Protective Effects And Mechanism Of ST2-104 On Aβ-induced Alzheimer’s Disease Rats’ Neurons Via CRMP2/NMDAR2B

Alzheimer’s Disease（AD） is an age-related, progressive neurodegenerative disorder, which is characterized by learning, spatial memory and cognitive dysfunction. With the development of social economy and medical technology, elderly population has increased dramatically and so dose the incident rate of AD. AD has become one of the most serious diseases which threatens the health of the elderly and has become a heavy burden to both the society and the family. However, current strategies against AD cannot improve the manifestation effectively and can only delay the progression of the disease. Therefore, it is of great importance to study the mechanism of AD and to develop new therapeutic strategies.The early symptoms of AD neuronal damage include dysfunction in learning, memory and cognition. And with progression of AD, patients start to show pathological changes such as the decrease in neurons and their synaptic connections, the deposition of amyloid beta protein and the production of cortical neurofibrillary tangles especially in the hippocampus. As shown in many studies, the neuron toxicity exerted by the production and aggregation of Aβ protein, which is the main cause of AD, lead to pathological changes including losses in hippocampus neurons, abnormal cellular structure and decrease in synapses.Glutamate, an excitatory neurotransmitter, involves in almost all of the central nervous system function. There are many glutamate receptors in the hippocampus, in particular, the N-methyl-D-aspartate receptors（NMDAR） are highly important to learning, memory and synaptic plasticity. There are five major subunits of NMDAR, NR1 and NR2（A-D）. Both two NR1 and NR2 subtype polymerizate to form a functional hole, which forms the main component of NMDAR, meanwhile glutamic/glycine binding sites are mainly in NR2 surface. NMDAR dysfunction is one of the key factors for AD, A beta deposition can act directly on the NMDAR, which leads to its continuous activation, and opens downstream N-type voltage-gated Ca V2.2 channel.The Ca²⁺ influx causes intracellular Ca²⁺ overload and leads to excitatory toxicity in postsynaptic cells and eventually neural dysfunctions. The toxicity of A beta can be blocked by NMDAR antagonists APV（2-amina-5-phosphopentanoi acid）. NR2 B specific blocker but not NR2 A was able to completely prevent Glu/Gly-induced toxicity. So that in this study we focus on NR2 B. NMDARs can be regulated by various factors. Amongst which the CRMP2（Collapsin response mediator 2）, a protein enriched in hippocampus and contributes to neuronal growth and development, can interact with NMDAR and affect its function. CRMP2 highly phosphorylated by CDK5 and GSK3β in AD brain. It was shown that the phosphorylation of CRMP2 can strengthen its interaction with NMDAR, activate of the downstream pathway, and result in neuronal damage. Therefore, the dysfunction of NMDAR plays a crucial role in the pathogenesis of AD and targeting the NMDAR can be a promising strategy for AD treatment.CRMP2-derived peptide, namely ST2-104, is a nona-arginine-conjugated CBD3 peptide combined with trans-membrane portion R9. It was confirmed that ST2-104 was a neuron-protective peptide in glutamate-mediated neurotoxicity by blocking the combination of CRMP2 and NMDAR in the brain trauma and cerebral hemorrhage. It was also shown that ST2-104 exhibited suppressive effect in rat with peripheral neuropathy. While the effect of ST2-104 on AD is not fully elucidated, there is an urgent need for further investigation.The present study seeked to evaluate the protective effect of ST2-104 in Aβ25-35 induced AD in rats and to explore its possible mechanism. To see whether ST2-104 exerted protective effect via blocking the interaction between CRMP2 and NMDAR2 B, or modulating CRMP2 phosphorylation and NMDAR2 B expression. After inducing AD via intracerebroventricular injection, high and low dose ST2-104 were given via tail vein injection. Morris water maze was used to evaluate the spatial learning and memory ability to make sure ST2-104 peptide would improve Aβ induced congnitive dysfunction. HE and Highman Congo staining indicated hippocampus lesion and amyloid deposition. Immunostaining and Western blot with anti-CRMP2, P-CRMP2, and NMDAR2 B were applied to investigate the influence of ST2-104 their expression and distribution in tissue. IP was used to detect the effect of ST2-104 on P-CRMP2 interaction with NMDAR2 B.The AD model group showed significantly prolonged escape latency（the fourth day P<0.05, the third day and fifth day P<0.01） in the morris water maze training. In the space exploration experiment, AD model group trajectory is disorderly, residence time and effective times through the original platform are reduced significantly. Severed loss of AD hippocampus neuron（P<0.01） and increased hippocampus orange splash blocks were observed, indicating deposition of amyloid protein. The level of CRMP2 phosphorylation was raised（P<0.01） and the expression of NMDAR2 B was increased（P<0.01） was observed. The interaction of P-CRMP2 with NMDAR2 B was enhanced（P<0.01）. ST2-104 and memantine could significantly improve the above parameters.Thus, ST2-104 might improve the cognitive ability and protect hippocampus neuron via reducing NMDAR2 B expression and P-CRMP2 level, reducing the interaction between P-CRMP2 and NMDAR2 B.