突触中突变亨廷顿蛋白通过抑制突触素Ⅰ磷酸化导致突触功能障碍
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摘要
亨廷顿病（Huntington’s disease，HD）是一种家族遗传性神经退行性疾病，临床上，以运动障碍、精神异常和痴呆为特点；病理学上，以纹状体和大脑皮质神经元选择性死亡为特征。HD由亨廷顿基因第一外显子中的CAG三核苷酸序列过度重复（>35）编码突变亨廷顿蛋白（mutant huntingtin，mHtt）所致。正常Htt主要表达、定位于神经元细胞质内，与细胞内的膜结构和许多细胞器如线粒体、高尔基体、内质网、内吞小体、自噬囊泡的功能密切相关。mHtt在神经元胞质和核内形成不溶性聚集物，且聚集物的数量随着年龄的增长逐渐增多。mHtt可通过影响基因表达、线粒体功能、细胞内运输、新陈代谢产生毒性作用。突触作为神经元特有的结构容易受到毒性蛋白的损害，突触功能障碍常见于多种神经退行性疾病中。尽管许多研究已经对HD中突触功能障碍有一定的探索，但mHtt是否以及如何直接导致HD的神经功能障碍的机制目前尚不明确，其对于神经细胞突触功能的影响也有待进一步研究。正因为神经元具有特有的突触结构，所以探讨突触mHtt是否可以导致年龄相关性的HD神经症状和早期死亡，以及突触mHtt是否直接引起突触神经递质释放功能的障碍，对区分mHtt对各个亚细胞区的功能影响至关重要。
本实验通过构建表达突触小体相关蛋白（synaptosomal-associated protein25，SNAP25）与Htt融合蛋白（SNAP25-Htt20Q/150Q）的质粒，制备一种将mHtt选择性地靶向定位在突触前细胞质中的新型HD转基因小鼠模型：SNAP25-Htt150Q小鼠和相应的SNAP25-Htt20Q对照小鼠，并应用这种新的HD转基因小鼠模型对突触mHtt的毒性及其相关机制进行研究，以期揭示突触mHtt在HD的致病机理中的作用。
1.突触Htt在SNAP25-Htt转基因小鼠神经元突触中选择性表达
采用小鼠胚胎原核注射方法建立转基因小鼠模型，挑选出可以稳定繁殖、转基因表达位置及表达量明确的SNAP25-Htt20Q/150Q品系。比较SNAP25-Htt20Q/150Q转基因小鼠品系转基因mHtt表达水平结果表明，SNAP25-Htt150Q表达水平低于SNAP25-Htt20Q中的表达水平。同时，与利用小鼠内源性亨廷顿基因启动子表达HttCAG150的基因敲入(knock in，KI)小鼠[1]相比，SNAP25-Htt150Q mHtt表达水平低于Htt CAG150全长mHtt内源性表达水平。
亚细胞器差速离心方法可以富集突触蛋白，用此方法提取SNAP25-Htt20Q/150Q转基因小鼠大脑皮质突触蛋白结果显示，两种融合蛋白都富集在突触前组分，并且该组分中也发现了Htt的降解片段。用抗Htt抗体(mEM48)对SNAP25-Htt150Q转基因小鼠脑片进行免疫组织化学染色表明，SNAP25-Htt150Q在大脑皮质和纹状体中形成细胞核外神经毡聚集物。电子显微镜(EM)结果也进一步证实mHtt聚集物存在于轴突和突触前细胞质中，而不存在于突触后细胞质和突触前膜。
Western Blot和免疫荧光双标结果显示，SNAP25-Htt150Q在神经突起中形成聚集物，而大多数Htt的聚集物不能被SNAP25抗体所标记，表明SNAP25-Htt150Q的蛋白质水解可能会导致mHtt片段缺失SNAP25表位并更容易聚集。尽管SNAP25靶向性地将mHtt定位在突触前，但可能有部分SNAP25-Htt150Q由于蛋白质水解作用导致融合蛋白发生断裂。
通过亚细胞器分离、免疫荧光染色和电子显微镜的手段，充分证明了mHtt转基因产物选择性地表达在转基因小鼠的突触前细胞质，并且主要由切割后的不含有SNAP25的mHtt片段形成聚集物。因此，本课题成功构建了将mHtt特异性表达在突触前细胞质中的一种新型HD小鼠模型。
2．SNAP25-Htt150Q转基因小鼠HD症状随年龄进行性加重
为了判断SNAP25-Htt150Q小鼠是否出现类似于大多数HD转基因小鼠模型的症状，对SNAP25-Htt转基因小鼠进行了行为学检测，发现老龄SNAP25-Htt150Q转基因小鼠出现典型的HD表型，如：体重减轻、抱屈反射和弓背外观。该表型呈年龄相关性地渐进性发展。SNAP25-Htt150Q转基因小鼠早于野生型和SNAP25-Htt20Q转基因小鼠死亡。旋转杆实验、声学刺激、明暗穿箱实验及平衡木行走实验均表明突触mHtt能导致小鼠出现进行性的运动能力下降。
3．突触mHtt引起神经病理学损害并抑制神经递质释放
对小鼠脑片进行星形胶质细胞标记蛋白胶质纤维酸性蛋白(GFAP)抗体染色，发现SNAP25-Htt150Q转基因小鼠皮质、纹状体和小脑各脑区GFAP染色明显增强，提示突触mHtt引起神经元损伤后反应性的神经胶质细胞增生。对GFAP的Western Blot检测也证实SNAP25-Htt150Q小鼠纹状体中星形胶质细胞明显增生。
应用电生理方法检测突触mHtt对突触电活动功能的影响发现，SNAP25-Htt150Q小鼠大脑切片的感觉运动皮质中成对脉冲刺激的振幅(PPF)及长时程增强(LTP)的信号强度明显降低；分离出SNAP25-Htt转基因小鼠的皮质纹状体脑片检测[3H]谷氨酸释放，结果表明SNAP25-Htt150Q脑片谷氨酸释放减少，而[3H]GABA释放并未受到影响，由此表明谷氨酸囊泡释放更容易受到突触mHtt损伤；采用PC12-Htt-exon1-20Q/150稳转细胞系评估突变mHtt是否影响囊泡释放发现，PC12稳转细胞系中[3H]多巴胺释放的测定结果显示mHtt能抑制PC12细胞神经递质多巴胺的释放。
4．突触mHtt与突触素I的C端相互作用增强
突触mHtt是选择性地定位在突触前细胞质，可与突触囊泡异常结合导致突触信号传递障碍。从野生型（wild type，WT）小鼠脑皮质中分离出突触小体蛋白，分别与PC12-Htt-exon1-20Q/150Q稳转细胞系来源的蛋白样品孵育后进行检测，发现相比于PC12-Htt-exon1-20Q，突触小体更容易与PC12-Htt-exon1-150Q结合。为了探索mHtt与突触蛋白的相互作用，用mEM48抗体对SNAP25-Htt转基因小鼠脑皮质突触蛋白进行免疫沉淀，并对沉淀得到的蛋白进行质谱分析，发现了578种蛋白质和1732种多肽，并证实SNAP25-Htt150Q可以沉淀更多的突触素I蛋白。应用SNAP25-Htt转基因小鼠、Htt CAG150KI小鼠脑组织及PC12-Htt-exon1-20Q/150Q稳转细胞系模型，经免疫共沉淀实验显示mHtt相比于正常Htt与突触素I相互作用更强。
突触素I有1a和1b两个亚型，在其各个结构域上含有大量的磷酸化位点，而其C端具有脯氨酸富集结构域这一特殊片段。用删除C端脯氨酸富集结构域的片段化突触素I (tsynapsin)与Htt-exon1-20Q/150Q共转染HEK293细胞后，免疫共沉淀分析显示，尽管突触素Ia和突触素Ib都与mHtt相互作用，但去除脯氨酸富集结构域后则消除了突触素I与mHtt的结合关系。
5．mHtt通过突触素I脯氨酸富集区的相互作用抑制突触素I磷酸化
突触素I参与囊泡锚定、融合、并从突触前膜释放神经递质中的功能，这一过程与突触素I的磷酸化水平直接相关。在HEK293细胞中表达不同片段长度的突触素I，检测mHtt是否影响突触素I的磷酸化水平。比较全长突触素Ib和截断C端的突触素I(tsynapsin) N端9号位点丝氨酸的磷酸化水平显示，Htt-150Q抑制突触素Ib中9号位点丝氨酸磷酸化水平，而与Htt-20/150Q共转染的tsynapsin中9号位点丝氨酸磷酸化水平无明显差异。因此，mHtt对突触素I磷酸化的抑制依赖于mHtt与突触素I的C端脯氨酸富集区结构域的相互作用。
对PC12-Htt稳转细胞系和SNAP25-Htt转基因小鼠脑蛋白进行Western Blot分析，检测突触素I不同位点的磷酸化抗体水平，结果显示，PC12-Htt-exon1-150Q细胞及SNAP25-Htt150Q转基因小鼠脑皮质中内源性突触素I的S9、S549和S603位点的磷酸化水平均明显降低。
以上研究表明，mHtt在突触前可通过与突触素I蛋白C端区域的异常相互作用使多个位点的磷酸化降低，从而抑制突触神经递质释放，导致神经病理学损害与相应神经症状，为探索治疗HD的方法提供了新的靶点。
Huntington’s disease (HD) is an inherited neurodegenerative disease.Clinically, Huntington’s disease is characterized by irrepressible motor dysfunction,psychiatric disturbances and cognitive deterioration to dementia. The pathologicalcharacteristic feature of HD is selectively neurodegeneration preferentially in striataland cortex neurons. HD is caused by CAG repeats (>35CAGs) in exon1ofhuntingtin gene which generates mutant huntingtin (mHtt) with a polyglutamine(polyQ) expansion. Normal huntingtin mainly expresses in cytoplasm of neuron,and is associated with membrane structure and organelle, such as mitochondrion,Golgi, endoplasmic reticulum, endosome and autophagic vacuoles. Mutanthuntingtin leads to formation of aggregates in cytoplasma and nucleus and thedisease progresses over time. We know that mutant htt in different subcellularregions affects multiple cellular functions. For example, mutant htt in the nucleuscan affect gene expression, mitochondrial function, intracellular trafficking, andmetabolism. Considering neuronal cells have unique, long neuronal processes andsynapses that may be vulnerable to toxic proteins, many studies have focused onsynaptic function, revealing that synaptic dysfunction is the common pathologicalevent in a variety of neurodegenerative diseases. Although synaptic dysfunction inHD mouse models has been well-documented, but whether and how synaptic mutant huntingtin directly mediates HD neuropathology remains to be determined.Since synapses are unique to neuronal cells, understanding whether synaptic mHttcould cause age-dependent progression of neurological symptoms and early death,or synaptic mHtt caused neuropathology and inhibited neurotransmitter release iscritical for unraveling the selective neuropathology seen in HD.
Therefore, we fused exon1htt containing normal (20Q) or150Q (150Q) tosynaptosomal-associated protein25(SNAP25) to generate SNAP25-htt transgenicmice: SNAP25-Htt150Q and SNAP25-Htt20Q as control. And we studied thesetransgenic mice which selectively express huntingtin in the presynaptic terminals, tounderstanding whether and how synaptic mutant huntingtin directly mediates HDneuropathology.
Transgenic mutant htt selectively accumulated in the presynapticterminals in transgenic mice. We used SNAP25-Htt20Q/150Q for pronuclearinjection of mouse embryos. Then we selected the SNAP25-Htt20Q/150Q lines thatcould be bred to F1generation and showed the clear expression of transgene.Genomic DNA and Western Blot analysis revealed that transgenic mutant htt wasexpressed at a lower level than control SNAP25-Htt20Q and was notoverexpressed compared to endogenous mutant htt.
To better assess the relative levels of transgenic htt in presynaptic terminals,we performed subcellular fractionation, which could enrich presynaptic proteins,such as SNAP25. The results showed that both SNAP25-Htt20Q andSNAP25-Htt150Q were enriched in the presynaptic fraction. Importantly, degradedhtt fragments were also present in the presynaptic fraction.
We performed immunofluorescent staining, which could better reveal thesubcellular localization of proteins. SNAP25-Htt150Q was clearly present as punctaoutside the nuclei of neuronal cells in the cortex and striatum. To verify that mutanthtt was indeed localized in the presynaptic terminals, we performed electronmicroscopy (EM). EM examination revealed the presence of htt aggregates in theaxons and presynaptic, but not postsynaptic terminals.
Since Western Blot had demonstrated that these htt fragments were notlabeled by anti-SNAP25, we further verified that cleaved htt fragments wereresponsible for forming synaptic aggregates, the majority of synaptic aggregateswere not labeled by anti-SNAP25. In the mouse brain and in the synapses oftransfected neurons, the negative staining of the majority of htt aggregatessuggested that the most htt aggregates in the mouse brain were formed by cleavedhtt fragments lacking SNAP25. Also, these aggregates were not associated with thepresynaptic plasma membrane, perhaps because they were formed by smallhuntingtin fragments without SNAP25.
Thus, using fractionation assays, immunofluorescent staining and EM, wefound convincing evidence that transgenic htt was selectively accumulated in thepresynaptic terminals in transgenic mice and this accumulation was age-dependentand largely mediated by cleaved htt fragments without SNAP25. Therefore, wegenerated novel SNAP25-Htt20Q/150Q transgenic mouse model that selectivelyexpress mutant huntingtin in synapses.
Age-dependent progression of neurological symptoms inSNAP25-Htt150Q transgenic mice. In order to value whether transgenicSNAP25-Htt150Q mice showed progressive neurological phenotypes similar toother HD transgenic mice, we observed the SNAP25-Htt150Q transgenic mice tofind some phenotype such as reduced body weight, clasping, and hunchbackappearance. These symptoms occurred in old mice in an age-dependent mannerand importantly, did not occur in SNAP25-Htt20Q mice that expressed a higherlevel of SNAP25-Htt20Q than SNAP25-Htt150Q, indicating that polyQ expansion,rather than tagging htt with SNAP25, was responsible for the neurologicalsymptoms. Strikingly, transgenic SNAP25-Htt150Q mice die earlier than WT andSNAP25-Htt20Q transgenic mice.
Using rotarod performance assessment, acoustic startle responses, thelight-dark box test and the beam-walking assay to test the transgenic micephenotype, our findings showed that SNAP25-Htt150Q mice recapitulated the neurological symptoms as other established HD transgenic mice.
Synaptic mutant huntingtin caused neuropathology and inhibitedneurotransmitter release. It remained unclear whether synaptic mutant htt couldcause early neuropathology, such as reactive gliosis which was an early sign ofneurodegeneration due to glial proliferation in response to neuronal injury. Wetherefore stained the brains of our HD mouse models using an antibody againstglial fibrillary acidic protein (GFAP), an astrocytic marker protein. We found thatthere was an obvious increase in GFAP staining in SNAP25-Htt150Q transgenicmouse brain regions, including the cortex, striatum, corpus callosum, andcerebellum. Western Blot and quantitation of the ratio of GFAP to GAPDH alsoverified the increased astrocytic gliosis in the SNAP25-Htt150Q mouse striatumversus WT and SNAP25-Htt20Q transgenic mouse brains. These results suggestedthat mutant htt in presynaptic terminals could mediate neuronal injury andneuropathological changes.
Electrophysiological studies of corticostriatal slices from mice had suppliedan approach to test glutamate release. Given the presynaptic localization oftransgenic mutant htt in SNAP25-Htt150Q mice, it was important to examinewhether mutant htt affected synaptic neurotransmitter release. Evaluation thestrength of synaptic transmission by measuring the field excitatory post-synapticpotentials (fEPSPs) in the sensorimotor cortex of the mouse brain slices, theamplitude of paired-pulse facilitation (PPF), and examination of the long-termpotentiation (LTP) of fEPSPs indicated that the expanded glutamine repeat inSNAP25-Htt150Q had specific toxicity, and the pre-synaptic function, especially thecapacity for glutamate release, which was impaired in SNAP25-Htt150Q mice.
To verify the reduction in glutamate release occurred in the striatum, weisolated the mouse brain striatal slices and performed glutamate release assaysusing[3H]glutamate. We found that glutamate release was selectively reduced in theSNAP25-Htt150Q slices, and[3H]GABA release did not appear to be significantlyimpacted, suggesting that vesicular release of glutamate was more vulnerable to synaptic mutant htt.
PC12cells provided a cellular model to measure vesicular dopamine releasethat was dependent on synapsin-1phosphorylation. We measured[3H]dopaminerelease from these PC12cells and found that mutant htt indeed inhibited dopaminerelease.
Mutant htt bound to C-terminal synapsin-1. Because it was selectivelylocalized in the presynaptic terminals, transgenic mutant htt may abnormally boundsynaptic vesicles to impair synaptic transmission. To test this hypothesis, we usedsynaptosomes isolated from WT mouse cortex, and then incubated them with thelysates of transfected PC12cells, which stably expressed either normal (20Q) orpolyQ expanded (150Q) exon1htt. We found that more mutant htt (150Q) thannormal htt (20Q) bound synaptosomes. To explore how mutant htt bound moresynaptosomes, we wanted to use htt immunoprecipitates and mass spectrometry toidentify synaptic proteins that bound mutant htt. We isolated synaptosomes fromSNAP25-Htt150Q mouse brains, and then immunoprecipitated synaptic proteinswith an antibody to htt (mEM48). The precipitated proteins were subjected to massspectrometry, which identified578proteins and1732peptides. This experimentrevealed that synapsin-1, a presynaptic vesicle protein that plays an important rolein neurotransmitter release from vesicles was a good candidate to associate withmutant htt, as it was increased in SNAP25-Htt150Q immunoprecipitates. To verifythat mutant htt associates with synapsin-1in vivo, we performed httimmunoprecipitation using brain tissues from our newly generated HD mouse, HDCAG150KI mouse and PC12stable cell lines. All of these htt immunoprecipitationsupported the idea that mutant htt in synapse bound more tightly to synapsin-1.
Synapsin-1consists of two isoforms,1a and1b, which contains a number ofphosphorylation sites and a proline-rich domain at their C-terminal region. Becausethe proline-rich domain can serve as a binding region for vesicle trafficking, wegenerated a truncated synapsin-1(tsynapsin) by deleting the C-terminal proline-richregion and expressed it with exon1-20Q or exon1-150Q htt in transfected HEK293 cells. Immunoprecipitation of htt revealed that, while both synapsin-1a and-1bcould associate with htt, depletion of the proline domain eliminated the associationof synapsin-1with htt.
Synaptic mutant htt inhibited synapsin-1phosphorylation.Phosphorylation of synapsin-1critically regulates the function of synapsin-1, and S9phosphorylation in the N-terminal region of synapsin-1is important for synapticvesicle neurotransmitter release. By expressing truncated synapsin-1lacking thisC-terminal region in HEK293cells, we then tested if mutant htt bound theC-terminal region of synapsin-1to affect synapsin-1phosphorylation. Comparingthe phosphorylation of N-terminal synapsin-1at serine9(S9) of full-lengthsynapsin-1b and truncated synapsin-1(tsynapsin), we found that while S9phosphorylation in full-length synapsin-1was inhibited by htt-150Q, the samephosphorylation in tsynapsin was not affected by htt-150Q as compared to htt-20Q.Thus, the inhibition of synapsin-1phosphorylation by mutant htt depended on theinteraction of mutant htt with C-terminal region of synapsin-1.
To investigate whether mutant htt could affect the phosphorylation ofendogenous synapsin-1, we performed Western Blot analysis of PC12cells and HDmouse brains with antibodies to different phosphorylated synapsin-1, as theabnormal interaction of mutant htt with synapsin-1could also affect otherphosphorylation sites of synapsin-1. We compared endogenous synapsin-1phosphorylation in PC12cells expressing exon-1150Q htt via Western Blot andspecific antibodies to different phosphorylation sites in synapsin-1and found areduction in S9, S549, and S603phosphorylation in synapsin-1. It was important tovalidate this finding in the HD mouse brain. Examination of SNAP25-Htt150Qtransgenic mouse brain tissues revealed that these phosphorylations were alsoreduced in the HD mouse brains.
Many genetic mouse models of Huntington’s disease (HD) had establishedthat mutant huntingtin accumulated in various sub cellular regions to affect a varietyof cellular functions, but whether and how synaptic mutant huntingtin directly mediates HD neuropathology remaind to be determined. We generated transgenicmice that selectively express mutant huntingtin in the presynaptic terminals.Despite its low level, synaptic mutant huntingtin caused age-dependentneurological symptoms and earlier death in mice, as well as defects in synapticneurotransmitter release. Mass spectrometry analysis of synaptic fraction andimmunoprecipitation of synapsin-1from HD CAG150KI mouse brain revealedmutant huntingtin bound to synapsin-1, a protein whose phosphorylation wascritical for neurotransmitter release. We found that polyQ expansion enhanced theinteraction of exon-1huntingtin with C-terminal region of synapsin-1to reducesynapsin-1phosphorylation. Our findings pointed to a critical role for synaptichuntingtin in the neurological symptoms of HD, providing a new therapeutic target.
本实验通过构建表达突触小体相关蛋白（synaptosomal-associated protein25，SNAP25）与Htt融合蛋白（SNAP25-Htt20Q/150Q）的质粒，制备一种将mHtt选择性地靶向定位在突触前细胞质中的新型HD转基因小鼠模型：SNAP25-Htt150Q小鼠和相应的SNAP25-Htt20Q对照小鼠，并应用这种新的HD转基因小鼠模型对突触mHtt的毒性及其相关机制进行研究，以期揭示突触mHtt在HD的致病机理中的作用。
1.突触Htt在SNAP25-Htt转基因小鼠神经元突触中选择性表达
采用小鼠胚胎原核注射方法建立转基因小鼠模型，挑选出可以稳定繁殖、转基因表达位置及表达量明确的SNAP25-Htt20Q/150Q品系。比较SNAP25-Htt20Q/150Q转基因小鼠品系转基因mHtt表达水平结果表明，SNAP25-Htt150Q表达水平低于SNAP25-Htt20Q中的表达水平。同时，与利用小鼠内源性亨廷顿基因启动子表达HttCAG150的基因敲入(knock in，KI)小鼠[1]相比，SNAP25-Htt150Q mHtt表达水平低于Htt CAG150全长mHtt内源性表达水平。
亚细胞器差速离心方法可以富集突触蛋白，用此方法提取SNAP25-Htt20Q/150Q转基因小鼠大脑皮质突触蛋白结果显示，两种融合蛋白都富集在突触前组分，并且该组分中也发现了Htt的降解片段。用抗Htt抗体(mEM48)对SNAP25-Htt150Q转基因小鼠脑片进行免疫组织化学染色表明，SNAP25-Htt150Q在大脑皮质和纹状体中形成细胞核外神经毡聚集物。电子显微镜(EM)结果也进一步证实mHtt聚集物存在于轴突和突触前细胞质中，而不存在于突触后细胞质和突触前膜。
Western Blot和免疫荧光双标结果显示，SNAP25-Htt150Q在神经突起中形成聚集物，而大多数Htt的聚集物不能被SNAP25抗体所标记，表明SNAP25-Htt150Q的蛋白质水解可能会导致mHtt片段缺失SNAP25表位并更容易聚集。尽管SNAP25靶向性地将mHtt定位在突触前，但可能有部分SNAP25-Htt150Q由于蛋白质水解作用导致融合蛋白发生断裂。
通过亚细胞器分离、免疫荧光染色和电子显微镜的手段，充分证明了mHtt转基因产物选择性地表达在转基因小鼠的突触前细胞质，并且主要由切割后的不含有SNAP25的mHtt片段形成聚集物。因此，本课题成功构建了将mHtt特异性表达在突触前细胞质中的一种新型HD小鼠模型。
2．SNAP25-Htt150Q转基因小鼠HD症状随年龄进行性加重
为了判断SNAP25-Htt150Q小鼠是否出现类似于大多数HD转基因小鼠模型的症状，对SNAP25-Htt转基因小鼠进行了行为学检测，发现老龄SNAP25-Htt150Q转基因小鼠出现典型的HD表型，如：体重减轻、抱屈反射和弓背外观。该表型呈年龄相关性地渐进性发展。SNAP25-Htt150Q转基因小鼠早于野生型和SNAP25-Htt20Q转基因小鼠死亡。旋转杆实验、声学刺激、明暗穿箱实验及平衡木行走实验均表明突触mHtt能导致小鼠出现进行性的运动能力下降。
3．突触mHtt引起神经病理学损害并抑制神经递质释放
对小鼠脑片进行星形胶质细胞标记蛋白胶质纤维酸性蛋白(GFAP)抗体染色，发现SNAP25-Htt150Q转基因小鼠皮质、纹状体和小脑各脑区GFAP染色明显增强，提示突触mHtt引起神经元损伤后反应性的神经胶质细胞增生。对GFAP的Western Blot检测也证实SNAP25-Htt150Q小鼠纹状体中星形胶质细胞明显增生。
应用电生理方法检测突触mHtt对突触电活动功能的影响发现，SNAP25-Htt150Q小鼠大脑切片的感觉运动皮质中成对脉冲刺激的振幅(PPF)及长时程增强(LTP)的信号强度明显降低；分离出SNAP25-Htt转基因小鼠的皮质纹状体脑片检测[3H]谷氨酸释放，结果表明SNAP25-Htt150Q脑片谷氨酸释放减少，而[3H]GABA释放并未受到影响，由此表明谷氨酸囊泡释放更容易受到突触mHtt损伤；采用PC12-Htt-exon1-20Q/150稳转细胞系评估突变mHtt是否影响囊泡释放发现，PC12稳转细胞系中[3H]多巴胺释放的测定结果显示mHtt能抑制PC12细胞神经递质多巴胺的释放。
4．突触mHtt与突触素I的C端相互作用增强
突触mHtt是选择性地定位在突触前细胞质，可与突触囊泡异常结合导致突触信号传递障碍。从野生型（wild type，WT）小鼠脑皮质中分离出突触小体蛋白，分别与PC12-Htt-exon1-20Q/150Q稳转细胞系来源的蛋白样品孵育后进行检测，发现相比于PC12-Htt-exon1-20Q，突触小体更容易与PC12-Htt-exon1-150Q结合。为了探索mHtt与突触蛋白的相互作用，用mEM48抗体对SNAP25-Htt转基因小鼠脑皮质突触蛋白进行免疫沉淀，并对沉淀得到的蛋白进行质谱分析，发现了578种蛋白质和1732种多肽，并证实SNAP25-Htt150Q可以沉淀更多的突触素I蛋白。应用SNAP25-Htt转基因小鼠、Htt CAG150KI小鼠脑组织及PC12-Htt-exon1-20Q/150Q稳转细胞系模型，经免疫共沉淀实验显示mHtt相比于正常Htt与突触素I相互作用更强。
突触素I有1a和1b两个亚型，在其各个结构域上含有大量的磷酸化位点，而其C端具有脯氨酸富集结构域这一特殊片段。用删除C端脯氨酸富集结构域的片段化突触素I (tsynapsin)与Htt-exon1-20Q/150Q共转染HEK293细胞后，免疫共沉淀分析显示，尽管突触素Ia和突触素Ib都与mHtt相互作用，但去除脯氨酸富集结构域后则消除了突触素I与mHtt的结合关系。
5．mHtt通过突触素I脯氨酸富集区的相互作用抑制突触素I磷酸化
突触素I参与囊泡锚定、融合、并从突触前膜释放神经递质中的功能，这一过程与突触素I的磷酸化水平直接相关。在HEK293细胞中表达不同片段长度的突触素I，检测mHtt是否影响突触素I的磷酸化水平。比较全长突触素Ib和截断C端的突触素I(tsynapsin) N端9号位点丝氨酸的磷酸化水平显示，Htt-150Q抑制突触素Ib中9号位点丝氨酸磷酸化水平，而与Htt-20/150Q共转染的tsynapsin中9号位点丝氨酸磷酸化水平无明显差异。因此，mHtt对突触素I磷酸化的抑制依赖于mHtt与突触素I的C端脯氨酸富集区结构域的相互作用。
对PC12-Htt稳转细胞系和SNAP25-Htt转基因小鼠脑蛋白进行Western Blot分析，检测突触素I不同位点的磷酸化抗体水平，结果显示，PC12-Htt-exon1-150Q细胞及SNAP25-Htt150Q转基因小鼠脑皮质中内源性突触素I的S9、S549和S603位点的磷酸化水平均明显降低。
以上研究表明，mHtt在突触前可通过与突触素I蛋白C端区域的异常相互作用使多个位点的磷酸化降低，从而抑制突触神经递质释放，导致神经病理学损害与相应神经症状，为探索治疗HD的方法提供了新的靶点。
Huntington’s disease (HD) is an inherited neurodegenerative disease.Clinically, Huntington’s disease is characterized by irrepressible motor dysfunction,psychiatric disturbances and cognitive deterioration to dementia. The pathologicalcharacteristic feature of HD is selectively neurodegeneration preferentially in striataland cortex neurons. HD is caused by CAG repeats (>35CAGs) in exon1ofhuntingtin gene which generates mutant huntingtin (mHtt) with a polyglutamine(polyQ) expansion. Normal huntingtin mainly expresses in cytoplasm of neuron,and is associated with membrane structure and organelle, such as mitochondrion,Golgi, endoplasmic reticulum, endosome and autophagic vacuoles. Mutanthuntingtin leads to formation of aggregates in cytoplasma and nucleus and thedisease progresses over time. We know that mutant htt in different subcellularregions affects multiple cellular functions. For example, mutant htt in the nucleuscan affect gene expression, mitochondrial function, intracellular trafficking, andmetabolism. Considering neuronal cells have unique, long neuronal processes andsynapses that may be vulnerable to toxic proteins, many studies have focused onsynaptic function, revealing that synaptic dysfunction is the common pathologicalevent in a variety of neurodegenerative diseases. Although synaptic dysfunction inHD mouse models has been well-documented, but whether and how synaptic mutant huntingtin directly mediates HD neuropathology remains to be determined.Since synapses are unique to neuronal cells, understanding whether synaptic mHttcould cause age-dependent progression of neurological symptoms and early death,or synaptic mHtt caused neuropathology and inhibited neurotransmitter release iscritical for unraveling the selective neuropathology seen in HD.
Therefore, we fused exon1htt containing normal (20Q) or150Q (150Q) tosynaptosomal-associated protein25(SNAP25) to generate SNAP25-htt transgenicmice: SNAP25-Htt150Q and SNAP25-Htt20Q as control. And we studied thesetransgenic mice which selectively express huntingtin in the presynaptic terminals, tounderstanding whether and how synaptic mutant huntingtin directly mediates HDneuropathology.
Transgenic mutant htt selectively accumulated in the presynapticterminals in transgenic mice. We used SNAP25-Htt20Q/150Q for pronuclearinjection of mouse embryos. Then we selected the SNAP25-Htt20Q/150Q lines thatcould be bred to F1generation and showed the clear expression of transgene.Genomic DNA and Western Blot analysis revealed that transgenic mutant htt wasexpressed at a lower level than control SNAP25-Htt20Q and was notoverexpressed compared to endogenous mutant htt.
To better assess the relative levels of transgenic htt in presynaptic terminals,we performed subcellular fractionation, which could enrich presynaptic proteins,such as SNAP25. The results showed that both SNAP25-Htt20Q andSNAP25-Htt150Q were enriched in the presynaptic fraction. Importantly, degradedhtt fragments were also present in the presynaptic fraction.
We performed immunofluorescent staining, which could better reveal thesubcellular localization of proteins. SNAP25-Htt150Q was clearly present as punctaoutside the nuclei of neuronal cells in the cortex and striatum. To verify that mutanthtt was indeed localized in the presynaptic terminals, we performed electronmicroscopy (EM). EM examination revealed the presence of htt aggregates in theaxons and presynaptic, but not postsynaptic terminals.
Since Western Blot had demonstrated that these htt fragments were notlabeled by anti-SNAP25, we further verified that cleaved htt fragments wereresponsible for forming synaptic aggregates, the majority of synaptic aggregateswere not labeled by anti-SNAP25. In the mouse brain and in the synapses oftransfected neurons, the negative staining of the majority of htt aggregatessuggested that the most htt aggregates in the mouse brain were formed by cleavedhtt fragments lacking SNAP25. Also, these aggregates were not associated with thepresynaptic plasma membrane, perhaps because they were formed by smallhuntingtin fragments without SNAP25.
Thus, using fractionation assays, immunofluorescent staining and EM, wefound convincing evidence that transgenic htt was selectively accumulated in thepresynaptic terminals in transgenic mice and this accumulation was age-dependentand largely mediated by cleaved htt fragments without SNAP25. Therefore, wegenerated novel SNAP25-Htt20Q/150Q transgenic mouse model that selectivelyexpress mutant huntingtin in synapses.
Age-dependent progression of neurological symptoms inSNAP25-Htt150Q transgenic mice. In order to value whether transgenicSNAP25-Htt150Q mice showed progressive neurological phenotypes similar toother HD transgenic mice, we observed the SNAP25-Htt150Q transgenic mice tofind some phenotype such as reduced body weight, clasping, and hunchbackappearance. These symptoms occurred in old mice in an age-dependent mannerand importantly, did not occur in SNAP25-Htt20Q mice that expressed a higherlevel of SNAP25-Htt20Q than SNAP25-Htt150Q, indicating that polyQ expansion,rather than tagging htt with SNAP25, was responsible for the neurologicalsymptoms. Strikingly, transgenic SNAP25-Htt150Q mice die earlier than WT andSNAP25-Htt20Q transgenic mice.
Using rotarod performance assessment, acoustic startle responses, thelight-dark box test and the beam-walking assay to test the transgenic micephenotype, our findings showed that SNAP25-Htt150Q mice recapitulated the neurological symptoms as other established HD transgenic mice.
Synaptic mutant huntingtin caused neuropathology and inhibitedneurotransmitter release. It remained unclear whether synaptic mutant htt couldcause early neuropathology, such as reactive gliosis which was an early sign ofneurodegeneration due to glial proliferation in response to neuronal injury. Wetherefore stained the brains of our HD mouse models using an antibody againstglial fibrillary acidic protein (GFAP), an astrocytic marker protein. We found thatthere was an obvious increase in GFAP staining in SNAP25-Htt150Q transgenicmouse brain regions, including the cortex, striatum, corpus callosum, andcerebellum. Western Blot and quantitation of the ratio of GFAP to GAPDH alsoverified the increased astrocytic gliosis in the SNAP25-Htt150Q mouse striatumversus WT and SNAP25-Htt20Q transgenic mouse brains. These results suggestedthat mutant htt in presynaptic terminals could mediate neuronal injury andneuropathological changes.
Electrophysiological studies of corticostriatal slices from mice had suppliedan approach to test glutamate release. Given the presynaptic localization oftransgenic mutant htt in SNAP25-Htt150Q mice, it was important to examinewhether mutant htt affected synaptic neurotransmitter release. Evaluation thestrength of synaptic transmission by measuring the field excitatory post-synapticpotentials (fEPSPs) in the sensorimotor cortex of the mouse brain slices, theamplitude of paired-pulse facilitation (PPF), and examination of the long-termpotentiation (LTP) of fEPSPs indicated that the expanded glutamine repeat inSNAP25-Htt150Q had specific toxicity, and the pre-synaptic function, especially thecapacity for glutamate release, which was impaired in SNAP25-Htt150Q mice.
To verify the reduction in glutamate release occurred in the striatum, weisolated the mouse brain striatal slices and performed glutamate release assaysusing[3H]glutamate. We found that glutamate release was selectively reduced in theSNAP25-Htt150Q slices, and[3H]GABA release did not appear to be significantlyimpacted, suggesting that vesicular release of glutamate was more vulnerable to synaptic mutant htt.
PC12cells provided a cellular model to measure vesicular dopamine releasethat was dependent on synapsin-1phosphorylation. We measured[3H]dopaminerelease from these PC12cells and found that mutant htt indeed inhibited dopaminerelease.
Mutant htt bound to C-terminal synapsin-1. Because it was selectivelylocalized in the presynaptic terminals, transgenic mutant htt may abnormally boundsynaptic vesicles to impair synaptic transmission. To test this hypothesis, we usedsynaptosomes isolated from WT mouse cortex, and then incubated them with thelysates of transfected PC12cells, which stably expressed either normal (20Q) orpolyQ expanded (150Q) exon1htt. We found that more mutant htt (150Q) thannormal htt (20Q) bound synaptosomes. To explore how mutant htt bound moresynaptosomes, we wanted to use htt immunoprecipitates and mass spectrometry toidentify synaptic proteins that bound mutant htt. We isolated synaptosomes fromSNAP25-Htt150Q mouse brains, and then immunoprecipitated synaptic proteinswith an antibody to htt (mEM48). The precipitated proteins were subjected to massspectrometry, which identified578proteins and1732peptides. This experimentrevealed that synapsin-1, a presynaptic vesicle protein that plays an important rolein neurotransmitter release from vesicles was a good candidate to associate withmutant htt, as it was increased in SNAP25-Htt150Q immunoprecipitates. To verifythat mutant htt associates with synapsin-1in vivo, we performed httimmunoprecipitation using brain tissues from our newly generated HD mouse, HDCAG150KI mouse and PC12stable cell lines. All of these htt immunoprecipitationsupported the idea that mutant htt in synapse bound more tightly to synapsin-1.
Synapsin-1consists of two isoforms,1a and1b, which contains a number ofphosphorylation sites and a proline-rich domain at their C-terminal region. Becausethe proline-rich domain can serve as a binding region for vesicle trafficking, wegenerated a truncated synapsin-1(tsynapsin) by deleting the C-terminal proline-richregion and expressed it with exon1-20Q or exon1-150Q htt in transfected HEK293 cells. Immunoprecipitation of htt revealed that, while both synapsin-1a and-1bcould associate with htt, depletion of the proline domain eliminated the associationof synapsin-1with htt.
Synaptic mutant htt inhibited synapsin-1phosphorylation.Phosphorylation of synapsin-1critically regulates the function of synapsin-1, and S9phosphorylation in the N-terminal region of synapsin-1is important for synapticvesicle neurotransmitter release. By expressing truncated synapsin-1lacking thisC-terminal region in HEK293cells, we then tested if mutant htt bound theC-terminal region of synapsin-1to affect synapsin-1phosphorylation. Comparingthe phosphorylation of N-terminal synapsin-1at serine9(S9) of full-lengthsynapsin-1b and truncated synapsin-1(tsynapsin), we found that while S9phosphorylation in full-length synapsin-1was inhibited by htt-150Q, the samephosphorylation in tsynapsin was not affected by htt-150Q as compared to htt-20Q.Thus, the inhibition of synapsin-1phosphorylation by mutant htt depended on theinteraction of mutant htt with C-terminal region of synapsin-1.
To investigate whether mutant htt could affect the phosphorylation ofendogenous synapsin-1, we performed Western Blot analysis of PC12cells and HDmouse brains with antibodies to different phosphorylated synapsin-1, as theabnormal interaction of mutant htt with synapsin-1could also affect otherphosphorylation sites of synapsin-1. We compared endogenous synapsin-1phosphorylation in PC12cells expressing exon-1150Q htt via Western Blot andspecific antibodies to different phosphorylation sites in synapsin-1and found areduction in S9, S549, and S603phosphorylation in synapsin-1. It was important tovalidate this finding in the HD mouse brain. Examination of SNAP25-Htt150Qtransgenic mouse brain tissues revealed that these phosphorylations were alsoreduced in the HD mouse brains.
Many genetic mouse models of Huntington’s disease (HD) had establishedthat mutant huntingtin accumulated in various sub cellular regions to affect a varietyof cellular functions, but whether and how synaptic mutant huntingtin directly mediates HD neuropathology remaind to be determined. We generated transgenicmice that selectively express mutant huntingtin in the presynaptic terminals.Despite its low level, synaptic mutant huntingtin caused age-dependentneurological symptoms and earlier death in mice, as well as defects in synapticneurotransmitter release. Mass spectrometry analysis of synaptic fraction andimmunoprecipitation of synapsin-1from HD CAG150KI mouse brain revealedmutant huntingtin bound to synapsin-1, a protein whose phosphorylation wascritical for neurotransmitter release. We found that polyQ expansion enhanced theinteraction of exon-1huntingtin with C-terminal region of synapsin-1to reducesynapsin-1phosphorylation. Our findings pointed to a critical role for synaptichuntingtin in the neurological symptoms of HD, providing a new therapeutic target.
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