like behavior),T maze(working memory),marble-burying test(repetitive,compulsion-like behavior),nestlet-shredding test(repetitive,compulsion-like behavior),Barnes maze(long-term spatial memory),accelerating rotarod(motor coordination),tail suspension test(depression-like behavior),forced swimming test(depression-like behavior),fear conditioning(emotional memory),metabolic caging(energy metabolism),and novel object recognition test(recognition memory).3.Analysis of the changes in synaptic structure and function of Rab10 mutant miceTo identify the brain regions responsible for the behavioral deficits,we screened five anxiety-related brain areas of Rab10T73V/T73V mice,namely the striatum(STR),amygdala(AMY),dorsal hippocampus(DH),ventral hippocampus(VH),and prefrontal cortex(PFC),by immunoblotting to analyze the expression levels of VAMP1,VAMP2,Syntaxin 1,SHANK2,Synaptophysin,PSD95 and other synaptic proteins.Then we took the responsible brain region,STR,for further analysis.To determine the types and domains of the synapses affected,we recorded the miniature inhibitory postsynaptic current(mIPSC)and miniature excitatory postsynaptic current(mEPSC)by whole-cell patch-clamp recordings.The density of synapses,the density and diameter of synaptic vesicles,and the length and width of postsynaptic density(PSD)were observed by transmission electron microscopy.Immunofluorescent staining of inhibitory presynaptic and postsynaptic markers GAD67 and Gephyrin,and the excitatory presynaptic and postsynaptic markers VGLUT1 and PSD95,were performed to analyze the density of inhibitory and excitatory synapses in STR.4.Analysis of the density of neurons and glial cells in Rab10 mutant miceIn order to evaluate the density of neurons in cerebral cortex,DH and STR,Nissl staining was performed for 3-month-old mutant mice,and panoramic images were taken using VS 120 Virtual Slide Microscope.Immunohistochemical staining of Ibal and GFAP was performed to identify the density of microglia and astrocytes in STR respectively.5.Analysis of the development of dendritic branches and spines in Rab10 mutant miceCoronal sections from 3-month-old mutant mice were stained using the FD Rapid GolgiStain kit.Panoramic images of z-stacks of Golgi-stained dendrites were taken at 20 ×magnification by VS 120 Virtual Slide Microscope for STR,DH,VH and AMY.Then,the number of branches,average dendritic length and total dendritic length were counted and measured.The mRNA and protein levels of dendritic marker MAP2 were measured by real-time quantitative polymerase chain reaction(RT-qPCR)and immunofluorescent staining.In addition,E16.5 primary cortical neurons were collected.After 3 days of culture,immunofluorescent staining of neurofilament and MAP2 were performed to calculate the number and length of axons and minor neurites.Subsequently,Golgi stained sections were photographed along the z-axis with 63 × oil lens,the density and the types of dendritic spines were analyzed.6.Differential interacting proteins of Rab10 mutant were detectedTotal protein lysate was collected from 3-month-old Rab10+/+ and Rab10T73V/T73V mice using natural cell lysis buffer,and Rab10 protein was immunoprecipitation(IP)from the tissue by Rab10 antibody.Then the IP samples were analyzed by Tandem Mass Tag(TMT)-based quantitative proteomics to find out the proteins differentially interact with wild type Rab10 and Rab10 T73V mutant.Finally,those proteins were subjected to bioinformatics analysis,to identify the molecular signaling pathways involved.Results:Part Ⅰ:The effect of Rab10 T73V mutation on mice1.Rab10 T73V mutant mice exhibit anxiety-like behaviorCompared to Rab10+/+mice,Rab10T73V/T73V mice displayed significantly reduced activity in the center of the open field.Similarly,in the elevated plus maze and the zero maze,the Rab10T73V/T73V mice spent less time in the open arms or open sections.Rab10T73V/T73V mice also exhibited significantly decreased activity in the light chamber during the light/dark exploration task.Of note,the Rab10T73V/T73V mice displayed remarkable impairments in repetitive,compulsion-like social behaviors,because they shredded conspicuously fewer nestlets in the nestlet shredding test and buried significantly fewer marbles in the marble burying test compared to the wild-type group.In contrast,no discernible abnormalities were detected between Rab10T73V/T73V and Rab10+/+ mice,in terms of locomotor activity,cognitive function,depression-like behaviors,motor coordination and energy metabolism.Thus,deletion of the Thr73 phosphorylation in Rab10 gives rise to anxiety-like behavior.2.Rab10 T73V mutant mice show synaptic dysfunctionAmong the five anxiety-related brain areas screened,only STR showed intergroup differences in the expression of synaptic proteins.More specifically,in the STR of the Rab10 T73V mutant mice,a slight increase was observed in the postsynaptic protein SHANK2,and three presynaptic proteins,namely the SNARE proteins VAMP1,VAMP2 and Syntaxin 1 were elevated to a remarkable degree.Moreover,in recordings from the striatal MSNs,we found that the frequency,but not the amplitude,of mIPSC was selectively reduced by the Rab10 T73V mutation.In contrast,we observed no changes in the amplitudes or frequencies of the mEPSC.Transmission electron microscopy revealed that the synaptic densities,thelength and thickness of PSD,the density of synaptic vesicles,and the vesicle diameters were quite similar in the STR of the two groups.Immunostaining demonstrated that Rab10 T73V mutation did not induce any significant alteration in excitatory and inhibitory synapses.Collectively,these data suggest that the Rab10 T73V mutation disturbs the inhibitory synaptic transmission in STR,while the structure of synapses is significantly affected by the mutation.3.Rab10 T73V mutant mice do not show any changes in the density of neurons,microglia and astrocytesNo alteration in neuronal density was detected between Rab10T73V/T73V mice and Rab10+/+mice in cerebral cortex,hippocampus and STR.At the same time,there was no significant difference in Ibal-labeled microglia and GFAP-labeled astrocytes between the two groups.Therefore,Rab10 T73V mutation does not significantly affect the density of neurons and glial cells in the brain of mice.4.Rab10 T73V mice display altered neuronal morphology in STRSignificant increases in neurite number and total dendritic length were observed in striatal MSNs of Rab10 T73V mice,with no change in the average dendritic length.The effect of Rab10 phosphorylation on neuronal morphology appeared to be restricted to STR,as no arborization abnormality was observed in DH,VH and AMY.Consistently,there was an increase in the dendritic marker MAP2 expression in the T73V STR.Taken together,these results show that T73V mutation elicits neurite over-branching phenotypes in striatal MSNs,and induces neuronal dysfunction.5.Rab10 T73V mice display altered neuronal morphology in STRNo differences were observed in spine density between Rab10T73V/T73V and wild-type mice.Subsequent spine classification found that mature spines(stubby,mushroom,and branched)were relatively more prevalent in Rab10T73V/T73V mice,and immature spines(filopodia,long thin and thin),especially thin spines,were less prevalent.Likewise,the maturation processes of dendritic spines were largely unaffected in other brain regions.This suggests that the Rab10 T73V mutation accelerates the maturation of dendritic spines in striatal MSNs.6.The T73V mutation alters the Rab10 interacting protein network associated with cytoskeleton assemblyIn order to explore the effect of Rab10 T73V mutation on the function of Rab10 itself,Rab10 protein in the STR of Rab10+/+and Rab10T73V/T73V mice was extracted by IP,and analyzed by TMT-based quantitative proteomics.There were 89 proteins interacting differentially with Rab10,among which 62 proteins had increased affinity with Rab10 T73V mutant and 27 proteins had decreased affinity.Further GO enrichment analysis revealed that most of these proteins were located in cytoskeleton,synapses,and dendritic spines.In addition,they were closely associated with neurofilament bundle assembly,actin filament capping,synaptic vesicle endocytosis,axonogenesis,and neuron projection development.These results suggest that the effects of Rab10 T73V mutation on neuronal dendritic branches,dendritic spines and synaptic function may result from the regulation of phospho-Rab10 on neuronal cytoskeleton.Part Ⅱ:The effect of Rab10 T73D mutation on mice1.Homozygous Rab10 T73D mutant mice display abnormal brain,lung,and kidney development,and die at or shortly after birthMost Rab10T73D/T73D mice died at birth or a few hours after birth,and only one Rab10T73D/T73D mouse was found among the 77 mice obtained at the time of weaning.In addition,compared with Rab10T73D/+and Rab10+/+neonatal mouse,the weight of brain and kidney of Rab10T73D/T73D neonatal mice significaitly decreased,while the weight of lung was significantly increased.HE staining found the collapsed alveolar and atelectasis in the lung of Rab10T73D/T73D neonatal mice,which might result in the death of neonatal mice.2.Heterozygous Rab10 T73D mutant mice behave largely normal at 9 months of ageAt the age of 3,5,7,9 months,Rab10T73D/+mice behaved nearly the same as Rab10+/+mice in open field test,elevated plus maze,Y maze,zero maze,novel object recognition test,and T maze.Thus,Rab10T73D/+mice did not exhibit any phenotypes in anxiety-like behavior and cognitive function at least till 9 months of age.3.Rab10 T73D mutation inhibits the formation of dendritic branches in brainThe only remaining 3-month-old Rab10T73D/T73D mouse and it wild-type littermate were subjected to Golgi staining.It was found that DH CA1 pyramidal neurons of Rab10T73D/T73D mice possessed fewer branches and exhibited shorter total dendrite length,but the average dendrite length was not affected.The number of basal dendrites and the total dendrite length also decreased by 13.68%and 16.68%respectively,although the difference did not reach statistical significance.The number of branches in Rab10 T73D mutant striatal MSNs was also reduced compared with that of the wild type,but the average and total dendrite length did not change.Rab10 T73D mutation also inhibited the development of axons of primary neurons.Compared with Rab10+/+neurons,the number of axons of Rab10T73D/T73D neurons was reduced,but the total length of axons did not change significantly.The number of of Rab10T73D/T73D neurons remained unchanged,while the total minor neurite length increased.These results suggest that Rab10 T73D mutation could impair dendrite ramification,and this effect is universal and not restricted to specific brain regions.4.Rab10 T73D mutation reduces the density of dendritic spines in brainCompared with Rab10+/+ mice,the density of dendritic spines in DH CA1 pyramidal neurons and striatal MSNs of Rab10T73D/T73D mice decreased significantly.Further classification revealed that the proportion of different types of dendritic spines remained unchanged,although the density of mature and immature dendritic spines of Rab10 T73D mutant neurons both decreased.Therefore,Rab10 T73D mutation mainly impairs the density of dendritic spines,but had little effect on the maturation process of dendritic spines.Conclusions and innovation:1.Rab10 Thr73 phosphorylation probably plays an important role in the regulation of the striatal circuits for anxiety,and the non-phosphorylatable T73V mutation of Rab10 could induce anxiety-like behaviors in mice.2.Rab10 Thr73 phosphorylation may affect the release of inhibitory presynaptic neurotransmitter by regulating the function of SNARE complex in the striatum.3.Rab10 Thr73 phosphorylation plays an important role in dendritic arborization and the development of dendritic spines.The non-phosphorylatable T73V mutation of Rab10 specifically enhances neurite arborization and accelerates spine maturation in striatal MSNs.While phosphomimetic Rab10 T73D mutation inhibits the formation of dendritic branches and spines in the brain.4.Non-phosphorylated Rab10 is necessary for the development of brain,lung and kidney.The phosphomimetic T73D mutation of Rab10 causes perinatal death of mice.5.In this study,we used non-phosphorylatable and phosphomimetic Rab10 mutant mouse models and revealed the physiological significance of Rab10 Thr73 phosphorylation in the brain.This study could provide cues for understanding any side effects that may be observed during the development of LRRK2-based clinical treatments,as well as novel diagnostic and therapeutic targets for treating neurodegenerative diseases,such as AD and PD. |
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