Changes in microtubule-associated protein tau during peripheral nerve injury and regeneration

Tau, a primary component of microtubule-associated protein, promotes microtubule assembly and/or disassembly and maintains the stability of the microtubule structure. Although the importance of tau in neurodegenerative diseases has been well demonstrated, wheth- er tau is involved in peripheral nerve regeneration remains unknown. In the current study, we obtained sciatic nerve tissue from adult rats 0, 1, 4, 7, and 14 days after sciatic nerve crush and examined tau mRNA and protein expression levels and the location of tau in the sciatic nerve following peripheral nerve injury. The results from our quantitative reverse transcription polymerase chain reaction analysis showed that compared with the uninjured control sciatic nerve, mRNA expression levels for both tau and tau tubulin kinase 1, a serine/ threonine kinase that regulates tau phosphorylation, were decreased following peripheral nerve injury. Our western blot assay results suggested that the protein expression levels of tau and phosphorylated tau initially decreased 1 day post nerve injury but then gradually increased. The results of our immunohistochemical labeling showed that the location of tau protein was not altered by nerve injury. Thus, these results showed that the expression of tau was changed following sciatic nerve crush, suggesting that tau may be involved in periph- eral nerve repair and regeneration.

Role of Microtubule-associated Protein Tau Phosphorylation in Alzheimer's Disease

As a major microtubule-associated protein, tau plays an important role in promoting microtubule assembly and stabilizing microtubules. In Alzheimer’s disease(AD) and other tauopathies, the abnormally hyperphosphorylated tau proteins are aggregated into paired helical filaments and accumulated in the neurons with the form of neurofibrillary tangles. An imbalanced regulation in protein kinases and protein phosphatases is the direct cause of tau hyperphosphorylation. Among various kinases and phosphatases, glycogen synthase kinase-3β(GSK-3β) and protein phosphatase 2A(PP2A) are the most implicated. Accumulation of the hyperphosphorylated tau induces synaptic toxicity and cognitive impairments. Here, we review the upstream factors or pathways that can regulate GSK-3β or PP2A activity mainly based on our recent findings. We will also discuss the mechanisms that may underlie tau-induced synaptic toxicity.

Effects of microtubule-associated protein tau expression on neural stem cell migration after spinal cord injury

Our preliminary proteomics analysis suggested that expression of microtubule-associated protein tau is elevated in the spinal cord after injury. Therefore, the first aim of the present study was to examine tau expression in the injured spinal cord. The second aim was to determine whether tau can regulate neural stem cell migration, a critical factor in the successful treatment of spinal cord injury. We established rat models of spinal cord injury and injected them with mouse hippocampal neural stem cells through the tail vein. We used immunohistochemistry to show that the expression of tau protein and the number of migrated neural stem cells were markedly increased in the injured spinal cord. Furthermore, using a Transwell assay, we showed that neural stem cell migration was not affected by an elevated tau concentration in the outer chamber, but it was decreased by changes in intracellular tau phosphorylation state. These results demonstrate that neural stem cells have targeted migration capability at the site of injury, and that although tau is not a chemokine for targeted migration of neural stem cells, intracellular tau phosphorylation/dephosphorylation can inhibit cell migration.

Visualizing the microtubule-associated protein tau in the nucleus

Although tau is mainly known as an axonal microtubule-associated protein,many studies indicate that it is not restricted to this subcellular compartment.Assessing tau’s subcellular distribution,however,is not trivial as is evident from transgenic mouse studies.When human tau is over-expressed,it can be immunohistochemically localized to axons and the somatodendritic domain,modeling what is found in neurodegenerative diseases such as Alzheimer’s disease.Yet,in wild-type mice,despite its abundance,tau is difficult to visualize even in the axon.It is even more challenging to detect this protein in the nucleus,where tau has been proposed to protect DNA from damage.To establish a framework for future studies into tau’s nuclear functions,we compared several methods to visualize endogenous nuclear tau in cell lines and mouse brain.While depending on the fixation and permeabilization protocol,we were able to detect nuclear tau in SH-SY5Y human neuroblastoma cells,we failed to do so in N2a murine neuroblastoma cells.As a second method we used subcellular fractionation of mouse tissue and found that in the nucleus tau is mainly present in a hypophosphorylated form.When either full-length or truncated human tau was expressed,both accumulated in the cytoplasm,but were also found in the nuclear fraction.Because subcellular fractionation methods have their limitations,we finally isolated nuclei to probe for nuclear tau and found that the nuclei were free of cytoplasmic contamination.Together our analysis identifies several protocols for detecting tau in the nucleus where it is found in a less phosphorylated form.

Expression changes in tau and microtubule-associated proteins in rat testicular interstitium

Objective: To examine the expression of the tau protein and mi-crotubule-associated proteins (MAP) in the testicular interstitium of aged and young rats. Methods: Sprague-Dawley male rats were divided into a young group (6 months) and an aged group (28 months) of 10 animals each. The two-step immunohistochemistry method with the antibody against tau protein and MAPa was performed with the testis tissues. Results: The immunoreactive cells of the testicular interstilial tau protein were significantly increased (P<0.01) and those of the MAP significantly decreased (P<0.01) in the aged than in the young rats. Conclusion: The changes in the expression of the tau protein and MAP may be related to the aging process of the testis.

Analysis of the autophagy gene expression profile of pancreatic cancer based on autophagy-related protein microtubule-associated protein 1A/1B-light chain 3

BACKGROUND Pancreatic cancer is a highly invasive malignant tumor. Expression levels of the autophagy-related protein microtubule-associated protein 1 A/1 B-light chain 3(LC3) and perineural invasion(PNI) are closely related to its occurrence and development. Our previous results showed that the high expression of LC3 was positively correlated with PNI in the patients with pancreatic cancer. In this study, we further searched for differential genes involved in autophagy of pancreatic cancer by gene expression profiling and analyzed their biological functions in pancreatic cancer, which provides a theoretical basis for elucidating the pathophysiological mechanism of autophagy in pancreatic cancer and PNI.AIM To identify differentially expressed genes involved in pancreatic cancer autophagy and explore the pathogenesis at the molecular level.METHODS Two sets of gene expression profiles of pancreatic cancer/normal tissue(GSE16515 and GSE15471) were collected from the Gene Expression Omnibus.Significance analysis of microarrays algorithm was used to screen differentially expressed genes related to pancreatic cancer. Gene Ontology(GO) analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis were used to analyze the functional enrichment of the differentially expressed genes. Protein interaction data containing only differentially expressed genes was downloaded from String database and screened. Module mining was carried out by Cytoscape software and ClusterOne plug-in. The interaction relationship between the modules was analyzed and the pivot nodes between the functional modules were determined according to the information of the functional modules and the data of reliable protein interaction network.RESULTS Based on the above two data sets of pancreatic tissue total gene expression, 6098 and 12928 differentially expressed genes were obtained by analysis of genes with higher phenotypic correlation. After extracting the intersection of the two differential gene sets, 4870 genes were determined. GO analysis showed that 14 significant functional items including negative regulation of protein ubiquitination were closely related to autophagy. A total of 986 differentially expressed genes were enriched in these functional items. After eliminating the autophagy related genes of human cancer cells which had been defined, 347 differentially expressed genes were obtained. KEGG pathway analysis showed that the pathways hsa04144 and hsa04020 were related to autophagy. In addition,65 clustering modules were screened after the protein interaction network was constructed based on String database, and module 32 contains the LC3 gene,which interacts with multiple autophagy-related genes. Moreover, ubiquitin C acts as a pivot node in functional modules to connect multiple modules related to pancreatic cancer and autophagy.CONCLUSION Three hundred and forty-seven genes associated with autophagy in human pancreatic cancer were concentrated, and a key gene ubiquitin C which is closely related to the occurrence of PNI was determined, suggesting that LC3 may influence the PNI and prognosis of pancreatic cancer through ubiquitin C.

Interventional effect of hirudin on the expression of microtubule-associated protein 2 in peripheral tissue of hematom of model rats with acute intracerebral hemorrhage

BACKGROUND： It is suspected that dissociation, destruction or synthetic disorder of microtubule-associated protein 2 （MAP-2） may participate in secondary injury of intracerebral hemorrhage （ICH）, and the reason may be related to thrombin in high concentration after ICH; therefore, the mechanism should be studied further. OBJECTIVE： To explore the effect of hirudin on expression of MAP-2 in peripheral tissue of hematom after ICH and changes of water content in brain tissue and analyze pathogenesis of thrombin in secondary injury after ICH. DESIGN ： Completely randomized grouping design and controlled animal study SEn-ING ： Department of Neurology, the First Affiliated Hospital of Jilin University MATERIALS ： The experiment was carried out in the Neurological Laboratory of the First Affiliated Hospital of Jilin University from April 2003 to April 2004. A number of 80 healthy Wistar rats, of both genders, aged 3-4 months, weighing 250-350 g, were randomly divided into 8 groups： normal control group, 6-hour ICH group, 1-day ICH group, 2-day ICH group, 3-day ICH group, 7-day ICH group, 3-day hirudin group and 7-day hirudin group with 10 in each group. Five rats from each group were selected to measure their water content, and the others were undertaken immunohistochemical stain. Hirudin was produced by Sigma Company, USA, and MAP-2 rabbit-rat polyclonal antibody was provided by Fuzhou Maixin Biotechnology Company Limited. METHODS： ① Model establishing and grouping intervention： Rats in simple ICH group were collected their blood from tails and then inserted with 50 μL non-anticoagulant auto-arterial blood into the cauda of the putamen in right brain within 5 minutes. Rats in hirudin groups were inserted with 10 U hirudin （which was diluted with saline to 20 μL） into local hematom regions within 5 minutes, and the needle was pulled out after 10 minutes. Rats in normal control group were untouched. ② Water content in peripheral tissue of hematom： Based on the ratio between dry weight and wet weight, brain tissue at bleeding side and in right frontal lobe was selected to measure dry and wet weights so as to calculate the water content [（wet weight - dry weight） /wet weight] × 100%.③ Positive expression of MAP-2： Based on immunohistochemical stain, positive MAP-2 cells were regarded as neurons and they were buffy morphological. Positive rate of MAP-2 was calculated, i.e., percentage of positive cells in each sight to total cells in all sights. ④ Statistical analysis： Data among groups were compared with one-way analysis of variance, averages were compared with SNK-q test by each other, and relation between water content and MAP-2 was analyzed with linear regression technique. MAIN OUTCOME MEASURES： Changes of water content and MAP-2 expression in peripheral tissue of hematorn at various time points after ICH and intervention of hirudin. RESULTS： All 80 rats were involved in the final analysis. ①Water content： Water content was increased at day 1, reached peak at day 3 and decreased at day 7. It was （72.31±0.32）%, （77.42±0.53）%, （78.44±0.28）%, （74.10±0.13）%, （74.85±0.51）% and （70.07±0.36）%, respectively in 1-day, 2-day, 3-day and 7-day ICH groups and 3-day and 7-day hirudin groups, which was higher than that in normal control group （63.85±0.41, q=-4.684 3 to -7.262 0, P〈 0.05）; that in 2-day and 3-day ICH groups was higher than that in 7-day ICH group （q=-3.053 4, -3.727 0, P 〈 0.05）; and that in 3-day and 7-day ICH groups was higher than that in hirudin groups at the same time points （q=-2.965 6, -2.726 4, P 〈 0.05）. ②Positive expression of MAP-2： Positive expression of MAP-2 was decreased at 6 hours after ICH, reached the lowest value at day 3 and increased at day 7. Positive rate was （78.60±0.42）%, （60.56±0.74）%, （44.60±0.26）%, （25.45±0.85）%, （32.55±0.64）%, （37.69＋0.76）%, （41.75±0.68）%, respectively in 6-hour, 1-day, 2-day, 3-day and 7-day ICH groups and 3-day and 7-day hirudin groups, which was lower than that in normal control group [（96.50±0.33）%, q= -3.074 5 to -8.128 5, P 〈 0.05]. In addition, positive cells of MAP-2 disappeared plentifully at 3-7 days after ICH, stain of positive cells were light, and only stain of plasma was positive. That in 3-day and 7-day hirudin groups was higher than that in ICH groups at the same time points （q= -3.391 8, -2.967 9, P 〈 0.05）. Moreover, positive cells of MAP-2 was formed slightly but deeply stained. ③ Results of linear regression： Water content was negatively related to MAP-2 changes at 7 days after ICH （r= -0.894 9, P〈 0.01）, i.e., water content was increased with decrease of MAP-2 expression. CONCLUSION ： The deterioration of MAP-2 may be involved in the pathogenesis of thrombin within the first week after ICH, and the local administration of hirudin can protect neurons.

Granulocyte colony-stimulating factor promotes growth of processes,growth associated protein 43 and microtubule-associated protein 2 expression in cultured rat retinal ganglion cells in vitro

Following granulocyte colony-stimulating factor （G-CSF） treatment,the growth of processes in cul-tured rat retinal ganglion cells （RGCs） in vitro,expression of growth associated protein 43,and expression of microtubule-associated protein 2 mRNA expression were significantly increased.In contrast,RhoA/Rock protein content was significantly reduced by G-CSF treatment.These results indicate that G-CSF promotes the growth of processes in RGCs and increases the expression of growth-associated protein 43 and microtubule-associated protein 2 mRNA by inhibiting the RhoA/Rock pathway,thereby benefiting axonal repair in RGCs exposed to hypoxia.

Interactions of the HSV-1 UL25 Capsid Protein with Cellular Microtubule-associated Protein

An interaction between the HSV-1 UL25 capsid protein and cellular microtubule-associated protein was found using a yeast two-hybrid screen and β-D-galactosidase activity assays. Immunofluorescence microscopy of the UL25 protein demonstrated its co-localization with cellular microtubule-associated protein in the plasma membrane. Further investigations with deletion mutants suggest that UL25 is likely to have a function in the nucleus.

The role of microtubule-associated protein 1B in axonal growth and neuronal migration in the central nervous system

In this review, we discuss the role of microtubule-associated protein 1 B （MAP1B） and its phosphorylation in axonal development and regeneration in the central nervous system. MAP1B exhibits similar functions during axonal development and regeneration. MAP1B and phosphorylated MAPIB in neurons and axons maintain a dynamic balance between cytoskeletal components, and regulate the stability and interaction of microtubules and actin to promote axonal growth, neural connectivity and regeneration in the central nervous system.

Phosphorylation of tau protein over time in rats subjected to transient brain ischemia

Transient brain ischemia has been shown to induce hyperphosphorylation of the micro- tubule-associated protein tau. To further determine the mechanisms underlying these processes, we investigated the interaction between tau, glycogen synthase kinase （GSK）-313 and protein phos- phatase 2A. The results confirmed that tau protein was dephosphorylated during brain ischemia; in addition, the activity of GSK-3β was increased and the activity of protein phosphatase 2A was de- creased. After reperfusion, tau protein was hyperphosphorylated, the activity of GSK-3β was de- creased and the activity of protein phosphatase 2A remained low. Importantly, the interaction of tau with GSK-3β and protein phosphatase 2A was altered during ischemia and reperfusion. Lithium chloride could affect tau phosphorylation by regulating the interaction of tau with GSK-3β and pro- tein phosphatase 2A, and improve learning and memory ability of rats after transient brain ischemia. The present study demonstrated that it was the interaction of tau with GSK-3β and protein phos- phatase 2A, rather than their individual activities, that dominates the phosphorylation of tau in tran- sient brain ischemia. Hyperphosphorylated tau protein may play an important role in the evolution of brain injury in ischemic stroke. The neuroprotective effects of lithium chloride partly depend on the inhibition of tau phosphorylation during transient brain ischemia.

鼠李糖乳杆菌对老年小鼠术后海马区小胶质细胞激活及Tau蛋白磷酸化的影响

【目的】探讨术前益生菌鼠李糖乳杆菌(LGG)灌胃对麻醉手术老年小鼠海马区小胶质细胞及Tau磷酸化的影响。【方法】18月龄C57BL/6J小鼠30只随机分为3组,10只/组:对照组,麻醉手术组,麻醉手术+鼠李糖乳杆菌组。生理盐水/LGG 109CFU 150μL灌胃,每日1次,连续20 d后接受异氟醚麻醉+剖腹探查手术,术后12 h免疫荧光染色检测海马区小胶质细胞激活状态,ELISA检测IL-6的浓度变化,Western blot检测Tau蛋白磷酸化位点Tau-pS202/pT205和total Tau蛋白表达变化。【结果】对照组海马区小胶质细胞呈静息状态,炎症因子IL-6浓度为(82.08±12.07)pg/mL。与对照组相比,麻醉手术组海马区小胶质细胞活化增生,胞体变大,突起缩短变粗,炎症因子IL-6上升至(123.7±5.72)pg/mL(P=0.000),磷酸化Tau-pS202/pT205蛋白表达量也明显增加(P=0.002)。而与麻醉手术组相比,麻醉手术+LGG组海马区小胶质细胞增生肥大不明显,炎症因子IL-6分泌减少至(96.68±9.59)pg/mL(P=0.008),磷酸化Tau-pS202/pT205蛋白表达量明显下降(P=0.002)。而3组total Tau蛋白表达水平差异无统计学意义。【结论】术前服用益生菌鼠李糖乳杆菌减轻麻醉手术导致的老年小鼠海马区小胶质细胞活化、炎症因子分泌增加、以及Tau蛋白磷酸化水平增加。

超声引导下腹横肌平面麻醉对无张力疝修补术患者血清Tau蛋白的影响

目的:探讨无张力疝修补术患者采用超声引导下腹横肌平面麻醉(TAPB)的临床效果及对血清蛋白的影响。方法:选取2018年2月-2019年2月于柳州市人民医院行无张力疝修补术的患者94例作为研究对象,采用随机数字表法分为对照组和观察组,各47例。对照组进行全身麻醉,观察组采用超声引导下TAPB,比较两组麻醉效果及对血清蛋白[β淀粉样蛋白(Aβ)-42、Tau]。结果:术后1、3 d,观察组简易精神状态检查量表评分高于对照组,差异有统计学意义(P<0.05)。观察组术后20 min、1 h、8 h及24 h的疼痛评分低于对照组,差异有统计学意义(P<0.05)。术后7 d,观察组Aβ-42水平高于对照组,Tau蛋白及Tau/Aβ-42水平低于对照组,差异有统计学意义(P<0.05)。结论:将超声引导下TAPB应用在无张力疝修补术患者中,可降低术后血清Tau蛋白水平,有助于预防认知功能障碍。

血浆中tau蛋白磷酸化表达在多奈哌齐治疗阿尔茨海默病中的机制研究

目的基于血浆中tau蛋白磷酸化探讨多奈哌齐对阿尔茨海默病(AD)的保护机制。方法2019年1月至2022年7月从齐齐哈尔市第一医院神经内科招募了两组参与者作为样本。在第一组样本中包括58名轻度至重度AD患者和20名健康老年对照组;另一组样本包括37名轻度至中度AD患者的样本,其中18名患者接受了24周的多奈哌齐治疗,19名患者接受了24周安慰剂治疗。从患者的血液标本中提取神经元衍生的细胞外囊泡(EV),采用酶联免疫吸附分析试剂盒对β淀粉样蛋白42(Aβ_(42))、P-T181-tau、P-S396-tau、总tau蛋白(t-tau)、神经颗粒蛋白(NRGN)水平进行分析。结果与对照组相比,AD患者的EV中Aβ_(42)、t-tau、P-T181-tau、P-S396-tau水平显著升高(P<0.05),NRGN水平显著降低(P<0.05)。在AD患者中,t-tau、NRGN和沉默转录因子(REST)的EV水平与简易智力状态检查量表(MMSE)和阿尔茨海默病合作研究–日常生活活动量表(ADCS-ADL)评分呈负相关(P<0.05),并与阿尔茨海默病评估量表–认知子量表的14项扩展版本(ADAS-cog+)评分呈正相关(P<0.05)。在为期24周的治疗期间,与安慰剂组患者相比,多奈哌齐组患者血浆中Aβ_(42)、t-tau、P-T181-tau和P-S396-tau的表达水平(EV水平)从基线水平到第24周结束时均呈显著降低趋势(P<0.05)。结论轻重度AD患者血浆EV中Aβ_(42)、t-tau、P-T181-tau和P-S393-tau水平升高。t-tau、NRGN和REST的水平增加与认知功能和生活能力的下降相关。多奈哌齐治疗可使轻中度AD患者血浆中t-tau、P-T181-tau和P-S396-tau的表达水平降低。

Effect of erythropoietin combined with hypothermia on serum tau protein levels and neurodevelopmental outcome in neonates with hypoxic-ischemic encephalopathy

Although hypothermia therapy is effective to treat neonatal hypoxic-ischemic encephalopathy,many neonatal patients die or suffer from severe neurological dysfunction.Erythropoietin is considered one of the most promising neuroprotective agents.We hypothesized that erythropoietin combined with hypothermia will improve efficacy of neonatal hypoxic-ischemic encephalopathy treatment.In this study,41 neonates with moderate/severe hypoxic-ischemic encephalopathy were randomly divided into a control group（hypothermia alone for 72 hours,n = 20） and erythropoietin group（hypothermia ＋ erythropoietin 200 IU/kg for 10 days,n = 21）.Our results show that compared with the control group,serum tau protein levels were lower and neonatal behavioral neurological assessment scores higher in the erythropoietin group at 8 and 12 days.However,neurodevelopmental outcome was similar between the two groups at 9 months of age.These findings suggest that erythropoietin combined with hypothermia reduces serum tau protein levels and improves neonatal behavioral neurology outcome but does not affect long-term neurodevelopmental outcome.

INHIBITION OF MELATONIN BIOSYNTHESIS ACTIVATES PROTEIN KINASE A AND INDUCES ALZHEIMER-LIKE TAU HYPERPHOSPHORYLATION IN RATS

Objective To investigate effect of inhibiting melatonin biosynthesis on activities of protein kinase A (PKA), glycogen synthase kinase-3 (GSK-3) and tau phosphorylation at PS214 and M4 epitopes using haloperidol, a specific inhibitor of 5-hydroxyindole-O-methyltransferase. Methods Brain ventricular and intraperitoneal injections were used for haloperidol administration, Western blots for tau phosphorylation, 32P-labeling for PKA and GSK-3 activity, and high performance liquid chromatograph for detection of serum melatonin levels. Results Haloperidol injection through the lateral ventricle and intraperitoneal reinforcement significantly stimulated PKA activity with a concurrent hyperphosphorylation of tau at M4 (Thr231/Ser235) and PS214 (Ser214) sites. Prior treatment of the rats using melatonin supplement for one week and reinforcement during the haloperidol administration arrested PKA activity and attenuated tau hyperphosphorylation. GSK-3 activity showed no obvious change after haloperidol injection, however, melatonin supplements and reinforcements during haloperidol infusion inactivated basal activity of GSK-3. Conclusion Decreased melatonin may be involved in Alzheimer-like tau hyperphosphorylation, and overactivation of PKA may play a crucial role in this process.

基于^(18)F-Florzolotau PET显像评估阿尔茨海默病脑内tau蛋白异常沉积

目的探讨新一代tau PET显像剂18F-Florzolotau在阿尔茨海默病(AD)不同发展阶段中的应用价值。资料与方法回顾性纳入2020年2月—2022年1月复旦大学附属华山医院β-淀粉样蛋白阳性的25例轻度认知功能障碍(MCI)与61例AD,患者均行18F-Florzolotau PET脑显像,并收集相关人口统计学与临床资料。使用SPM双样本t检验比较两组患者预处理后的18F-Florzolotau PET图像,以显著差异(P<0.001)脑区为感兴趣区提取标准化摄取值比值(SUVR);同时利用尺度亚轮廓/主成分分析模型分别建立MCI、AD的tau蛋白异常沉积相关模式(MCItauRP、ADtauRP)并计算对应的表达值。采用受试者工作特征曲线评价MCItauRP、ADtauRP表达值以及SUVR的分类性能。结果AD组与MCI组患者相比,主要在双侧颞顶叶脑区tau蛋白异常沉积增加(P<0.001),此差异脑区感兴趣区内AD组SUVR高于MCI组(Z=-3.164,P<0.001);AD组与MCI组患者各MCItauRP、ADtauRP表达值差异有统计学意义(t=3.72,Z=-3.51;P均<0.001),且AD组患者表达值均高于MCI组;MCItauRP、ADtauRP表达值以及SUVR鉴别AD与MCI的准确度分别为61.63%、65.12%和65.12%,敏感度分别为88.00%、96.00%和100.00%,特异度分别为50.82%、52.46%和50.82%。结论新一代tau PET能够检测及区分AD、MCI患者脑内tau蛋白沉积,但分类准确度不高,未来需要进一步寻找更加理想的分析方法。

Neuroprotective effect of heat shock protein 70 Inhibition of Tau protein expression

BACKGROUND： Previous studies have shown that heat shock protein 70 （HSP70） has neuroprotective effects by decreasing phosphorylation of Tau protein, thereby reducing the expression of Tau protein and proper aggregation. OBJECTIVE： To observe and verify expressional changes of HSP70 and Tau in retinal ganglion cells following stretch injury to the right optic nerve in rats, and to determine the effect of heat stress pretreatment on HSP70 and Tau protein expressions. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the Laboratory of Neurology Research Institute of the First Affiliated Hospital of Chongqing Medical University from March to June 2006. MATERIALS： Instant SABC immunohistochemistry kit, as well as mouse anti-HSP70 and rabbit anti-Tau polyclonal antibodies, were purchased from Wuhan Boster Bioengineering Limited, China. METHODS： A total of 57 male, Wistar rats were randomly assigned to 4 groups： control （n = 3）; 150-180 g stretch force was induced in the right optic nerves in stretch-only group （n = 18） to establish optic nerve stretch injury model; heat stress was applied to 18 animals in heat-stress treatment group; 18 rats in the heat-stress pretreatment plus stretch group were subjected to identical stretch injury as stretch-only group after 24-hour heat-stress pretreatment. According to sacrifice time, the groups were assigned to 6 subgroups at different time points of 4, 8, and 16 hours, and 1,3, and 5 days, with 3 rats in each subgroup. No treatment was performed in the control group except anesthesia. MAIN OUTCOME MEASURES： Morphological changes of optic nerves and retinal ganglion cells following stretch injury were observed by light microscopy following hematoxylin-eosin staining. HSP70 and Tau protein expression levels were observed in retinal ganglial cells from each group using im munohistochemistry. RESULTS： （1） Compared with the control group, morphological axonal and retinal ganglial cell changes, as well as a decreased number of retinal ganglial cells, were identified in the stretch-only group （P 〈 0.01）. Pathological damage in optical nerve and retinal ganglial cells were not remarkable in the heat-stress pretreatment plus stretch group, with no statistical difference in the number of retinal ganglial cells compared with the control group （P 〉 0.05）. （2） Compared with the control group significantly increased HSP70 expression in retinal ganglial cells occurred in the stretch-only, heat-stress treatment, and heat-stress pretreatment plus stretch groups （P 〈 0.05 or P 〈 0.01 ）. The peak of HSP70 expression was earlier in the heat-stress pretreatment plus stretch group compared with the stretch-only and heat-stress treatment groups, and was expressed over a longer period of time compared with the heat-stress treatment group. Compared with the control group, Tau expression in the retinal ganglial cells rapidly increased 4-16 hours following stretch injury in the stretch-only group （P 〈 0.05 or P 〈 0.01）, and obviously decreased in the heat-stress pretreatment plus stretch group （P 〈 0.05 or P 〈 0.01 ）. CONCLUSION： Tau expression increased following stretch injury, with an earlier expression peak than HSP70, which indicated that stretch injury-induced HSP70 expression was not strong or quick enough to sufficiently protect the nerve. A much more enhanced HSP70 expression, with an earlier peak and longer expression period, was observed in rats subjected to stretch injury following heat stress, which demonstrated that HSP70 exhibited neuroprotective functions by reducing abnormal aggregation of Tau.

Neurofibrillary tangles in Alzheimer＇s disease： elucidation of the molecular mechanism by immunohistochemistry and tau protein phospho-proteomics

As a key contributor to memory storage, the synapse is one of the earliest affected neuronal components in Alzheimer＇s disease （AD）. Under physiological conditions, the synaptic con- nections between neurons undergo activity-dependent func- tional and morphological re-organisation. This dynamic, ＇plastic＇ neural ability critically depends on the structural integrity of the synapse. Thus, proteins that are implicated in preserving the organisation and dynamics of synaptic connections, including microtubules of the cytoskeleton and associated proteins, have attracted much focus for their involvement in the malfunction- ing AD synapse.

Role of Notoginsenoside Rg1 in Improving Spatial Cognitive Ability and Lowering Phosphorylation Level of Tau Protein in AD Model Rats

[Objectives] To study the effects and mechanism of notoginsenoside Rg1 on the spatial learning and memory and phosphorylated tau protein in the AD( Alzheimer's Disease) model rat. [Methods]The AD model rat was replicated by injection of Aβ_(25-35) in the left lateral ventricles of SD rats. The low dose( 25 mg/kg),middle dose( 50 mg/kg) and high dose( 100 mg/kg) notoginsenoside Rg1 was used for intragastric administration,respectively,two times every day. After 4 weeks,the Morris water maze test was done to detect the learning and memory capacity,and the immunoblotting,immunohistochemical methods were used to detect the changes in the phosphorylation level and distribution of tau protein in hippocampus of the rats. [Results] After the intracerebroventricular injection of Aβ_(25-35),the learning and memory capacity of the model rats was significantly lower than the learning and memory capacity of the normal control rats. The immunoblotting test results showed that the phosphorylation level of tau protein threonine 231 site( Thr231) in hippocampus was significantly increased,and the nonphosphorylation level was significantly decreased. The morphological testing results showed that the phosphorylation level of tau protein Thr231 of AD model rats was increased markedly in region of DG,CA1 and CA3 of the hippocampus. The intervention of the middle dose notoginsenoside Rg1 could significantly improve the learning and memory capacity of the model rats in Morris water maze. The notoginsenoside Rg1 in three different doses could all reduce the phosphorylation level of tau protein Thr231 in the hippocampal DG,CA1,CA3 regions,and there were no significant differences among the three doses. [Conclusions]The notoginsenoside Rg1 could improve Aβ_(25-35)-induced spatial learning and memory impairment of the AD model rats,and decreased the phosphorylation level of tau protein in hippocampus.