The Suppressive Effects Of MANF On The Neurotoxicity Induced By Aβ And Hyperphosphorylated Tau

Alzheimer’s disease (AD) is a progressively neurodegenerative disease. The twohallmarkers of AD include extracelluar senile plaques mainly composed ofβ-amyloid (Aβ) and intracellular neurofibrillary tangles (NFTs) containinghyperphosphorylated tau. Aβ peptide is derived from larger amyloid β-proteinprecursor (APP). APP is processed by various pathways of proteolytic cleavage, andthe Aβ fragments are the cleavage products of β-and γ-secretases. Variousconditions lead to misfolding of three-dimensional structure of Aβ peptide and thenAβ peptide aggregates, which is hard to be removed by protease and deposits intissues, and finally induces neuron apoptosis. tau protein, a component ofmicrotubule, plays an important role in promoting the assembling of tubulin intomicrotubules and stabilizing the structure of the microtubule. There are6predominant isoforms and more than30Ser/Thr phosphorylation sites within tau protein. During the progress of AD, tau is abnormally phosphorylated, andhyperphophorylated tau further aggregates and forms NFTs with loss of the abilityto bind microtubules. Indeed, the abnormal tau phosphorylation is one of theimportant etiological causes of neuron loss and neurodegenerative condition. Inneurodegenerative diseases, such as AD and Parkinson’s disease, activated ERstress and UPR signaling have been detected. In AD, the misfolding of Aβ andabnormally hyperphophorylated tau can induce ER-stress.Mesencephalic astrocyte-derived neurotrophic factor (MANF), also known asarginine-rich, mutated in early stage tumors (ARMET), is an ER stress-inducibleprotein and belongs to neurotrophic factors (NTFs) family. MANF has been shownto promote survival and recovery of neurons under various pathological conditons.In our previous study, we found that MANF protein was upregulated by variousforms of ER stress in several cell lines as well as by cerebral ischemia of rat, andoverexpression of MANF inhibited neurons and non-neuronal cell proliferation andimproved cell viability under serum-free conditions and tunicamycin treatment. Ithas been found that there was a neural protective effect of MANF gene on animalmodel of PD, but the relevant function of MANF in AD are still unclear.Objective:To investigate the possible roles of MANF in protecting neurons from thetoxicity induced by Aβ and phosphorylated tau.Materials and methodsFull length MANF cDNA with Flag Tag was subcloned into pCIneo vector,named as pCIneo-MANF-Flag. For transient knockdown experiment, RNAinterference (RNAi) target sequences for MANF/Armet (sense：5’-GGA CCUCAA AGA CAG AGA UTT-3’; antisense：5,-GCA GAU CGA CCU GAG CACATT-3’) was selected, and then subcloned into plentiLox3.7vector, andrecombinant plasmid was named ‘pLentilox3.7-MANF-siRNA’. Expression and purification of recombinant MANF protein were performed. Primary cultures ofcortical neurons were prepared from E18embryos of timed-pregnantSprague-Dawley rats, and then identified by NueN immuno-staining. Human andmouse derived neural cell lines, SH-SY5Y cells and N2a cells were cultured.Various neuron cells were treated by Aβ to induce neurotoxicity. PI staining wasused for identifying dead cells under Aβ treatment. Immunofluorescent Stainingwas performed to assess the expression level of MANF, Bip/GRP78and CHOPgene in SH-SY5Y cells and N2a cells under Aβ treatment. SH-SY5Y cells weresubjected to recombinant MANF protein and N2a cells were transfected withpCIneo-MANF-Flag or pLentilox3.7-MANF-siRNA, and after Aβ treatment, thecell viability were determined by MTT assay. Aβ-induced N2a cell apoptosis wasmeasured by the Terminal deoxynucleotidyl transferase (TdT)-mediateddUTP-fluorescein nick-end labelling (TUNEL) method. Western blot was used toanalysis the expression level changes MANF and apoptosis associated molecular inN2a cells after plasmid transfection. To observe the effect of MANF gene onabnormally hyperphosophrylation of tau protein, MTT assay and Western blot werealso used to determine the protect effect of MANF on neurotoxicity induce byhyperphosophrylated tau.Results:1. Aβ induces ER stress and neuronal cell deathUsing human neuroblastoma SH-SY5Y cells, we initially assessed neuronal celldeath and ER stress after Aβ exposure. Propidium iodide (PI), a membrane-impermeant dye, was used to distinguished dead cells from viable cells. PI stainingwas performed at24hrs after Aβ25-35exposure (from6.25μM to50μM). Theresults showed that Aβ increased the percentage of dead cells in a dose-dependentmanner. CHOP/GADD153, an ER stress inducible bZIP transcription factor, wasused as a marker of ER stress-induced programmed cell death. Double-labeled immunofluorescent staining showed that the expression of CHOP/GDADD153was remarkably increased after Aβ25-35exposure for24hrs, compared withuntreated control group and serum-free group. Tunicamycin, an ER stress inducer,was used here as a positive control. To confirm this result, the cortical neuronsisolated from pregnant18-day embryos of Sprague-Dawley (SD) rats were culturedfor10days. The well differential neurons were identified with neuron marker NeuN.Similarly, CHOP/GADD153was upregulated by Aβ25-35and tunicamycin inprimarily cultured rat neurons. The expression of BiP/GRP78, an ER chaperoneserving as a master controller of ER stress response and UPR, was also increased byAβ25-35and tunicamycin in primary cultured rat neurons. These results indicatethat ER stress contributes to Aβ-induced neuronal cell death.2. Aβ upregulates MANF expression in neuronal cellsOur previous study found that MANF was an ER stress inducible protein. Weare wondering if Aβ exposure also up-regulates MANF expression. To test this, weinitially observed MANF expression in SH-SY5Y cells after Aβ exposure. It wasfound that MANF expression was obviously increased under Aβ25-35(10μM)exposure for24hrs, as well as after treatment with tunicamycin, compared withuntreated control group and with serum-free group. Meanwhile, MANF was alsoup-regulated by Aβ in the primarily cultured neurons. These results suggest that Aβexposure induces MANF expression in neuronal cells.3. MANF contributes to neuron protection against Aβ toxicity.MANF belongs to neurotrophic factors family, which is an important group ofsecreted proteins. We found above that Aβ induced MANF expression in neuronalcell line and cultured primary neurons. Therefore, we hypothesize that induction ofMANF is protective for cells while they suffer insults, such as Aβ. To prove this,we evaluated the effects of treatment with recombinant human MANF, overexpression of MANF by transfection of cDNA, and knockdown of endogenousMANF with siRNA on Aβ-treated neuronal cells. Recombinant MANF protein wasexpressed and purified as we previously described. SH-SY5Y cells were pretreatedwith various concentrations of recombinant MANF protein (0.5μg,1μg, and2μg)for4hrs prior to adding Aβ25-35. Twenty-four hr after treatment with Aβ25-35(10μM), cell viability was determined by using the MTT assay. The results showed thatrecombinant MANF prevented cells from death induced by Aβ25-35in adose-dependent manner. The time-course of MANF at the dose of2μg on cellviability was showed.N2a cells were transiently transfected with plasmids pCIneo-MANF orplentiLox3.7-MANF-siRNA to over-express MANF or knockdown endogenousMANF. The blank vectors were used as the controls. It was found that MANFover-expression inhibited the decrease of cell viability induced by Aβ1-42exposure.On the contrary, knockdown of endogenous MANF with siRNA promoted thedecrease of cell viability induced by Aβ. Meanwhile, the cell lyses were performedfor a Western blot analysis and MANF protein was blotted. These results indicatethat MANF prevents from Aβ toxicity and promotes cell survival.4. MANF attenuates apoptosis and caspase-3activationThe above results showed that Aβ exposure promotes the MANF geneexpression through activating ER stress, and overexpression of MANF gene ortreatment with recombinant MANF protein attenuates Aβ-induced neuronal celldeath. Therefore, we further elucidated the molecular mechanisms involved. Todetect whether MANF protect against Aβ toxicity through inhibiting ERstress-mediated apoptosis, we performed a TUNEL assay. A typical molecularcharacteristic of apoptosis is genomic DNA cleavage into internucleosomalsegments attributed to the specific activation of an endogenous endonuclease. Thisapoptosis specific genomic DNA cleavage was also assessed by the TUNEL assay. A transient transfection to over-expression or knockdown MANF gene in N2a cellswas performed as above described. MANF gene over-expression decreased thenumber of TUNEL-positive cells under Aβ exposure, whereas MANF geneknockdown obviously increased it. The relative percentage of TUNEL-positivecells was shown. Western blotting analysis showed that CHOP expression andcaspase-3cleavage were increased after Aβ exposure, MANF over-expression andknockdown attenuated and aggravated CHOP expression and caspase-3activation,respectively. The results indicate that MANF inhibits ER stress-mediated apoptosis,which may contribute to the neuron protection.5. MANF inhibits tau hyperphosphorylation in cultured neuronal cellsPretreatment with recombinant human MANF for4hrs before OA exposure for24hrs, the viability of N2a was increased and the cellular level ofhyperphosphorylated tau was decreased. Similaly, MANF cDNA transfectioninhibited OA-induced tau hyperphosphorylation and cell impairment in N2a cells,whereas MANF shRNA trancfection had an opposite effect.ConclusionThese results confirmed that MANF prevents from Aβ-induced toxicitythrough inhibiting ER stress-mediated neuronal apoptosis and inhibits tauhyperphosphorylation, suggesting that MANF may serve as a potentially therapeutictarget for the neurodegenerative diseases, including AD.