The Mechanism Of CSF1R Involved In The Occurrence Of ALSP And Drug Intervention Research

Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia(ALSP),a subgroup of adult-onset leukodystrophy,is a progressive neurodegenerative white matter disease caused by mutations in the colony stimulating factor 1 receptor(CSF1R)gene,which is highly expressed on microglia in the Central Nervous System(CNS).The main clinical manifestations of ALSP are executive dysfunction,memory decline,personality changes,movement disorders and epilepsy,etc.However,there are no therapeutic approaches for ALSP so far.Although the human CSF1R gene was recognized as the site of point mutations underlying ALSP,how CSF1R deficiency involves in the pathogenesis of ALSP and the effective therapies for ALSP remain elusive.In this study,Csf1r+/-(heterozygous,HE)mouse model was generated by the CRISPR/Cas9 system.By mating HE mice,Csf1r+/-(wild type,WT),HE and Csf1r-/(homozygous,HO)perinatal littermates and the primary microglia were obtained,respectively.WT,HE and HO microglia were double stained with Iba1,the specific marker of microglia,and CD68,the phagocytic marker of microglia.Compared with WT microglia,HE microglia generally showed an activated state,which was further confirmed in HE mouse brain.Six-month-old HE mouse showed abnormalities in motor,cognition,emotion and smell.Minocycline(Mino)is a widely used tetracycline antibiotic that could suppress neuroinflammation in a variety of rodent models of neurodegenerative diseases.Given the over activation of microglia in HE mouse brain,it’s interesting to explore whether Mino can rescue the abnormal phenotype of HE mice by inhibiting microglial activation.WT and HE microglia were treated with 0.02 μM Mino for 0,6,12 or 24 hours.Ibal and CD68 fluorescent double-staining showed that Mino effectively inhibited the activation of HE microglia in vitro.We further observed the expression of Tnf-α and Il-1β using quantitative real-time PCR,and found that they were both up-regulated in HE microglia compared with those in WT microglia.Furthermore,the mRNA levels of Tnf-α and Il-1βwere obviously decreased in Mino-treated HE microglia when compared with those in Mino-treated WT microglia,which were further confirmed in HE and WT mouse brain.Thus,Mino can effectively inhibit the activation and inflammation levels in HE mouse microglia both in vivo and in vitro.Given the strong response to Mino in the prevention of microglial activation,we next analyzed if Mino restores the pathological and behavior phenotypes in HE mice.Using magnetic resonance imaging(MRI)assay,we found that Mino attenuated the enlarged lateral ventricle and the thin corpus callosum in HE mouse brain.Simultaneously,the pathological phenotypes such as the demyelination and the decreased synaptic density in HE mouse brain were all improved by Mino,as visualized by transmission electron microscope(TEM)assay.We next studied the impact of Mino on motorial behavior.The motorial ability was evaluated using the beam-walking test.Compared to Saline treatment,HE mice were able to pass the balance beam in a shorter time and the number of slips was obviously decreased in response to Mino administration.We then studied the impact of Mino on spatial working memory.The spatial working ability was evaluated using the T-maze test.Remarkably,Mino treatment in HE mice resulted in better memorial performance compared to those in Saline-treated ones as the Mino-treated HE mice were able to navigate and identify new objects in a shorter time.Furthermore,open field test and lightdark transtition test were conducted to study the impact of Mino on anxious behavior in HE mice.Significantly,the time staying in the bright place and white box was reduced in Mino-treated HE mice compared to that in Saline-treated mice.Depression-like behavior in HE mice was also observed by sucrose preference test.Compared to Saline treatment,Mino remarkably increased the sucrose intake of HE mice indicating that Mino improved the depressive behavior of HE mice.Finally,the olfactory ability of HE mouse was observed through the buried food test.Compared to Saline treatment,the time for HE mice to find buried food was significantly decreased in response to Mino treatment.These results showed that the deficiencies of motorial,memorial,emotional and olfactory abilities in HE mice were all improved by Mino administration.In summary,this study suggested that HE mouse model generated by the CRISPR/Cas9 system is sufficient to simulate the clinical phenotypic characterization of ALSP and the tetracycline antibiotic Mino can be a potential therapeutic candidate for ALSP.