Central Nervous System Effect Of Microglial VPS35 Knockout And Its Role In Ischemic Brain Injury

The retromer complex is a multimeric protein complex which facilitates intracellular trafficking of a variety of transmembrane proteins.Vacuolar protein sorting-associated protein35?VPS35?is a critical component of retromer's cargo recognition subunit and has been implicated in neurodegenerative disease pathology,including Alzheimer's disease?AD?.Without functional VPS35,trafficking of retromer cargo is often impaired,compromising cargo function and/or downstream signaling events.VPS35 expression is ubiquitous and can be found throughout the central nervous system?CNS?.Microglia express VPS35,and microglia from AD patients exhibit reduced VPS35 expression.We sought to determine how microglial VPS35 loss-of-function might contribute to neurodegenerative disease pathology.Ischemic stroke is one of the most common brain diseases,accounting for 70%of cerebrovascular diseases.It is the fourth leading cause of death in the United States and Europe,and a major cause of adult disability.It is thus of considerable interest to investigate the cellular and molecular mechanisms of ischemic brain damage.Although many progresses have been achieved,it remains to be a detrimental disease without an effective treatment in clinic.Thus,it is necessary to further investigate its pathological mechanisms and intervention targets for the development of a better therapy.Therefore,we attempt to explore the role of microglia VPS35 in neurodegenerative diseases and ischemic brain injury.This study is mainly divided into the following two parts.In the first part,we tried to explore the role of microglia VPS35 in neurodegenerative diseases.RT-PCR was used to detect the expression of VPS35 gene at E17,P7,P14,P21 and P60 time points.It was found that VPS35 was not only expressed in neurons,but also widely expressed in cell bodies and processes of microglia.The expression of VPS35 varied in different age stages of mice?E17-P60?,peaked on the 14th day of P14,then decreased gradually.The level of VPS35 increased significantly after LPS stimulation of microglia,suggesting that VPS35 is related to LPS-stimulated microglia response.Then we developed VPS35^CX3CR1 mice by Cre-LoxP gene editing system for the very first time,a model which induces microglial-specific VPS35 depletion.Microglial VPS35 loss-of-function upregulated microglial density in a region-specific manner,which we determined to be an effect of increased microglial differentiation/survival.Upon further inspection,we found evidence of upregulated microglial inflammatory activity in the hippocampus,including increased levels of proinflammatory cytokine interleukin-6.Microglial density was increased within the subgranular zone?SGZ?of VPS35^CX3CR1 mice,so we sought to determine if microglial VPS35loss-of-function has any effect on hippocampal neurogenesis.In order to detect the proliferation of microglia,we used BrdU?5-Bromo-2-deoxyUridine,5-bromo-deoxyuridine?as a marker of cell proliferation.The results of brain slice staining after 24 hours showed that there was no significant difference in the proliferation of microglia between knockout group and control group.These results suggest that increased microglia are not the source of proliferation.After 7 days of BrdU labeling,we detected the differentiation or migration of microglia.The results indicated that BrdU labeling increased significantly in VPS35 knockout group.These results suggest that the increased microglia may come from increased differentiation or migration.While we found decreased doublecortin+?DCX?immature neurons,an increase was observed in the differentiation/survival of neural progenitor cells?NPCs?.Further analysis suggested the cell cycle exit of VPS35^CX3CR1 hippocampal NPCs may be compromised.To examine morphology of newborn neurons,hippocampi were labeled with a retroviral vector,revealing impaired dendritic development in newborn hippocampal VPS35^CX3CR1 neurons.Aberrant regulation of hippocampal neurogenesis in VPS35^CX3CR1 mice was associated with a depressive behavioral phenotype and long-term memory impairment.Then,primary microglia and primary neurons were co-cultured to observe the changes of phagocytic function of microglia lacking VPS35 and its effects on neurons.Through a variety of behavioral experiments,it was confirmed that mice knocked out of microglia VPS35 had long-term memory impairment and behavioral manifestations similar to anxiety.In the second part,microglia VPS35 has been found to affect microglia activation,and microglia activation is closely related to inflammation.Therefore,we used the ischemic brain injury model with neuroinflammation as the main pathological changes to explore whether microglia VPS35 plays a role in it.We used the microglial VPS35 cKO?conditionally knocking out?mice and examined their response to photo thrombotic cortical stroke injury.Cortical brain injury response was assessed by a combination of methods,TTC?2,3,5-triphenyltetrazolium chloride,2,3,5-triphenyltetrazolium chloride?staining,Nissl's staining,immunohistochemistry and immunofluorescence were used to evaluate the size of the lesion site and the severity of neuronal cell death after ischemic brain injury in mice.The results showed that the infarct area of cerebral cortex in VPS35^CX3CR1 mice decreased significantly 3 days after injury,and the number of dead neurons was significantly lower than that in the control group.Mice were also subjected to neurological functional behavior tests in different time point post injury.Immunofluorescence and Western blot were used to detect the difference of TMEM119?Damage Associated Microglia?marker in the first stage and LPL?Damage Associated Microglia?marker in the second stage after ischemic injury.The results showed that TMEM119 positive microglia increased significantly in the VPS35 knockout group in the periinfarct area,while LPL positive microglia decreased significantly in the VPS35 knockout group.It is suggested that knockout of VPS35 in microglia can interfere with the production of DAM in two stages.We provide evidence that microglial VPS35-loss regulates microglial polarization in mouse cortical brain in response to ischemia/stroke.The Ischemia/stroke-induced injury response is attenuated by microglial VPS35-deficiency.Further studies have observed a reduction in CX3CR1 receptor levels in microglial VPS35-deficient cortical brain in response to ischemia/stroke,implicating CX3CR1 as a potential cargo of VPS35.These findings implicate a novel microglial-specific role of VPS35 that might contribute to neurodegenerative disease pathogenesis.The full extent of this role remains to be determined,as well as the mechanisms underlying our observations.It also remains to be determined what role microglial VPS35 might play in other brain regions,and how microglial VPS35 depletion might contribute to other aspects of neurodegenerative disease pathology.These results reveal an unrecognized function of microglial VPS35 in suppressing cortical microglial?M1 to M2?polarization change,and pro-inflammatory?M1?microglia are likely to be?toxic?for cortical ischemic brain injury response.In summary,our study reveals,for the very first time,that VPS35 is essential for maintaining microglial homeostasis.When the VPS35 function of microglia is destroyed in CNS,the effect is not only the microglia itself,but also the key process of neurogenesis by interfering with the differentiation and proliferation of peripheral neural stem cells and neural precursor cells.In addition,we found that the loss of microglia VPS35 could play a protective role in the model of cortical ischemic brain injury.The protective effect may be due to the change of activation polarity of microglia after ischemic injury,which enhances neuroprotective effect and alleviates neuroinflammatory reaction.At the same time,CX3CR1may be a potential cargo protein of microglia VPS35 in ischemic brain injury response,but further research is needed to verify it.Exploring the molecular mechanism of disease pathology is the first step in disease treatment and discovery of therapeutic methods.Our findings provide important experimental evidence for exploring disease-related cargo proteins and mediators in neurodegenerative pathways.