The Effect Of Aβ1-42 On Expression Of T514 Phosphorylated CRMP2 In SH-SY5Y Cells

BackgroudAlzheimer's disease (AD) is one of the most common neurodegnerative disorders. The disease is defined by histopathologic features, especially the accumulation ofβ-amyloid peptides (Aβ) that forms senile plaques.β-amyloid peptides 1-42 (Aβ1-42) which are much more self-aggregated pron and daminging than other produced Aβpeptides are the major constituents of early senile plaques in AD brain. The collapsin response mediator proteins (CRMPs) belong to a family of dihydropyrimidinase-related neuronal phosphoproteins which consists of five isoforms, CRMP1-5. CRMPs are most highly expressed during the neurogenic period of brain and play an important role in neural development. CRMP2 is expressed in mature neural system. Previous study demonstrated that CRMP2 regulate axon elongation, through its interaction with tubulin to promote microtubule assembly. Phosphorylation of Thr514 abolishes the ability of CRMP2 to bind to tubulin heterodimers.ObjectiveTo establish an injured model of all-trans retinoic acid induced neuroblastoma cells SH-SY5Y. To investigatethe effect of aggregated Aβ1-42 on cells viability, neurite growth, microtubule structure and intracellular expression of T514 phosphorylated CRMP-2.MethordEstablishing cells model by adding aggregated Aβ1-42 in the culture medium of all-trans retinoic acid induced neuroblastoma cells SH-SY5Y. MTT methord was employed to identify the viability of SH-SY5Y cells. Cytochemistry was used to measure neurite length of induced SH-SY5Y cells. The intracelluler microtubule structure and experssion of T514 phosphorylated CRMP-2 was detected by immunofluorescence. Westen blot was used to detect the influence of Aβ1-42 on total expression of T514 phosphorylated CRMP-2.Result1. Cultured human neurobastoma cells SH-SY5Y were induced with 10μmol/L all-trans retinoic acid. After 7 days'differention, obvious morphological changes can be observed under the microscope. Cells no longer clustered into a group and split much slower. Polygonal cells body transformed into fusiform. Formed one or a few long axons, cells were obvious polarity. In control group, cells remained epithelioid and didn't form long axon.2. The MTT assay result showed that all concentrations (0.1 nmol/L, 1nmol/L, lOnmol/L, 0.1μmol/L, 1μmol/L) of aggregated A(31-42 decreased the redox activity of SH-SY5Y cells, but only 0.1μmol/L and 1μmol/L had significant toxic effect, compared with control (p<0.05) Selected 0.1μmol/L and 1μmol/L aggregated Aβ1-42 for the next steps of our experiments.3. Cytochemistry showed that 1μmol/L aggregated A(31-42 caused a significant reduction in neurite length over a 24h incubation period (p<0.05). There was no significant reduction in neurite length in 0.1μmol/L Aβ1-42 induced groups (p>0.05)4. Immunofluorescence result showed that in control group there was homogeneous distribution of green fluorescence (on behalf of FITC-a-tubulin immunoreactive product) both in the cytoplasm and axon. The red fluorescence (TRITC labeled T514 phosphorylation of CRMP2 immunoreactive products mainly distributed in the cell body. After the treatment with aggregated Aβ1-42, cell axon became retracted. Microtubule network structure was blurred with more distribution of red fluorescence in the cell axon. The statistic analysis showed that there was no significant difference of the average area of green fluorescence between different groups. Compareed with control group and 0.1μmol/L treatment group, the average green fluorescence intensity of cells was significantly lower (p<0.05) in 1μmol/L treatment group. Red fluorescence were enhanced both in the cell plasma and axon area, compared with the control group. There were significant differences of the red fluorescence area and intensity between treatment group(0.1μmol/L and 1μumol/L) and cnontrol group (p<0.05).5. Westen blot result showed that the total expression of T514 phosphorylated CRMP-2 in both experimental groups (0.1μmol/L and 1μmol/L) maintain significant higher level compared with control group (p<0.01). And the expression of T514 phosphorylated CRMP-2 increased when the concentration of aggregated Aβ1-42 increased (p<0.05). The result indicated that aggregated Aβ2-42 affect intracellular experission of phosphorylated CRMP-2.ConclusionAβ1-42 can increase intracellular expression of T514 phosphorylated CRMP-2 and have negtive effects on cells viability, microtubule structure and neurite outgrowth.