alzheimer 's disease ( ad ) is the most common neurodegenerative disorder that leads to dementia .
the neuropathology of ad is characterized by extracellular neuritic plaques , intracellular neurofibrillary tangles , synapse loss , glial cell activation and neuronal death .
the neuritic plaques are primarily composed of amyloid  ( a ) protein , which is derived from the sequential cleavage of amyloid  precursor protein ( app ) via -secretase and then -secretase . the abnormal accumulation of a initiates neuronal dysfunction and plays an important role in ad pathogenesis . a40 and a42 are the major species of a in the brain . of these two species ,
a42 is the more toxic form ; furthermore , it is hydrophobic , prone to aggregation and predominant in neuritic plaques . under physiological conditions
, the majority of app is cleaved by -secretase within its a region to preclude a generation [ 2 - 4 ] .
the reduction of a disintegrin and metalloprotease 10 ( adam10 ) , the central component of -secretase , promotes ad pathogenesis by shifting -cleavage to -cleavage of app .
in addition , the dysfunction of the a clearance mediated by a-degrading enzymes ( e.g. , insulin degrading enzyme ; ide ) is crucial for the accumulation of a in the ad brain .
vitamin a ( va ) is one of the most essential micronutrients in humans . in the central nervous system ( cns ) ,
va and its active metabolite retinoic acid ( ra ) are involved in several essential biological processes including early development of brain structure and function , neuronal patterning , proliferation and differentiation , neurite outgrowth and synaptogenesis .
ra primarily exerts its biological effects by activating retinoic acid receptors ( rars ) and/or retinoid x receptors ( rxrs ) , which act on the retinoic acid responsive elements ( rares ) in the promoter regions of target genes .
both rars and rxrs have three subtypes :  ,  and . over 500 genes might be regulated by ra .
vitamin a deficiency ( vad ) is a serious public health problem throughout the developing world and is primarily caused by chronically low va intake .
the first evidence was provided by studies that reported decreased serum va and -carotene in patients with ad [ 9 - 12 ] .
a nutritional analysis of the risk factors for ad also revealed that low dietary va or -carotene is associated with an increased risk of ad .
furthermore , the depletion of va or the dysfunction of rars results in a deposition , impairment of hippocampal long - term synaptic plasticity and memory deficits in rodents ; these effects can be restored via va or rar agonist replenishment [ 14 - 22 ] .
currently , severe vad has been largely brought under control ; however , more widespread marginal states of deficiency have been revealed , designated as mild or marginal vitamin a deficiency ( mvad ) . mvad primarily affects pregnant / lactating women and preschool children [ 23 - 25 ] .
furthermore , lahiri first proposed the latent early - life associated regulation ( learn ) model , suggesting that environmental risk factors ( e.g. , dietary factors ) perturb gene regulation in a long - term fashion , beginning at early developmental stages , which plays a vital role in the etiology of ad [ 26 , 27 ] .
thus , we hypothesized that long - term mvad , beginning early in life , might regulate the ad - associated gene and protein expression , thereby increasing the risk for developing ad .
the present study established an mvad rat model from the embryonic period to the sacrifice .
aggregated a1 - 42 was injected into the hippocampus of these rats to investigate the effects of mvad on ad pathogenesis .
this study demonstrates aggravated hippocampus - dependent memory deficits in these mvad rats following a1 - 42 injection .
our work suggests that mvad , beginning during the embryonic period , enhances the risk of developing ad .
all wistar rats used in this study were obtained from the experimental animals center of beijing , china .
the rats were housed in a room with a constant airflow system , controlled temperature ( 22 - 24c ) and a 12-hour light / dark cycle .
the female rats were randomly divided into a control group and an mvad group and fed a va - normal ( van ) diet ( 6,500 iu va per kilogram of the basic diet ) and an mvad diet ( 400 iu va per kilogram of the basic diet ) , respectively , for 4 weeks to generate maternal control and mvad rats . after confirming that the serum retinol levels matched the recommended standards for mvad ( 0.70 - 1.05
mol / l ) , the female rats were mated with the van males . after giving birth ,
the male weanlings were subjected to experiment and were maintained throughout the study on a van or mvad diet .
this study was conducted in strict accordance with the recommendations of the national institutes of health s guide for the care and use of laboratory animals .
the animal experimentation ethical committee of chongqing medical university in chongqing , china , approved the protocol .
the preparation of the aggregated a142 and the brain stereotaxic surgery were performed as previously described .
a1 - 42 ( sigma - aldrich , usa ) was dissolved in 0.01 m phosphate - buffered saline ( pbs ) , ph 7.4 , and then vigorously agitated ( 200 rpm ) at room temperature for 36 hours .
sediments were dissolved with pbs to a 10 m stock , aliquoted , and stored at -80c .
after the baseline morris water maze tests were conducted with 18-week - old pups , the pups in both the control and mvad groups were randomly divided into the a group ( full dose of aggregated a1 - 42 ) , the 1/2a group ( half dose of aggregated a1 - 42 ) and the vehicle group .
all subjects were anaesthetized , and the injections were made bilaterally into the ca3 area of the dorsal hippocampus with the injectates deposited slowly over 10 minutes .
the stereotaxic coordinates were 2.6 mm lateral and 3.5 mm posterior to the bregma , and 3.7 mm ventral .
the injections were 5.0 l of 1x10 m aggregated a1 - 42 suspension , 5.0 l of 0.5x10 m aggregated a1 - 42 suspension and 5.0 l of pbs for the a group , the 1/2a group and the vehicle group , respectively .
the maternal rats at preconception and five to six pups from each group at postnatal 24 hours , 4 weeks , 8 weeks , 12 weeks , 18 weeks , 22 weeks and 26 weeks were randomly selected to detect serum retinol levels .
the pups younger than 8 weeks old were decapitated to collect the blood ; and the blood from the pups at the age of 8 weeks and older were collected via the caudal vein .
all pups were decapitated after the last behavioral tests to detect serum retinol levels and confirm the success of the animal models .
serum retinol was measured using high performance liquid chromatography ( hplc ) as previously described .
briefly , 200 l of serum was deproteinized with an equal volume of dehydrated alcohol .
the retinol was extracted from the serum by adding 1,000 l of hexane and evaporated with nitrogen gas .
the retinol residue was dissolved in 100 l of the mobile phase mixture ( methanol : water=97:3 ) .
then , 20 l of the dissolved liquid was tested in the hplc apparatus ( dgu-20as , shimadzu corporation , kyoto , japan ) on a silica column with a 315-nm ultraviolet photodiode array detector .
all procedures were performed in a dark room to protect the samples from light at the children s nutrition research center in the children s hospital of chongqing medical university .
total rna was extracted from the hippocampus of rats using tri reagent ( sigma ) and reverse - transcribed into cdna using the primescript rt reagent kit ( takara ) according to the manufacturer s instructions .
real - time pcr was conducted using a sybr premix ex tag ( perfect real time ; takara ) and a bio - rad real - time pcr system .
the primer sequences for rar , rar , rar , adam10 , ide and glyceraldehyde 3-phosphate dehydrogenase ( gapdh ) are listed in table 1 .
learning and memory at baseline was examined via the morris water maze tests at 18 weeks of age , and the rats were re - tested at 30 days and 80 days after the injection of aggregated a1 - 42 to evaluate their spatial learning and memory abilities as previously described .
the test was performed in a 1.5-m ( in diameter ) pool with a 10-cm ( in diameter ) platform .
the procedure consisted of 1 day of visible platform tests and 4 days of hidden platform tests , plus a probe trial 24 hours after the last hidden platform test . in the visible platform test
performed on the first day , the rats were tested for 5 continuous trials , with an inter - trial interval of 60 minutes . for the hidden platform tests ,
rats were trained for 5 trials , with an inter - trial interval of 60 minutes .
if the rat was unable to find the platform , then it was placed on the platform and allowed to stay for 20 seconds . on the last day of the test
, each rat was subjected to the probe trial in which the platform was removed .
animal movement was tracked and recorded using any - maze tracking software ( stoelting ) .
serum retinol data between the mvad and control groups prior to a1 - 42 injection were analyzed by student s t test .
after a1 - 42 injection , either the serum retinol data or mrna data were analyzed with the general linear model univariate anova for the main effects of diet ( control or mvad ) , a1 - 42 injection ( vehicle , 1/2a or a ) , and their interactions ( two - way anova ) ; all significant main effects were further analyzed using bonferroni post hoc tests .
behavioral data were analyzed by a two - way anova with repeated measures , with diet as the between - subjects factor and learning day as the within - subjects factor .
correlation analyses between the adam10/ide and rars mrna levels were conducted by calculating the pearson s correlation coefficient ( r ) .
all statistical analyses were performed using the statistical package for the social sciences ( spss 17.0 ) software .
all wistar rats used in this study were obtained from the experimental animals center of beijing , china .
the rats were housed in a room with a constant airflow system , controlled temperature ( 22 - 24c ) and a 12-hour light / dark cycle .
the female rats were randomly divided into a control group and an mvad group and fed a va - normal ( van ) diet ( 6,500 iu va per kilogram of the basic diet ) and an mvad diet ( 400 iu va per kilogram of the basic diet ) , respectively , for 4 weeks to generate maternal control and mvad rats . after confirming that the serum retinol levels matched the recommended standards for mvad ( 0.70 - 1.05
mol / l ) , the female rats were mated with the van males . after giving birth ,
the male weanlings were subjected to experiment and were maintained throughout the study on a van or mvad diet .
this study was conducted in strict accordance with the recommendations of the national institutes of health s guide for the care and use of laboratory animals .
the animal experimentation ethical committee of chongqing medical university in chongqing , china , approved the protocol .
the preparation of the aggregated a142 and the brain stereotaxic surgery were performed as previously described .
a1 - 42 ( sigma - aldrich , usa ) was dissolved in 0.01 m phosphate - buffered saline ( pbs ) , ph 7.4 , and then vigorously agitated ( 200 rpm ) at room temperature for 36 hours .
sediments were dissolved with pbs to a 10 m stock , aliquoted , and stored at -80c .
after the baseline morris water maze tests were conducted with 18-week - old pups , the pups in both the control and mvad groups were randomly divided into the a group ( full dose of aggregated a1 - 42 ) , the 1/2a group ( half dose of aggregated a1 - 42 ) and the vehicle group .
all subjects were anaesthetized , and the injections were made bilaterally into the ca3 area of the dorsal hippocampus with the injectates deposited slowly over 10 minutes .
the stereotaxic coordinates were 2.6 mm lateral and 3.5 mm posterior to the bregma , and 3.7 mm ventral .
the injections were 5.0 l of 1x10 m aggregated a1 - 42 suspension , 5.0 l of 0.5x10 m aggregated a1 - 42 suspension and 5.0 l of pbs for the a group , the 1/2a group and the vehicle group , respectively .
the maternal rats at preconception and five to six pups from each group at postnatal 24 hours , 4 weeks , 8 weeks , 12 weeks , 18 weeks , 22 weeks and 26 weeks were randomly selected to detect serum retinol levels .
the pups younger than 8 weeks old were decapitated to collect the blood ; and the blood from the pups at the age of 8 weeks and older were collected via the caudal vein .
all pups were decapitated after the last behavioral tests to detect serum retinol levels and confirm the success of the animal models .
serum retinol was measured using high performance liquid chromatography ( hplc ) as previously described .
briefly , 200 l of serum was deproteinized with an equal volume of dehydrated alcohol .
the retinol was extracted from the serum by adding 1,000 l of hexane and evaporated with nitrogen gas .
the retinol residue was dissolved in 100 l of the mobile phase mixture ( methanol : water=97:3 ) .
then , 20 l of the dissolved liquid was tested in the hplc apparatus ( dgu-20as , shimadzu corporation , kyoto , japan ) on a silica column with a 315-nm ultraviolet photodiode array detector .
all procedures were performed in a dark room to protect the samples from light at the children s nutrition research center in the children s hospital of chongqing medical university .
total rna was extracted from the hippocampus of rats using tri reagent ( sigma ) and reverse - transcribed into cdna using the primescript rt reagent kit ( takara ) according to the manufacturer s instructions .
real - time pcr was conducted using a sybr premix ex tag ( perfect real time ; takara ) and a bio - rad real - time pcr system .
the primer sequences for rar , rar , rar , adam10 , ide and glyceraldehyde 3-phosphate dehydrogenase ( gapdh ) are listed in table 1 .
learning and memory at baseline was examined via the morris water maze tests at 18 weeks of age , and the rats were re - tested at 30 days and 80 days after the injection of aggregated a1 - 42 to evaluate their spatial learning and memory abilities as previously described .
the test was performed in a 1.5-m ( in diameter ) pool with a 10-cm ( in diameter ) platform .
the procedure consisted of 1 day of visible platform tests and 4 days of hidden platform tests , plus a probe trial 24 hours after the last hidden platform test . in the visible platform test
performed on the first day , the rats were tested for 5 continuous trials , with an inter - trial interval of 60 minutes . for the hidden platform tests ,
rats were trained for 5 trials , with an inter - trial interval of 60 minutes .
if the rat was unable to find the platform , then it was placed on the platform and allowed to stay for 20 seconds . on the last day of the test
, each rat was subjected to the probe trial in which the platform was removed .
animal movement was tracked and recorded using any - maze tracking software ( stoelting ) .
serum retinol data between the mvad and control groups prior to a1 - 42 injection were analyzed by student s t test .
after a1 - 42 injection , either the serum retinol data or mrna data were analyzed with the general linear model univariate anova for the main effects of diet ( control or mvad ) , a1 - 42 injection ( vehicle , 1/2a or a ) , and their interactions ( two - way anova ) ; all significant main effects were further analyzed using bonferroni post hoc tests .
behavioral data were analyzed by a two - way anova with repeated measures , with diet as the between - subjects factor and learning day as the within - subjects factor .
correlation analyses between the adam10/ide and rars mrna levels were conducted by calculating the pearson s correlation coefficient ( r ) .
all statistical analyses were performed using the statistical package for the social sciences ( spss 17.0 ) software .
to investigate the effects of embryonic - onset mvad following an a1 - 42 injection on spatial learning and memory , we first generated a maternal mvad model by feeding female adult rats an mvad diet for 4 weeks . compared with control rats ,
the serum retinol levels were significantly reduced in the mvad maternal rat model ( 0.790.07 mol / l vs. 1.330.20 mol / l , p<0.05 ; fig .
after confirming that the maternal model was established , the females were mated with normal males , and the male offspring were used for the subsequent experiments .
the serum retinol levels of the pups at 4 , 8 and 12 weeks of age were significantly lower in the mvad group than in the control animals ( p<0.05 ; fig .
after a half- or full - dose injection of aggregated a1 - 42 ( 1/2a or a ) , only significant main effects of diet ( p<0.05 ; fig .
1c ) without a main effect of a or a significant dieta interaction were observed at all ages of 18 , 22 and 26 weeks ( p>0.05 ; fig .
1c ) , indicating that the a1 - 42 injection did not significantly affect the serum retinol content while the mvad diet markedly lowered the serum retinol levels .
the rats were sacrificed immediately after the last behavioral tests at 80 days post - aggregated a1 - 42 injection , and the serum retinol levels were measured .
a two - way anova revealed a significant main effect of diet in the absence of a main effect of a or a significant dieta interaction ( diet : f(1,59)=95.47 , p<0.001 ; a : f(1,59)=2.59 , p>0.05 ; dieta : f(2,59)=0.72 , p>0.05 ) .
post hoc bonferroni tests indicated that serum retinol levels were significantly reduced in pbs- , 1/2a- and a-injected mvad rats compared with those of their respective controls ( p<0.001 ; fig .
since there was no significant difference in serum retinol levels among the pbs , 1/2a and a rats , we combined the pbs , 1/2a and a groups together for statistical analysis .
the results further confirmed that mvad treatment decreased the serum retinol levels compared with those of the control group ( 0.940.05 mol / l vs. 1.200.053
these data demonstrate that the mvad model , which began during the embryonic period , was successfully established .
we also detected the a1 - 42 levels in the hippocampus of both mvad and control groups after the last behavioral tests at 80 days post - a1 - 42 injection .
western blot analysis showed that a1 - 42 expression was significantly elevated after 1/2a or a injection compared with the pbs vehicle injection in either the mvad or control rats ( p<0.01 ; supplemental fig .
s1 ) , confirming that a1 - 42 was successfully injected . to determine whether mvad affects spatial learning and memory ,
morris water maze tests were used for both the mvad and control pups after an aggregated a1 - 42 injection .
first , we examined learning and memory at baseline ( prior to the a1 - 42 injection at 18 weeks old ) . on the visible platform tests , all rats exhibited similar escape latencies and path lengths ( data not shown ) , indicating that mvad did not affect the rats mobility or vision .
on the hidden platform tests , the mvad pups exhibited similar escape latencies relative to the control group ( p>0.05 ; fig .
, no significant differences were found between the mvad and control groups with regard to escape latency ( p>0.05 ; fig .
2b , left and middle ) , whereas a-injected mvad rats showed a significantly prolonged escape latency compared with that of their respective controls ( p<0.01 ; fig .
2b , right ) . however , 80 days after the injection of vehicle pbs and 1/2a , the mvad pups showed markedly longer escape latencies than those of controls ( p<0.01 ; fig . 2c , left and middle ) , whereas the escape latency of mvad rats was similar to that of controls after the a injection ( p>0.05 ; fig . 2c ,
these results suggest that mvad significantly exacerbated the cognitive deficits induced by a half dose of a1 - 42 during late stages of rat development and that a full dose of a1 - 42 could accelerate this aggravation . to further investigate the effect of mvad on app processing enzymes
, we examined the hippocampal gene expression of rars , including rar , rar and rar , as well as the ad - associated gene expression of adam10 and ide 80 days after the injection of a1 - 42 .
a two - way anova of rar , rar , rar and adam10 revealed significant main effects of diet , a and dieta interaction ( p<0.05 ; fig .
furthermore , significant main effects of a and dieta interaction ( p<0.05 ) in the absence of a significant main effects of diet were observed for ide ( p>0.05 ; fig .
, post hoc bonferroni tests showed that a full dose of a1 - 42 injection appeared to dramatically upregulate the mrna levels of rar , rar and adam10 in the control group ( p<0.001 ) ; however , upregulation occurred to a much lesser extent in the mvad group with no significant differences ( p>0.05 ; fig .
in addition , the mrna levels of both rar and ide were increased by the half dose of a1 - 42 but reduced by the full dose of a1 - 42 in control rats ( p<0.01 ) ; however , no significant differences were observed in the mvad rats following a1 - 42 injection ( p>0.05 ; fig .
pearson s correlation analysis showed that adam10 mrna was positively correlated with both rar and rar mrna ( rrar*adam10=0.9946 , p<0.0001 ; rrar*adam10 = 0.9964 , p<0.0001 ; fig .
a positive correlation was also found between rar and rar mrna ( rrar*rar=0.9951 , p<0.0001 ; fig .
4c ) , whereas no significant correlation was found between adam10 and rar mrna levels ( rrar*adam10=-0.2444 , p=0.13 ) .
furthermore , ide mrna was positively correlated with rar mrna ( rrar*ide=0.6780 , p<0.0001 ; fig .
4d ) but negatively correlated with both rar and rar mrna ( rrar*ide=-0.5118 , p<0.001 ; rrar*ide=-0.5152 , p<0.001 ; fig .
these findings suggest that mvad suppressed a-induced adam10 expression through rar and rar and ide expression through rar.
to investigate the effects of embryonic - onset mvad following an a1 - 42 injection on spatial learning and memory , we first generated a maternal mvad model by feeding female adult rats an mvad diet for 4 weeks . compared with control rats ,
the serum retinol levels were significantly reduced in the mvad maternal rat model ( 0.790.07 mol / l vs. 1.330.20 mol / l , p<0.05 ; fig .
after confirming that the maternal model was established , the females were mated with normal males , and the male offspring were used for the subsequent experiments .
the serum retinol levels of the pups at 4 , 8 and 12 weeks of age were significantly lower in the mvad group than in the control animals ( p<0.05 ; fig .
after a half- or full - dose injection of aggregated a1 - 42 ( 1/2a or a ) , only significant main effects of diet ( p<0.05 ; fig .
1c ) without a main effect of a or a significant dieta interaction were observed at all ages of 18 , 22 and 26 weeks ( p>0.05 ; fig .
1c ) , indicating that the a1 - 42 injection did not significantly affect the serum retinol content while the mvad diet markedly lowered the serum retinol levels .
the rats were sacrificed immediately after the last behavioral tests at 80 days post - aggregated a1 - 42 injection , and the serum retinol levels were measured .
a two - way anova revealed a significant main effect of diet in the absence of a main effect of a or a significant dieta interaction ( diet : f(1,59)=95.47 , p<0.001 ; a : f(1,59)=2.59 , p>0.05 ; dieta : f(2,59)=0.72 , p>0.05 ) .
post hoc bonferroni tests indicated that serum retinol levels were significantly reduced in pbs- , 1/2a- and a-injected mvad rats compared with those of their respective controls ( p<0.001 ; fig .
since there was no significant difference in serum retinol levels among the pbs , 1/2a and a rats , we combined the pbs , 1/2a and a groups together for statistical analysis .
the results further confirmed that mvad treatment decreased the serum retinol levels compared with those of the control group ( 0.940.05 mol / l vs. 1.200.053
these data demonstrate that the mvad model , which began during the embryonic period , was successfully established .
we also detected the a1 - 42 levels in the hippocampus of both mvad and control groups after the last behavioral tests at 80 days post - a1 - 42 injection .
western blot analysis showed that a1 - 42 expression was significantly elevated after 1/2a or a injection compared with the pbs vehicle injection in either the mvad or control rats ( p<0.01 ; supplemental fig .
s1 ) , confirming that a1 - 42 was successfully injected . to determine whether mvad affects spatial learning and memory ,
morris water maze tests were used for both the mvad and control pups after an aggregated a1 - 42 injection .
first , we examined learning and memory at baseline ( prior to the a1 - 42 injection at 18 weeks old ) . on the visible platform tests , all rats exhibited similar escape latencies and path lengths ( data not shown ) , indicating that mvad did not affect the rats mobility or vision .
on the hidden platform tests , the mvad pups exhibited similar escape latencies relative to the control group ( p>0.05 ; fig .
, no significant differences were found between the mvad and control groups with regard to escape latency ( p>0.05 ; fig .
2b , left and middle ) , whereas a-injected mvad rats showed a significantly prolonged escape latency compared with that of their respective controls ( p<0.01 ; fig .
2b , right ) . however , 80 days after the injection of vehicle pbs and 1/2a , the mvad pups showed markedly longer escape latencies than those of controls ( p<0.01 ; fig . 2c , left and middle ) , whereas the escape latency of mvad rats was similar to that of controls after the a injection ( p>0.05 ; fig . 2c ,
these results suggest that mvad significantly exacerbated the cognitive deficits induced by a half dose of a1 - 42 during late stages of rat development and that a full dose of a1 - 42 could accelerate this aggravation .
to further investigate the effect of mvad on app processing enzymes , we examined the hippocampal gene expression of rars , including rar , rar and rar , as well as the ad - associated gene expression of adam10 and ide 80 days after the injection of a1 - 42 .
a two - way anova of rar , rar , rar and adam10 revealed significant main effects of diet , a and dieta interaction ( p<0.05 ; fig .
furthermore , significant main effects of a and dieta interaction ( p<0.05 ) in the absence of a significant main effects of diet were observed for ide ( p>0.05 ; fig .
, post hoc bonferroni tests showed that a full dose of a1 - 42 injection appeared to dramatically upregulate the mrna levels of rar , rar and adam10 in the control group ( p<0.001 ) ; however , upregulation occurred to a much lesser extent in the mvad group with no significant differences ( p>0.05 ; fig .
in addition , the mrna levels of both rar and ide were increased by the half dose of a1 - 42 but reduced by the full dose of a1 - 42 in control rats ( p<0.01 ) ; however , no significant differences were observed in the mvad rats following a1 - 42 injection ( p>0.05 ; fig .
pearson s correlation analysis showed that adam10 mrna was positively correlated with both rar and rar mrna ( rrar*adam10=0.9946 , p<0.0001 ; rrar*adam10 = 0.9964 , p<0.0001 ; fig .
a positive correlation was also found between rar and rar mrna ( rrar*rar=0.9951 , p<0.0001 ; fig .
4c ) , whereas no significant correlation was found between adam10 and rar mrna levels ( rrar*adam10=-0.2444 , p=0.13 ) .
furthermore , ide mrna was positively correlated with rar mrna ( rrar*ide=0.6780 , p<0.0001 ; fig .
4d ) but negatively correlated with both rar and rar mrna ( rrar*ide=-0.5118 , p<0.001 ; rrar*ide=-0.5152 , p<0.001 ; fig . 4e and f ) .
these findings suggest that mvad suppressed a-induced adam10 expression through rar and rar and ide expression through rar.
va and its derivatives , the retinoids , play vital roles in the development , regeneration and maintenance of the cns in both developing and mature brains . despite increasing evidence
suggests that va nutrition is related to neurodegenerative diseases including ad , the mechanisms of vad and mvad in the pathogenesis of ad remain unclear . in the developing countries ,
the dietary pattern is primary an insufficient amount of dominated by grain and vegetables , with meat and va - rich foods .
furthermore , mvad is far more prevalent than vad ; however , it has been grossly neglected because its clinical symptoms are obscure . in many countries ,
including china , postnatal rather than prenatal supplementation is recommended for cases of vad to avoid the teratogenic effect of excess va during pregnancy .
our previous work also revealed that twice as many mvad cases than vad cases exist in the elderly ( 25.8% vs. 13.3% ) .
though highly prevalent of mvad among these people , human studies on mvad during pregnancy and the child s development are difficult to be conducted .
thus , the present study established the mvad rat model by partially depriving va in diets from preconception to the sacrifice of offspring , which was considered to be a long - term status of mvad .
the serum retinol content was found to be significantly  reduced in this mvad model in both mother and offspring , confirming that the model was successfully established .
some previous studies have found that the deprivation of va or disruption of rars induces memory impairment in rodents [ 16 , 17 , 33 ] ; however , these relationships have not been investigated in an ad mode .
in addition , few studies have investigated the involvement of mvad in the development of ad . to investigate the long - term effects of mvad on ad pathogenesis ,
the current study established the ad model in mvad rats through the bilateral injection of aggregated a142 into the ca3 area of the hippocampus in male rats using a single intrahippocampal injection .
this rodent model represents a valid tool for investigating ad because the injection can induce synaptic and memory dysfunction and can facilitate an ad - like pathology [ 34 , 35 ] .
nevertheless , there have been few studies establishing the ad model on this long - term mvad model .
behavioral deficits were not apparent during the first 30 days following aggregated a injection ; however , significant deficits became apparent approximately 60 to 80 days after injection [ 30 , 36 ] .
moreover , the current study employed morris water maze tests at 30 and 80 days post - injection with aggregated a1 - 42 . given that both mvad and a142 administration can impair behavioral performance , we injected rats with not only a full dose of a142 but also a half dose .
we found that the half dose of a142 induced learning and memory deficits compared with the vehicle pbs injection in both the mvad and control rats at 80 days post - injection but not at 30 days post - injection ( supplemental fig .
s2 ) , confirming that the half dose of a142 induced cognitive impairments during the later stages of development .
we also found no significant differences in spatial learning and memory between the mvad and control rats after 30 days of either vehicle pbs injection or a half dose of a1 - 42 injection .
however , at 80 days post - injection of vehicle pbs or a half dose of a1 - 42 , the mvad rats showed significant memory deficits compared with the control animals .
the delayed behavioral deficits induced by mvad might be because of the time required for the neurons to become damaged following a half dose of a1 - 42 injection and the age - related decline in the rats ability to protect their neurons from a-induced neurotoxicity .
however , the memory deficits induced by a full dose of a1 - 42 in the mvad rats were apparent at 30 days but not 80 days post - injection .
previous studies have demonstrated that intracerebral fibrillar a1 - 42 induces proliferation and activation of microglia [ 37 , 38 ] and ra suppresses microglial activation in an ad mouse model , so it is likely that mvad accelerates the activation of microglia following a1 - 42 injection .
a full dose of a1 - 42 might as well facilitate the damage of neurons .
furthermore , the age - related decline of anti - neurotoxicity properties might play an important role in the pathogenesis of ad .
numerous in vitro and in vivo studies have associated ra and its signaling to app processing genes or protein expression .
many recent studies have demonstrated that an ra or rar agonist can reduce a production by activating non - amyloidogenic -secretase [ 18 , 40 , 42 - 44 ] .
adam10 , the central component of -secretase , was identified as a key molecule in ra - mediated anti - ad mechanisms .
in addition , ra can increase a clearance by enhancing the expression of the a-degrading enzyme ide [ 20 , 46 ] .
as the current study showed , mvad did not induce the gene expression of adam10 or ide following a1 - 42 injection , suggesting that mvad might impair the regulatory feedback of adam10 and ide induced by a1 - 42 injection , resulting in an incompetent protective response .
we found that both rar and rar were positively correlated with adam10 , which somewhat corroborates previous studies reporting that rar and rar increased adam10 expression in vivo and in vitro [ 43 , 44 , 47 ] .
in addition , a positive correlation was observed between rar and ide in the current study .
this result supports a recent study reporting that rar and rar agonists enhanced the expression of ide .
moreover , we found that rar and rar were negatively correlated with ide , which is inconsistent with a previous in vitro study showing that ra - elicited rar induced ide expression .
our study of an elderly population also revealed a positive correlation between adam10 and rar and between ide and rar. together , these data indicated that different ad - associated enzymes were regulated by different rars .
additional studies are needed to gain insight regarding ra signaling and ad - associated gene expression .
our present study showed that mvad , beginning from embryonic period , contributed to dysregulation of the alzheimer - associated gene expression as well as exacerbated memory impairment late in life .
similar viewpoints have been demonstrated in the learn model , which was established to integrate environmental risk factors and the developmental basis of ad .
this model indicates that accumulated environmental hits , such as dietary factor and toxicological exposure , produce latent epigenetic changes ( e.g. , dna methylation ) in a long - term fashion , beginning at early developmental stages , until a pathological threshold is reached , resulting in the onset of ad [ 27 , 48 , 49 ] . in addition , deficiency of vitamin b12 or folate has been shown to result in gene promoter methylation with upregulation of ad - associated genes .
all these results suggested that environmental risk factors play a vital role in the etiology of late - life disorders , including ad .
it was also reported that improving the environment could restore the impaired hippocampal neurogenesis and cognitive function in ad .
all these studies supported our hypothesis that va malnutrition could bring about dysregulation of ad related genes which remain latent for many years until aging , poor mid - life diet or a1 - 42 disturbance , resulting in sustained alterations in these genes to promote ad progression .
1 ) it was reported that the maternal nutritional and metabolic environment is critical in determining long - term health and viability with brain function being most sensitive to influences in the embryonic period .
disturbed maternal nutrition may contribute to deficits in offspring s development and health , resulting in offspring at increased risk of child and adult diseases [ 51 - 53 ] .
however , whether maternal mvad affects the cognitive function of the offspring and increases the risk of ad in the offspring was not investigated in the present study .
2 ) additional research is required to explore the therapeutic potential of va in mvad - induced ad pathogenesis and the critical windows of therapy .
mvad beginning during the embryonic period did not induce the ad - associated gene expression of adam10 and ide via rars following a1 - 42 injection in rats .
our results suggest that long - term mvad results in an increased risk of ad .
long - term nutritional remediation , such as monitoring va status and correcting low va levels , during the early stage of life might reduce the risk for ad late in life .
evidently , more research on the therapeutic potential of va in mvad - induced ad pathogenesis is of crucial importance and may provide novel targets for the prevention or treatment of ad .
supplementary material is available on the publisher s web site along with the published article .
 background : although clinical vitamin a deficiency ( vad ) , which is a public health problem developing throughout the world , has been well controlled , marginal vitamin a deficiency ( mvad ) is far more prevalent , especially among pregnant women and preschool children in china . 
 increasing evidence suggests that vad is involved in the pathogenesis of alzheimer s disease ( ad ) . 
 however , whether mvad , beginning early in life , increases the risk of developing ad has yet to be determined.objective : the goal of this study was to investigate the long - term effects of mvad on the pathogenesis of ad in rats.method : an mvad model was generated from maternal mvad rats and maintained with an mvad diet after weaning . 
 the males were bilaterally injected with aggregated amyloid  ( a)142 into the ca3 area of the hippocampus , and the ad - associated cognitive and neuropathological phenotypes were examined.results : we found that mvad feeding significantly aggravated a1 - 42-induced learning and memory deficits in the morris water maze test . 
 mvad did not induce the mrna expression of retinoic acid receptors ( rars ) , a disintegrin and metalloprotease 10 ( adam10 ) or insulin - degrading enzyme ( ide ) in a1 - 42-injected rats . 
 moreover , rar and rar mrna were positively correlated with adam10 mrna , whereas rar mrna was positively correlated with ide mrna.conclusion : our study suggests that mvad beginning from the embryonic period perturbs the ad - associated genes , resulting in an enhanced risk of developing ad . 
INTRODUCTION
MATERIAL AND METHODS
Animals and Diets
Aggregated A
Serum Retinol Assay
Real-time Polymerase Chain Reaction (RT-PCR)
Morris Water Maze Test
Statistical Analysis
RESULTS
MVAD Exacerbates Memory Deficits in A
MVAD Suppresses A
DISCUSSION
CONCLUSION
SUPPLEMENTARY MATERIAL
CONFLICT OF INTEREST