alzheimer 's disease ( ad ) is the most common form of dementia and represents a serious economic and social burden worldwide . the familial form of ad ( fad ) is caused by autosomal dominant mutations that increase levels of the 42 amino acid isoform of the amyloid - beta 42 ( a42 ) peptide [ 1 , 2 ] . the primary genetic risk factor for ad is inheritance of the apoe4 gene for apolipoprotein e ( apoe ) , compared to apoe3 , with apoe2 reducing risk [ 35 ] . the mechanism(s ) by which apoe and a affect pathogenesis of the disease is unclear ( reviewed [ 35 ] ) . however , evidence suggests that apoe may isoform - specifically modulate a-induced neurotoxicity [ 4 , 5 ] . to address potential mechanisms in vivo , several transgenic ( tg ) mouse models have been developed to assess the structural and functional interactions between apoe and a. however , each of these models has potential drawbacks that affect the interpretation and physiological relevance of the results . this paper will summarize the current models resulting from the introduction of human - apoe into a-tg mice . the continued development and characterization of both apoe and apoe / a-tg mouse models is critical to understanding the apoe - isoform effects on ad pathology . traditional diagnosis of ad is based on pathology that includes extracellular amyloid plaques , composed primarily of a42 , intraneuronal neurofibrillary tangles , hyperphosphorylated tau , neuroinflammation , and neuronal cell loss . a is a 3943 amino acid ( 4  kda ) peptide produced via sequential proteolysis of amyloid precursor protein ( app ) by -secretase / bace followed by -secretase ( composed of presenilins [ ps ] 1 and 2 ) , to produce a peptides primarily 40 and 42 amino acids in length . genetic and experimental evidence indicates that a42 is the cause of ad pathogenesis [ 1 , 2 ] : ( 1 ) fad , although only 35% of all ad cases is caused by autosomal dominant mutations in app , ps1 , or ps2 that increase levels of a42 or the a42  :  40 ratio ; ( 2 ) down syndrome is caused by trisomy of chromosome 21 ( the location of the app gene ) , and is characterized by plaque deposition and dementia by the age 40 ; ( 3 ) a42 is neurotoxic in vitro and in vivo . a , particularly the more toxic a42 , aggregates to form a variety of higher - order assemblies including oligomers , protofibrils , fibrils , and amyloid plaques . amyloid plaques themselves exist in different conformations including compact plaques ( composed of a dense thioflavin - s- ( thios- ) positive core ) , neuritic plaques ( identified as thios - positive plaques surrounded by a ring of dystrophic neurites ) , and diffuse plaques ( characterized by amorphous wisps of amyloid that lack a central core and are not neurotoxic ) [ 710 ] . while total plaque burden does not directly correlate with dementia , it is an indication of increased a42 levels , and compact or dense core plaques may be disease relevant [ 7 , 12 ] . apoe is the only apolipoprotein synthesized within the blood - brain barrier ( bbb ) and is the primary apolipoprotein associated with lipoprotein particles in the central nervous system ( cns ) , as peripheral apoe is unable to cross the bbb or blood - cerebrospinal fluid barrier . while the majority of apoe in the cns is secreted by glial cells , particularly astrocytes , neuronal production of apoe has been observed under specific pathological conditions . cns lipoproteins are critical for lipid homeostasis , particularly as cholesterol and phospholipids are required for neuronal growth , repair , and synaptogenesis ( reviewed [ 35 ] ) . in humans , three apoe polymorphic alleles exist ( apoe2 , apoe3 , and apoe4 ) which encode three protein isoforms ( apoe2 , apoe3 , and apoe4 ) . although human - apoe ( h - apoe ) is a 299 amino acid protein , the three apoe - isoforms differ by a single amino acid substitution at residues 112 or 158 : apoe2 contains cys , apoe3 contains cysarg , and apoe4 contains arg . in the general population , apoe3 is the most common allele ( 77% ) , followed by apoe4 ( 15% ) and apoe2 ( 8% ) . compared to apoe3 , inheritance of one or two copies of the apoe4 allele increases the risk for developing ad by 4- and 12-fold , whereas apoe2 decreases risk by 2- and 4-fold [ 5 , 14 ] . in addition to ad , apoe4 is a risk factor for cerebral amyloid angiopathy ( caa ; amyloid deposition in blood vessels ) and impairs recovery from cerebral insults such as stroke , cerebral hemorrhage , and brain injury . while apoe knock - out mice ( apoe ) and h - apoe - tg models demonstrate that apoe affects neuronal viability independent of a-induced neurotoxicity , the focus of this paper is on the synergism between the h - apoe isoforms and a on neuropathology . to address the latter in vivo , apoe / a-tg models were subsequently developed to specifically address the isoform - specific effects of h - apoe on a deposition . several approaches have been used to make tg mouse models to assess the function of apoe . apoe tg - mice were initially used to help understand the role of apoe in the brain [ 15 , 16 ] although the homology between mouse ( m- ) and h - apoe is 70% , and mice express only a single isoform , comparable to apoe4 at residues 112 and 158 . apoe mice have been used as the background for several h - apoe - tg mouse lines . heterologous promoters have been used to drive the expression of h - apoe in glia or neurons . examples include glial fibrillar acidic protein ( gfap ; glial ) [ 22 , 5557 ] , transferrin ( neuronal ) , platelet - derived growth factor ( pdgf ; neuronal ) , neuron - specific enolase ( nse ; neuronal ) , and thymocyte differentiation antigen 1 ( thy1 ; neuronal ) . however , these models have limitations inherent in the use of a heterologous promoter and specific to apoe : ( 1 ) the expression of protein by a heterologous promoter is not regulated as it would be by the endogenous promoter ; ( 2 ) the inserted copy number of the transgene can not be regulated by a traditional tg mouse approach ; ( 3 ) while the cell - specific expression of apoe in the brain is controversial [ 6063 ] , the majority of evidence suggests that glia , not neurons , are the primary cell type to express apoe [ 13 , 6467 ] ; ( 4 ) by using the m - apoe background and inducing apoe expression via cns - specific promoters , lack of peripheral apoe becomes a variable of potential importance when interpreting results from these mice ; ( 5 ) evidence suggests that in both humans and apoe - tr mice , apoe4 levels are significantly lower than apoe2 or apoe3 [ 6873 ] . knock - in or targeted - replacement ( tr ) mice were developed that express h - apoe under the control of the endogenous mouse promoter and provide an alternative to heterologous expression of h - apoe . in apoe - tr mice , the coding domain for each of the h - apoe isoforms replaces the coding domain for m - apoe . thus , in apoe - tr mice , glial cells express h - apoe in a native conformation at physiologically regulated levels , and in the same temporal and spatial pattern as endogenous m - apoe . thus , the interpretation of apoe isoform - specific results is determined by the nature of the apoe - tg mouse model . as discussed , a number of apoe - tg mouse models have been developed with apoe expression under the control of different promoters . the general phenotypes of apoe mice and three examples of the most widely studied apoe - tg mice ( gfap - apoe , nse - apoe , and apoe - tr ) are briefly discussed ( table 1 ) .   compared to wild type , apoe mice have decreased excitatory transmission , spine density , and dendritic length [ 1618 ] . these changes may underlie behavioral deficits , as apoe mice are cognitively impaired [ 15 , 19 , 20 ] . however , lack of peripheral apoe can have profound effects on plasma lipid homeostasis , potentially leading to a number of confounding variables , including metabolic disease and increased risk for cardiovascular disease , compromising the ability to compensate for oxidative stress or inflammation , deficits that can effect the vasculature of the brain . in addition , the relevance of these mice is unclear as there are no apoe humans . apoe provides the background for a number of the h - apoe - tg mice . gfap is an intermediate filament protein , expressed at high levels by glia in the cns . to target apoe expression to glia , mice expressing h - apoe under the control of the gfap promoter were crossed with apoe mice [ 17 , 22 , 23 ] . at the cellular level , gfap - apoe4 mice show increased ca1 cellular atrophy and decreased spine density compared to gfap - apoe3 mice . in addition , compared to gfap - apoe3 mice , gfap - apoe4 mice demonstrate impaired cognition and increased anxiety ; common symptoms in ad patients [ 17 , 23 , 24 ] . interestingly , cognitive impairment is evident at an earlier age in female compared to male gfap - apoe4 mice [ 23 , 24 ] . a limitation of this model is that the effect of apoe isoform on neuroinflammatory responses can not be interpreted , as apoe is expressed under the control of a promoter that is induced by neuroinflammation . neuronal apoe expression remains controversial , although expression has been identified under conditions of stress . to investigate the role of neuronal expression , apoe - tg mice expressing h - apoe under the control of the nse promoter kainic acid was used to induce apoe expression in these mice and resulted in the protection of nse - apoe3 mice from age- and kainic acid - induced presynaptic and dendritic degeneration compared to nse - apoe4 mice . in addition , female nse - apoe4 mice demonstrate impaired cognition compared to female nse - apoe3 mice . in contrast this is similar to the early development of cognitive deficits in gfap - apoe4 . to investigate the function(s ) of apoe in a more physiologically relevant apoe - tg model , apoe - tr mice were developed to express each h - apoe isoform under the control of the endogenous m - apoe promoter [ 2835 ] . although apoe protein levels in brain homogenates of these mice were initially described as being similar for each h - apoe isoform , subsequent analysis demonstrated that apoe4 levels are significantly lower in plasma , csf , and brain homogenates than apoe2 and apoe3 [ 28 , 30 ] , similar to the levels of apoe isoforms in humans [ 6873 ] . compared to apoe3-tr mice , apoe4-tr mice demonstrate decreased spine density and dendrite length , reduced excitatory transmission , and long - term potentiation ( table 1 ) . again , similar to the cognitive deficits observed in female gfap - apoe4 mice and nse - apoe4 mice , female apoe4-tr mice are cognitively impaired compared to female apoe3-tr mice [ 31 , 32 ] interestingly , both apoe3- and apoe4-tr females are cognitively impaired when compared to apoe - isoform - matched males . although amyloid plaques are a hallmark of ad , the mechanisms underlying a-induced toxicity remain unknown . to help determine the effects of a and amyloid deposition on neurotoxicity in vivo , a-tg mouse models have been produced that express human fad mutations . these models include but are not limited to : pdapp ( app ) , tg2576 ( app ) , j9 ( ps1 , app ) , as well as 5xfad ( app , ps1 ) ( reviewed in ) . these a-tg mice express either app mutations alone ( pdapp , tg2576 ) or in combination with ps mutations ( j9 , 5xfad ) . plaque development generally begins at 6 months of age in these mice ( with the exception of 5xfad ) , and the onset of cognitive deficits is dependent on the model ( e.g. , pdapp at 6 months , tg2576 at 9 months ) . because the app mutation is linked primarily to caa , the tg2576 mice also develop a deposition around blood vessels , particularly leptomeningeal and cortical vessels . however , one limitation of a-tg models is the relative lack of neuronal loss as observed in ad ( reviewed ) although there are some exceptions ( e.g. , 5xfad , appps1ki , and tba2 mice ) . initially , the effect of apoe on a deposition was investigated using apoe crossed with a-tg mice ( table 2 ) . specifically , apoe mice were crossed with pdapp [ 7779 ] or tg2576 mice . in both models , the absence of apoe significantly delayed thios - positive plaque deposition by 6 months and decreased a levels in the hippocampus and cortex , as measured biochemically from brain homogenates and by a immunoreactivity [ 42 , 77 ] ( table 2 ) . apoe affects plaque deposition in a gene - dose - dependent manner , as plaque levels were intermediate in apoe / pdapp compared with apoe / pdapp and apoe / pdapp mice . in addition , the a40 caa present in the tg2576 mice was absent in the apoe / tg2576 mice . the initial studies demonstrating that the lack of m - apoe delayed plaque deposition in a-tg mice led to the question of what would be the effect of introducing h - apoe into apoe / a-tg mice ( table 2 ) . to initially address this issue , gfap - apoe mice was crossed with apoe / pdapp mice [ 41 , 42 ] . surprisingly , the introduction of h - apoe did not result in the expected reduction in the age of onset of a pathology , rather the presence of h - apoe further delayed a deposition . amyloid accumulation is delayed from 6 to 12 months in apoe / pdapp mice , and to 15 months in gfap - apoe / pdapp mice ( table 2 ) . once plaque pathology returned , the greatest plaque burden was found in gfap - apoe4/pdapp mice , compared with gfap - apoe2/pdapp and gfap - apoe3/pdapp mice . one potential confounding factor in these mice , as well as the nse - apoe mice described below , is the equal expression of the h - apoe isoforms , in contrast to human data where inheritance of an apoe4 allele results in lower apoe levels [ 68 , 69 , 73 ] . to determine the effect of neuronal h - apoe expression on a pathology , nse - apoe similar to gfap - apoe / pdapp - tg mice , the introduction of h - apoe to j9 mice delayed plaque pathology from 8 to 19 months , with deposition greater in the nse - apoe4/j9 than nse - apoe3/j9 . the physiologic advantages of using apoe - tr mice to study the function(s ) of apoe in vivo led to the generation of apoe - tr / pdapp   and apoe - tr / tg2576 mice   ( table 2 ) . the resulting data confirmed that h - apoe delayed plaque deposition . in apoe - tr / pdapp mice , plaque deposition was delayed from 6 months to 18 months of age . an apoe isoform - specific effect on a pathology was also observed , with thios staining , a immunoreactivity , and a levels in brain homogenates highest with apoe4 . in tg2576 mice , plaque deposition initiates at 9 months of age , while in apoe - tr / tg2576 mice there is minimal plaque staining at 15 months . interestingly , at 15 months , the isoform effect in these mice is primarily on caa ( e4 > e3 ) , as amyloid deposition in the parenchyma is minimal . the major drawback to the apoe / a-tg crosses described thus far is the significant h - apoe - induced delay in a pathology . for example , in apoe - tr / pdapp mice , plaque deposition is not identified until mice are 18 months of age ( table 2 ) . this substantial delay precludes timely analyses of apoe isoform - specific effects on early aspects of a pathology . one approach to address this temporal delay is to introduce an additional insult , such as traumatic brain injury ( tbi ) , kainic acid , nitric - oxide - synthase-2- ( nos2- ) knock - out , or by blocking a degradation via neprilysin inhibition . although no amyloid deposition is present in 12-month - old gfap - apoe / pdapp mice , tbi at 9 months leads to amyloid deposition at 12 months , which is greater with apoe4 compared to apoe3 . kainic acid decreases synaptophysin and map-2 staining in apoe and nse - apoe tg mice , with the effect more pronounced with apoe4 than apoe3 . nitric oxide , produced by inducible nos ( encoded for by the nos2 gene ) , is an important signaling and redox factor that plays a key role in neuroinflammation and neurodegeneration . nos2 mice have been crossed with multiple a-tg mouse models , and results demonstrate increased tau phosphorylation and neuronal loss in nos2/a-tg mice . in the tg2576/nos2 mice , inhibition of neprilysin , an enzyme that degrades extracellular a , with thiorphan , induces fibrillization and deposition of a and in wild - type mice . thiorphan treatment of apoe - tr mice leads to aggregation of mouse a 1 week after treatment , with higher a deposition in apoe4-tr compared to apoe3-tr mice [ 84 , 85 ] . thus , thiorphan treatment of h - apoe - a-tg mice represents a potential method of accelerating human a deposition . an alternative method to address the h - apoe - induced temporal delay in a accumulation is to cross apoe - tr mice with a-tg mice that have rapid - onset a pathology , such as 5xfad , 3xtr [ 86 , 87 ] , appps1ki , appps1 , or tgcrnd . for example , because 5xfad mice develop plaques by 2 months , the prediction is that apoe - tr/5xfad - tg mice develop plaques around 6 months and 12 months before other human - apoe / a-tg mice ( ladu lab , unpublished observations and [ 5053 ] ) . the approaches described herein will lead to more tractable apoe / a-tg - models to assess the apoe isoform - specific effects on a pathology . human apoe - tg mouse models demonstrate that , compared to apoe3 , apoe4 increases markers of neurodegeneration and cognitive impairment . initially , to determine the effect of apoe on a pathology , a-tg mice were crossed with apoe mice , resulting in a significant delay in plaque deposition . surprisingly , the introduction of h - apoe to several apoe / a-tg mouse models further delayed plaque deposition . this temporal delay restricts the usefulness of the current apoe / a-tg mice for investigating the process of a accumulation and the resulting neurotoxicity . to accelerate a deposition , current apoe / a-tg models could be treated with an additional insult such as tbi , crossed with other tg models of neurodegeneration ( nos2 ) , or treated with drugs that decrease a degradation . alternatively , the development of a pathology could be accelerated by crossing a-tg models with a rapid onset of a pathology , such as 5xfad mice with apoe - tr mice . transgenic models such as these provide tractable models for identifying biomarkers and the efficient initial validation of therapeutic targets .
apolipoprotein e ( apoe ) and apoe / amyloid- ( a ) transgenic ( tg ) mouse models are critical to understanding apoe - isoform effects on alzheimer 's disease risk . compared to wild type , apoe/ mice exhibit neuronal deficits , similar to apoe4-tg compared to apoe3-tg mice , providing a model for a-independent apoe effects on neurodegeneration . to determine the effects of apoe on a-induced neuropathology , apoe/ mice were crossed with a-tg mice , resulting in a significant delay in plaque deposition . 
 surprisingly , crossing human - apoe - tg mice with apoe//a-tg mice further delayed plaque deposition , which eventually developed in apoe4/a-tg mice prior to apoe3/a-tg . 
 one approach to address hapoe - induced temporal delay in a pathology is an additional insult , like head injury . 
 another is crossing human - apoe - tg mice with a-tg mice that have rapid - onset a pathology . 
 for example , because 5xfad mice develop plaques by 2 months , the prediction is that human - apoe/5xfad - tg mice develop plaques around 6 months and 12 months before other human - apoe / a-tg mice . 
 thus , tractable models for human - apoe / a-tg mice continue to evolve .