【作者】 李欣；

【导师】 郑红花；

【作者基本信息】 厦门大学 ， 药理学， 2021， 硕士

【摘要】 成年起病的白质脑病伴轴索球样变和胶质细胞色素沉着(adult-onset leukoencephalopathy with axonal spheroids and pigmented glia,ALSP)是一种遗传性进行性的罕见神经退行性疾病,临床主要表现为执行功能障碍、记忆能力衰减、人格改变、运动障碍和癫痫等,迄今尚无有效治疗措施。目前已有多篇文献研究指出集落刺激因子 1 受体(colony stimulating factor 1 receptor,CSF1R)是ALSP的致病基因。CSF1R在中枢神经系统中高表达于小胶质细胞上,但是,CSF1R如何引起ALSP疾病发生,其机制尚未阐明。在本研究中,我们采用CRISPR/Cas9技术构建Csf1r+/-(heterozygous,HE)小鼠以模拟临床ALSP疾病表型,通过将HE小鼠雌雄交配,获取原代分离培养Csf1r+/+(wildtype,WT)、HE、Csf1r-/-(homozygous,HO)三种基因型小胶质细胞,以小胶质细胞特异性标记蛋白Iba1和活化吞噬标记蛋白CD68免疫荧光双标染色,观察到HE小胶质细胞普遍呈现激活状态;这一现象在HE小鼠中进一步得到证实,即HE小鼠脑中小胶质细胞亦较其同窝对照WT小鼠呈现显著激活状态。动物行为学实验表明,HE小鼠于6月龄即出现运动、认知、情绪及嗅觉等方面的异常。米诺环素(Minocycline,Mino)是一种四环素类广谱抗生素,能有效抑制小胶质细胞活化,在多种神经退行性疾病模型中发挥有效的抗炎、抗凋亡和神经保护作用。那么Mino能否通过抑制HE小鼠小胶质细胞的活化而达到治疗目的呢?因而,我们首先用0.02 μM Mino分别处理WT和HE小胶质细胞0、6、12、24小时,观察了 Mino对两种基因型小胶质细胞的影响,通过小胶质细胞特异性标记蛋白Iba1和活化吞噬标记蛋白CD68免疫荧光共定位实验,我们发现Mino可以有效抑制HE小胶质细胞的活化。我们进一步通过实时定量荧光PCR(quantitative real-time PCR,qRT-PCR)实验观察促炎因子Tnf-α、Il-1β的转录水平表达,发现HE小胶质细胞Tnf-α和Il-1β的转录水平表达较同窝对照WT小胶质细胞显著上调,用Mino药物处理可以有效降低HE小胶质细胞Tnf-α、Il-1β的转录水平表达,说明Mino可以有效改善HE小胶质细胞促炎因子的释放。接着以50 mg/kg/d剂量腹腔注射(intraperitoneal injection,i.p.)小胶质细胞活化抑制剂Mino干预治疗WT和HE小鼠一个月,通过Iba1和CD68免疫荧光双标实验和实时定量荧光PCR实验,发现Mino在体内水平中也可有效抑制HE小鼠小胶质细胞的活化,降低促炎因子Tnf-α和Il-1β的表达。综上,Mino在体内外水平中均可有效抑制HE小鼠小胶质细胞的活化,降低炎症水平。接下来,通过磁共振成像(magnetic resonance imaging,MRI)技术发现HE小鼠侧脑室增大和胼胝体变薄的情况,而在Mino干预治疗后可以有效抑制HE小鼠的这种影像学改变;采用透射电子显微镜(transmission electron microscope,TEM)观察到Mino可以显著改善HE小鼠髓鞘丢失以及突触密度减少等病理表型。最后,我们探究了 Mino药物干预治疗后对HE小鼠行为学的影响。通过平衡木实验观察发现Mino可以有效降低HE小鼠通过平衡木的时间及打滑次数,说明Mino可以有效改善HE小鼠运动能力方面的缺陷;通过T迷宫实验观察发现Mino可以有效减少HE小鼠在转换臂内的转换次数百分比,说明Mino可以有效改善HE小鼠工作记忆能力;接下来通过旷场和黑白箱实验观察发现Mino可以明显增加HE小鼠在明亮处以及白箱中停滞的时间,说明Mino可以有效改善HE小鼠的焦虑情绪;通过糖水实验观察也发现经Mino干预治疗后,HE小鼠对糖水的偏好量增多,说明Mino同样可以改善HE小鼠的抑郁行为;通过埋藏食物实验观察发现用Mino干预治疗后,HE小鼠找到埋藏食物的时间减少,说明HE小鼠嗅觉方面的缺陷也得到改善。综上,我们发现,HE小鼠在运动、认知、情绪及嗅觉方面均有缺陷,采用Mino腹腔注射一个月进行药物干预治疗后,HE小鼠在运动、认知、情绪及嗅觉等方面的缺陷均显著改善。综上所述,我们的研究结果表明,CRISPR/Cas9技术构建的HE小鼠可以模拟临床ALSP疾病表型,该模型中小胶质细胞呈现出与人类ALSP疾病类似的小胶质细胞活化情况,用小胶质细胞抑制剂米诺环素处理,可以显著改善HE小鼠的运动能力、学习记忆、抑郁、焦虑及嗅觉等方面的缺陷。本研究将为ALSP疾病的治疗提供新的方案,亦为临床上老药新用提示新的方向。更多还原

【Abstract】 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.更多还原