Tau+/c1q-/-Double Transgenic Mouse Model Construction And The Research Of C1q’s Effect On Tau Protein Phosphorylation

Part ITau+/clq-/-double transgenic mouse model constructionBackground:Neurofibrillary tangles (NFTs) is one of the main pathological features of Alzheimer’s diseases (AD). Hyperphosphorylated Tau is the major component of neurofibrillary tangles (NFTs) in AD. Tau protein is an important microtubule associated protein, widely distributed in the central nervous system. The normal physiological function of this microtubule associated protein is to promote the accumulation of microtubulin to form microtubules and maintain microtubule function stability. When Tau protein is abnormally hyperphosphorylated, it will lose the function of stabilizing microtubules, lead to the formation of NFTs and thus participate in the pathogenesis and development of a variety of neurodegenerative diseases. The pathogenesis of AD is not clear. In recent years, researchers found intracranial chronic inflammatory reaction which is dominated by glial cells plays an important role in the process of AD. Complement is a system of thirty kinds of soluble proteins, receptors, and regulators that protect the host from infection and function as immune effectors and regulator. The complement system is an important part of humoral immunity, which is also participated in the inflammatory response. The complement cascade is initiated by one of three pathways:the classical, alternative, or lectin pathways. In general, complement components are in the blood and can not go through the blood brain barrier (BBB). Studies have shown that intracranial complement components are produced by neurons, microglia, astrocytes, oligodendrocytes and endothelial cells. Recent studies found that intracranial complement system is closely associated with AD. Researchers found that components of the complement classic pathway, C1, C2and C4and eventually formed membrane attack complex (MAC) express in AD brains. In addition to the classic pathway’s components, Strohmeyer et al, found that the complement alternative pathway is also associated with the pathological damage of AD. Lanzrein found the mannan combine agglutinin did not have significant difference in peripheral blood of AD patients and normal controls, while in the cerebrospinal fluid, mannan combine agglutinin is significantly reduced in AD patients group, supporting the idea that lectin pathway is also involved in the pathogenesis of AD. Yet the role of each component of complement system in Alzheimer’s disease (AD) process is still unclear. The first component of complement classical pathway C1, is a calcium dependency complexes which are composed of one c1q molecules, two c1r and two c1s molecules, c1q is the important component of C1. clq combines with the immune complex and activate complement classical pathway. Recent studies found c1q has various functions. Some scholars believe that c1q plays a toxic effect in AD:c1q can combine with A beta peptide and promote the nucleation process of senile plaques. While some scholars think that c1q protect patients from AD. Pisalyaput et al, found that the c1q can enhance microglial cells phagocytize A beta, and protect neurons from A beta neurotoxicity through the in vitro experiments. There have been no reports about the effects of c1q protein on Tau protein recently. B6C3-Tg (Prnp-MAPT*P301S) PS19mice express the P301S mutant human microtubule-associated protein tau, MAPT, under the direction of the mouse prion protein, Prnp, promoter. The expression of the mutant human MAPT is five-fold higher than the expression of the endogenous mouse MAPT protein. Hyperphosphorylated, insoluble mutant human MAPT protein in the brain accumulates with age causing decreased microtubule binding. At three months of age, transgenic mice exhibit clasping and limb retraction when lifted by the tail, which progresses to limb weakness. By10months of age the mice exhibit a hunched back and paralysis, followed by inability to feed. Transgenic mice have a median lifespan of approximately nine months with approximately80%dying by12months. Histological analysis reveals neuron degeneration in hippocampus and ventricular dilatation (brain atrophy) by eight months of age, although significant neuron degeneration in the hippocampus occurs at approximately nine months of age. Neuron loss spreads to the amygdala, neocortex and entorhinal cortex by12months of age. Defective translocation of endoplasmic reticulum proteins in affected neurons is observed as early as three months of age. The onset of neurofibrillay tangle formation in the neocortex, amygdala, hippocampus, brain stem and spinal cord is five months of age. These transgenic mice display neuroinflammation with microglial activation and astrogliosis. B6C3-Tg (Prnp-MAPT*P301S) PS19mice are the ideal animal models in research of Tau protein, clq-/-mice which c1qA chains were knockout in these mice are widely used in the study of c1q. In order to investigate the effect and its mechanism of clq protein on Tau protein, we establish the Tau+/c1q-/-double transgenic mice model firstly.Objective:To establish the Tau+/c1q-/-double transgenic mice model, and provide the experimental basis for the study of effect of c1q protein on Tau protein and its mechanism.Methods:B6C3-Tg (Prnp-MAPT*P301S) PS19mice cross with c1q-/-mice to generate F1mice, c1q+/-and Tau+/c1q+/-mice two genotypes. c1q+/-mice cross with Tau+/c1q+/-mice again to obtain F2generation mice.Results:The genotypes of the first generation are clq+/-and Tau+/c1q+/-two genotypes. Genotyping analysis of the second generation mice eventually obtained c1q+/+, c1q+/-, c1q-/-, Tau+/c1q+/+, Tau+/c1q+/-, Tau+/c1q-/-,6kinds of genotype mice. We select Tau+/c1q-/-mice group as experimental group and c1q+/+(WT), Tau+/c1q+/+(Tau+) groups as control groups.Conclusion:We construct Tau+/c1q-/-transgenic mice model successfully. Part IIPhosphorylated Tau protein expression in Tau+/c1q-/-double transgenic miceBackground:The major neuropathological findings of Alzheimer’s disease (AD)-neurofibrillary tangles (NFT) and senile plaques (SP)-have been known for many years. Abnormally hyperphosphorylated Tau protein is the main component of NFT. The normal function of Tau protein is to promote microtubule formation and maintain stability of microtubules. The function of Tau protein is regulated by its phosphorylation state. Hyperphosphorylated Tau protein changes its conformation, and losts functions of promoting protein aggregation into microtubules and stabling microtubule function, causing microtubule depolymerization, cytoskeleton damage. Therefore, hyperphosphorylated Tau protein is one of the most important pathological changes in AD, is likely to be a core element in its pathogenesis.Objective:To explore Tau protein phosphorylation in Tau+/c1q-/-double transgenic mice.Methods:Western blot method was used for detection of phosphorylated Tau protein.Results:Compared with control group, the expression of phosphorylated Tau protein (PHF1) tend to be reduced in the cerebral cortex of3months old Tau+/c1q-/-double transgenic mice. In6months mice groups, phosphorylated Tau protein (PHF1, PHF6, PHF13, p-Tau ser396, CP13, AT8) expression was significantly decreased in cortex of Tau+/c1q-/-double transgenic mice. Further study found that, the expression of phosphorylated Tau protein (PHF1, PHF6, PHF13, p-Tau ser396, CP13, AT8) in cerebral cortex of10months old Tau+/c1q-/-double transgenic mice was also decreased significantly, compared with control groups.Conclusion:Deficiency of c1q can significantly reduce the expression of phosphorylated Tau protein in Tau+/c1q-/-double transgenic mice. Part ⅢSynaptic protein expression in Tau+/c1q-/-double transgenic miceBackground:Synapse lose is one of the early pathological changes in AD. The lose of hippocampal synapse can be observed in patients with mild cognitive impairment (MCI). Synapse lose is closely related to the severity of the dementia.Objective:To explore synaptic proteins expression in Tau+/c1q-/-double transgenic mice.Methods:Western blot method was used to detect the hippocampus presynaptic membrane protein (SYP, SNAP25) and postsynaptic membrane protein (G1uR1, NMDAR1, PSD95) expression in Tau+/c1q-/-double transgenic mice and control mice groups.Results:In10months old mice groups, the expression of SYP and SNAP25in Tau+transgenic mice was decreased compared to those in WT mice; while in Tau+/c1q-/-double transgenic mice group, the expression of SYP and SNAP25is obviously recovered compared with those in Tau transgenic mice, but still less than those in WT mice. No obvious changes were observed about postsynaptic membrane proteins (GluR1NMDAR1PSD95) in Tau+/c1q-/-double transgenic mice and control mice groups.Conclusion:Deficiency of clq can protect synaptic proteins from lose in Tau+/c1q-/-double transgenic mice. Part IVNeuropathologic changes in Tau+/clq-/-double transgenic miceBackground:Extracellular neuritic plaques, intraneuronal neurofibrillary tangles (NFTs), the lose of cortical and hippocampal neurons and synapses and glial cell activation are the classical hallmark pathologies of AD. In previous work, we found that deficiency of c1q protein can significantly reduce the expression of phosphorylated Tau protein in Tau+/c1q-/-double transgenic mice.Objective:To explore neuropathologic changes in Tau+/c1q-/-double transgenic mice.Methods:Immunohistochemistry method was used to detect phosphorylated Tau protein expression, neurofibrillary tangles (NFT) and glial cell activation.Results:Compared with control groups, phosphorylated Tau protein expression and neurofibrillary tangles (NFT) and glial cell activation were significantly reduce or alleviate in Tau+/c1q-/-double transgenic mice.Conclusion:Deficiency of c1q can alleviate neuropathologic changes in Tau+/c1q-/-double transgenic mice. Part VBehavioral tests in Tau+/c1q-/-double transgenic miceBackground:A highly specific animal model is the prerequisite for researches on AD pathogenesis and development of effective drugs. An ideal AD animal model should reproduce its main neuropathological changes (neurofibrillary tangles and senile plaques) and show the corresponding behavioral change (such as memory damage). Learning and memory are the highest functions of the brain-two important parts of the intelligence. Learning refers to the experience (behavior, perception, thinking) acquisition or development while memory refers to the preservation and reproduction of experience. They are two different but closely related neurobiological processes, and human and animal behavior is very complicated process under the control of the central nervous system. People design a variety of experiment methods to analyze this complex process. Barnes maze test is a kind of better method for assessing animal spatial learning and memory ability. Rotorod test can test animal’s movement and coordination ability.Objective:To analyze learning, memory and movement abilities in Tau+/c1q-/-double transgenic mice and control groups.Methods:Barnes maze test and Rotorod test.Results:Barnes maze test found that compared with Tau+transgenic mice group, the short-term and long-term memory abilities of Tau+/c1q-/-double transgenic mice are improved significantly. Rotorod test found that no significant difference in exercise ability between Tau+transgenic mice group and Tau+/c1q-/-double transgenic mice group.Conclusion:Deficiency of c1q can significantly improve the short-term and long-term memory ability of Tau+/c1q-/-double transgenic mice. Part ⅥMechanism research of clq protein effects Tau protein phosphorylationBackground:Tau protein hyperphosphorylation is caused by protein kinases. According to the characteristics of the catalytic phosphorylation, protein kinase can be divided into the proline directed protein kinases (PDPK) including glycogen synthase kinase3β(GSK-3p), cyclin dependent kinase-5(CDK5), miotgen activated protein kinases (MAPK) and non-proline directed protein kinase (non-PDPK) including microtubule affinity regulating kinase (MARK), protein kinase A (PKA), protein kinase C (PKC) and calmodulin protein kinase II (CaMK II).Objective:To explore the possible molecular mechanisms of clq protein effects Tau protein phosphorylation.Methods:Western blot method was used to detect ERK1/2, P38, JNK, CDK5, GSK-3beta and CaMK II, PKA, PKC, GSK-3alpha, CREB protein kinase phosphorylation expression in the cortical area of Tau+/c1q-/-double transgenic mice and control mice groups. Enzyme linked immunosorbent assay (ELISA) method was used for detection of PKA activity and cAMP expression quantity in the cortical area of Tau+/c1q-/-double transgenic mice and control mice groups. Results: Compared with the Tau+transgenic mice, phosphorylated forms of ERK, p38, JNK, PKC, and CaMK II kinases have no obvious changes in the cortical area of Tau+/c1q-/-double transgenic mice; while phosphorylated forms of GSK3beta, PKA kinases were significantly reduced in the cortical area of Tau+/c1q-/-double transgenic mice. Using the method of ELISA, we found that, compared to Tau+transgenic mice, PKA kinase activity and cAMP expression were significantly decreased in Tau+/c1q-/-double transgenic mice. These results suggest that cAMP-PKA pathway plays an important role for Tau protein phosphorylation in Tau+/c1q-/-double transgenic mice. Conclusion:cAMP-PKA pathway plays an important role for Tau protein phosphorylation in Tau+/c1q-/-double transgenic mice. Part ⅦIn vitro study of clq protein effects Tau protein phosphorylationBackground:In our previous work, we set up the Tau+/c1q-/-double transgenic mice model, using Western blot and immunohistochemistry method confirmed that deletion of c1q protein in mice can reduce phosphorylated Tau protein expression, partially recover synaptic proteins expression and improve the learning and memory ability in the Tau+/clq-/-double transgenic mice.Objective:To explore the effect of overexpression of clq protein in vitro on Tau protein phosphorylation.Methods:Cotransfection of wild type Tau441(pCDNA3.1+) plasmid and clqA (pCMV6-AC) plasmid in SH-SY5Y cell model, using Western blot method to detect the expression of phosphorylated Tau protein from the experimental group and control group.Results:Compared with control group, the expression of phosphorylated Tau proteins PHF1, PHF13and CP13were significantly increased in the Tau+c1qAplasmids cotransfection group; while total Tau protein in cells (DA9) have no obvious difference.Conclusion:Overexpression of c1q protein in vitro can increase the Tau protein phosphorylation.