Dysregulated Amyloid Precursor Protein (APP) Expression And ?-Secretase Processing Of APP Involved In Manganese-induced Cognitive Impairment

Objective:Excessive manganese(Mn)exposure can cause cognitive impairment in adults and children.Yet,little is known about the linking Mn exposure and cognition in older age,and the mechanisms underlying Mn-induced cognitive impairment are far from clear.Cognitive decline is also a common characteristic feature of aging and AD.Strong evidence implicates APP and it's secretase processing-affected synaptic function is the key pathogenesis of AD.Epidemiological studies have found that environmental pollutants are related to aging-related cognitive impairment and AD.However,whether APP and its secretase processing are involved in Mn-induced cognitive impairment remains unknown.The objective of this research was to investigate the effects of chronic Mn exposure on cognitive function in the elderly,and to explore the role of APP and its?-and?-secretase processing mediated synaptic function in Mn-induced cognitive impairment.Methods:Between 2016 and 2017,a total of 375 older men were recruited from an area in Hechi City,Guangxi,China.All of the participants complete a baseline questionnaire and a cognitive assessment questionnaire(Mini-Mental State Examination,MMSE);provided urine and peripheral venous blood samples for further inductively coupled plasma mass spectroscopy(ICP-MS)and ELISA analysis,respectively.Then analyzed the association between urinary Mn(U-Mn)and the cognition as well as the association between U-Mn and the plasma APP levels.A total of 32 SPF grade mice were ranked by weight and randomly divided into four groups(n=8 per group),and respectively treated with 0,25,50,or 100 mg/kg Mn²⁺by gastric gavage once daily for three months to construct a model of Mn-induced cognitive impairment.The mouse primary hippocampal neurons were exposure to 0,100,200,and 400?M Mn²⁺for 24 h,and the N2a cells were exposure to 0,200,400,and 800?M Mn²⁺for24 h,respectively.The difference of protein content between 0 and 400?M Mn-treated mouse primary hippocampal neurons was determined using isobaric tags for relative and absolute quantitation(i TRAQ).The m RNA levels of APP,ADAM10,and BACE in Mn-treated mouse,primary hippocampal neurons and N2a cells were determined using q RT-PCR.The protein levels of APP,ADAM10,BACE1,A?40,A?42,PSD95 and synapsin1 in Mn-treated mouse,primary hippocampal neurons and N2a cells were determined using Western blot.The protein levels of s APP?,s APP?,A?40 and A?42 in Mn-treated mice plasma and primary hippocampal neurons,s APP?and s APP?in Mn-treated N2a cells,as well as the activity of ADAM10 and BACE1 in Mn-treated mouse primary hippocampal neurons and N2a cells were determined using ELISA.We further developed stable APP OE and APP sh RNA N2a cells.After exposure to 800?M Mn²⁺for 24 h,the protein levels of APP,ADAM10,BACE1,A?40,A?42,PSD95,and synapsin1were determined using Western blot.Results:1.A total of 29.07%older men were identified as cognitive impairment.The median MMSE score in cognitive impairment group(18)was lower than that in cognitive normal group(27)(P<0.05).The median of U-Mn levels in cognitive impairment group(0.256?g/g)was higher than that of cognitive normal group(0.211?g/g)(P<0.05).The median plasma APP levels in cognitive normal and cognitive impairment groups were 529.76 and 478.42pg/m L,respectively(P>0.05).2.When U-Mn levels were analyzed as tertile,after adjusting for age,marital status,nationality,education,BMI,smoking status,family smoking exposure status,drinking status,and hypertension,the binary logistic regression analysis results showed that,the risk of cognitive impairment in the high U-Mn group was 2.092 times of that in low Mn group(95%CI:1.156-3.788,P=0.015);the plasma APP levels in the high U-Mn group was 0.391 times of that in low Mn group(95%CI:0.268-0.887,P=0.019).Besides,the risk of cognitive impairment increased and plasma APP levels decreased as U-Mn levels increased in a dose-dependent manner(P for trend=0.023 and 0.018,respectively).3.The hippocampus Mn in the Mn-treated groups were all higher than that of the control(P<0.05).In the place navigation test,the escape latency of mice on day 3,4 and 5 were all shorter than that of the day 1(P<0.05),though no significantly different was observed between the control and Mn-treated mice.In the spatial probe test,the number of platform crossings and the swimming path length in the target quadrant of mice treated with 100 mg/kg Mn was lower than that of the control(P<0.05).The rotarod test results shown that the latency to fall on day 3,4 and 5 were all longer than that of the day 1(P<0.05),though no significantly different was observed between the control and Mn-treated mice(P>0.05).4.For i TRAQ analyses,we obtained a total of 4,756 quantification proteins,which including APP,APLP1,APLP2 and synaptic marker proteins(synapsin,syntaxin,synaptotagmin,synaptopsin,CASK,dynamin,and VAMP).Moreover,427 DEPs were identified between 0 and 400?M Mn-treated mouse primary hippocampal neurons,of which 251 were down-regulated and 176 were up-regulated.5.No significant difference of APP m RNA levels was observed in Mn-treated mice hippocampi when compared to the control(P>0.05).However,the APP m RNA levels in 400?M Mn-treated mouse primary hippocampal neurons and 800?M Mn-treated N2a cells were all lower than that of the control(P<0.05).The protein levels of APP in 50 and 100 mg/kg Mn-treated mice hippocampi,in 200 and 400?M Mn-treated mouse primary hippocampal neurons,and in 800?M Mn-treated N2a cells were all lower than that of the control(P<0.05).6.The ADAM10 m RNA levels in 50 and 100 mg/kg Mn-treated mice hippocampi,100,200,and 400?M Mn-treated mouse primary hippocampal neurons as well as 800?M Mn-treated N2a cells were all lower than that of the control(P<0.05).The hippocampi ADAM10 protein levels and plasma s APP?protein levels in 50 and 100 mg/kg Mn-treated mice,the protein levels of ADAM10 and s APP?in 400?M Mn-treated mouse primary hippocampal neurons as well as 400 and 800?M Mn-treated N2a cells were all lower than that of the control(P<0.05).The ADAM10 activity in 400?M Mn-treated mouse primary hippocampal neurons as well as 200,400 and 800?M Mn-treated N2a cells were all lower than that of the control(P<0.05).7.The BACE m RNA levels in 100 mg/kg Mn-treated mice hippocampi,100,200,and 400?M Mn-treated mouse primary hippocampal neurons,as well as 400 and 800?M Mn-treated N2a cells were all lower than that of the control(P<0.05).Nevertheless,no significant changes of BACE1,s APP?,A?40,and A?42 protein levels were observed in Mn-treated mice,mouse primary hippocampal neurons and N2a cells(P>0.05).Morever,no significant changes of BACE1 activity were observed in Mn-treated mouse primary hippocampal neurons and N2a cells(P>0.05).8.Western blot results shown that the PSD95 and synapsin1protein levels in 50 and 100 mg/kg Mn-treated mice hippocampi,200 and 400?M Mn-treated mouse primary hippocampal neurons,and 800?M Mn-treated N2a cells were lower than that of the control(P<0.05).Immunostaining results further also showed significantly lower fluorescence intensities of PSD95 and synapsin1 in200 and 400?M Mn-treated mouse primary hippocampal neurons as well as 400and 800?M Mn-treated N2a cells,respectively(P<0.05).9.Furthermore,lower APP,ADAM10,PSD95,and synapsin1 protein levels were observed in APP OE and APP sh RNA N2a cells after exposed to 800?M Mn for 24 h(P<0.05),while no significant changes of BACE1,A?40,and A?42 protein levels were observed(P>0.05).Conclusions:1.Chronic Mn exposure induced cognitive impairment in older men.2.Mn exposure inhibited APP and it's?-secretase processing,but not?-secretase processing,results in synaptic deficits and cognitive impairment.