Neurotoxicity Of Apolipoprotein E4 And Its Possible NMDAR Mechanism: Electrophysiological And Intracellular Calcium Imaging Study

Alzheimer's disease(AD),the most common form of dementia in the elderly,is a primary irreversible neurodegenerative disorder characterized by a progressive impairment of learning, memory and cognitive function,as well as neuropathological features such as senile plaques(SPs) and neurofibrillary tangles(NFTs).With the increase of average age of population,prevalence of AD is expected to quadruple over the next 50 years.Unfortunately,it is still short of effective therapeutic approaches to cure this disease.Thus,a better understanding of the molecular events underlying AD will be no doubt important for combating this affliction.Apolipoprotein E(apoE),a 34 kDa protein,is highly expressed in the brain.As one of only two serum apolipoproteins found in the cerebrospinal fluid(apoA-1 is the other),apoE possesses a number of biological properties.In addition to its role in the transport of lipid particles,apoE appears to be involved in many important life processes in the central nervous system such as neural development,learning and memory,regeneration and remodeling.In humans,three major isoforms of apoE(apoE2,apoE3,and apoE4) are encoded respectively by three allelic variants(ε2,ε3,andε4) of a single gene on chromosome 19.Increasing investigations reveal that inheritance ofε4 allele gene represents high occurrence and low age of onset of AD.It has been widely accepted that APOEε4 is a susceptibility gene especially for late-onset familial AD and sporadic AD.Meanwhile,apoE is found present in both SPs and NFTs,two characteristic hallmarks of AD,indicating that apoE is involved in the pathological processes of AD.Based on the clinical observations,a great many of studies have been conducted to elucidate the role of apoE in the pathogenesis of AD.However,the molecular basis underlying the mechanisim of apoE in AD is rather complicated and far from being understood until now.Neuroplasticity failure including synaptic dysfunction that precedes neuronal loss has acquired validity as a possible mechanism in early stage of AD.Hippocampal long-term potentiation(LTP),a persistent enhancement of excitatory synaptic transmission induced by high-frequency stimulation (HFS),has been accepted as a popular electrophysiologicai model for the cellular basis of learning and memory,owing to the close relationship between the hippocampal LTP impairment and the animal cognitive deficients.However,the studies about effects of apoE isoforms on LTP in vitro were rather controversial.Some researches indicated a beneficial function of apoE on LTP,while other experiments showed a detrimental influence of apoE on LTP.Especially,it is still unclear whether and how apoE isoforms affect LTP in vivo.Authough the mechanisms underlying the neuronal impairment in AD remain unclear, increasing scientific evidence suggests that glutamate receptors-mediated neurotoxicity is involved in the pathogenesis of AD.Of three glutamate ionotropic receptor subtypes,the NMDA receptor functions more necessarily in synaptic plasticity because of its high permeability to Ca²⁺ ions.It has been found that the levels and binding of NMDA receptors(NMDAR) are remarkably reduced in the brain of AD patients,while the function of NMDAR has been pathologically activated.However,it is unclear whether the excessive activation of NMDAR is resulted from the insult of apoE,and it is short of a direct electrophysiological proof showing the NMDA-induced currents are modulated by apoE.Calcium ion is one of the most important intracellular second messengers in the brain,being essential for a variety of neuronal functions such as neuronal development,synaptic transmission and plasticity,and the regulation of various metabolic pathways.Accumulating evidences demonstrate that the disturbance in calcium homeostasis is one of the earliest molecular changes occured in AD patients,supporting the calcium hypothesis firstly proposed by Khachaturian that sustained dysregulation of intracellular calcium signaling is of prominent importance in the pathogenesis of AD.As for the relationship between apoE and calcium signaling,the results reported are not coincident.The exact effects of apoE isoforms on neuronal calcium homeostasis need to be further clarified.Thus,in the present study,we aimed to(1) clarify the substantial roles of apoE on hippocampal LTP in vivo and elucidate its possible mediated pathway with field potential recording technique;(2) observe the direct effects of acute application of different isoforms of apoE on NMDA-induced whole-cell currents by using patch clamp technique,to further justify whether NMDARs are the targets of apoE;(3) investigate the effects of apoE4 on intracellular calcium concentration by using calcium fluorescent image,and try to clarify the possible mechanism by which apoE4 produces detrimental influences on calcium homeostasis. Partâ… :Apolipoprotein E4 Suppresses Long-Term Potentiation in Rat Hippocampal CA1 Region In VivoThe present study examined isoform-specific effects of apoE on in vivo LTP in rat hippocampal CA1 region and the possible NMDAR mechanism by acute intracerebroventricular (i.c.v.) injection of recombinant human apoE4 and apoE3 and recording hippocampal field excitatory postsynaptic potential(fEPSP).The results showed that pretreatment with apoE4(0.5～2μg) did not affect the basal synaptic transmission,but significantly suppressed the HFS-induced LTP in a dose-dependent manner.The average amplitude of fEPSPs after application of 0.5μg apoE4 decreased significantly from 210.0±10.7%,167.7±5.9%and 165.4±5.1%in control group to 192.1±8.6%,147.7±3.9%and 145.6±3.9%respectively at 1 min,30 min and 60 min after HFS(P＜0.01);the values dropped to 172.1±2.0%,129.3±5.1%and 121.1±3.9%for 1μg apoE4 application(P＜0.001),and further decreased to 151.8±3.9%,108.0±4.3%and 103.5±3.8%in 2μg apoE4 group(P＜0.001).In contrast to apoE4,apoE3,the most common type of human apoE,had no visible effect on HFS-induced LTP compared with vehicle injection.At the same time,apoE4 did not affect paired-pulse facilitation (PPF) ratio.Further,MK801,a noncompetitive antagonist of NMDAR significantly suppressed the HFS-induced LTP in the hippocampal CA1 area in vivo when applied alone,but did not further enhance apoE4-induced LTP impairment when co-applied with apoE4.The values of fEPSP amplitude in co-application group were 146.2±2.3%,108.1±3.9%and 100.8±3.1%,showing no significant statistical difference(P＞0.05) at three time points as compared to the values(151.1±4.7%, 107.5±5.1%and 101.0±2.6%) in apoE4 alone application group.These results suggest that apoE exerts isoform-specific effects on hippocampal LTP,with apoE4 impairing the induction of hippocampal LTP,and apoE3 maybe maintaining the induction of LTP.These findings give rise to the hypothesis that postsynaptic NMDA receptors,but not presynaptic transmitter release is involved in the suppression of LTP induced by apoE4.Partâ…¡:Apolipoprotein E4 Increases NMDA-Induced Currents Both in Acutely Isolated Rat Hippocampal Neurons and in Cultured Primary Rat Cortical NeuronsThe present study investigated the effects of acute application of different isoforms of apoE, apoE4 and apoE3,on NMDA-induced currents in both acutely isolated rat hippocampal neurons and cultured primary rat cortical neurons,by using whole-cell patch clamp technique.We demonstrated for the first time that NMDA-induced currents in acutely isolated rat hippocampal neurons and in cultured primary rat cortical neurons could be rapidly and significantly potentiated by pretreatment with a physiologically relevant concentration of recombinant human apoE4(100 nM).10 seconds and 30 seconds of pretreatment with 100 nM apoE4 increased the NMDA-induced current density,from 100%in control to 126.9±3.9%and 161.7±4.6%respectively in isolated hippocampal neurons(P＜0.01).Similarly,the relative current density was significantly enhanced by apoE4 in primary rat cortical neurons,from 100%in control to 144.2±14.0%in 30 second pretreatment(P＜0.01) and 170.3±13.9%in 60 sencond pretreatment(P＜0.01),respectively. On the contrary,pretreatment with the same concentration of recombinant human apoE3 had no significantly effects on NMDA-induced currents.These data suggest that apoE may exert an isoform-specific effect on the activity of NMDA receptor.ApoE4 encoded byε4 allel gene may be closely linked to the pathogenesis of AD by pathological activation of NMDAR and increasing in the intracellular Ca²⁺ level.The effective potentiation of NMDAR activities by physiological level of apoE4 supports the hypothesis we supposed that NMDA receptor is another target highly sensitive to apoE4.Partâ…¢:Effects of ApoE Isoforms on Neuronal Cytoplasmic Calcium HomeostasisThe present study investigated the acute and chronic effects of different apoE isoforms(apoE4 and apoE3) exposure on intracellular resting calcium level and calcium response to NMDA in cultured primary rat cortical neurons,by using confocal fluorescent imaging technique.The results showed that acute application of apoE4(100 nM and 300 nM) significantly increased intracellular calcium level and the calcium response to NMDA.20 min after applying apoE4,compared with the vehicle group(100.4±8.1%).The relative fluorescent intensity in 100 nM apoE4 group increased to 137.7±6.9%(P＜0.01);it further increased to 154.1±7.6%(P＜0.01) in 300 nM apoE4 group.Acute exposure of neurons to apoE4 significantly enhanced the calcium response to NMDA(100μM),the peak relative fluorescent density increased from 317.9±8.6%in control group to 335.8±9.7%(P＜0.05) in 100 nM group and 360.8±8.4%(P＜0.05) in 300 nM group respectively.Chronic apoE4(100 nM and 300 nM) pretreatment also significantly increased the resting intracellular calcium,but surprisingly decreased NMDA-induced calcium response.48 hours after apoE4 were applied,[Ca²⁺]_i significantly increased from 100.0±7.0%in vehicle group to 172.5±6.4%(P＜0.01) in 100 nM group and 201.2±9.3%(P＜0.01) in 300 nM group.The relative peak fluorescent density of calcium response to NMDA decreased from 317.9±8.6%in vehicle group to 200.8±9.7%(P＜0.01) and 154.8±8.4%(P＜0.01) respectively in 100 nM and 300 nM apoE4 group.All these apoE4-induced changes in calcium signaling were partly blocked by pretreatment with MK801,a noncompetitive NMDAR antagonist.Acute 100 nM apoE4-induced [Ca²⁺]_i elevation was suppressed from 137.7±6.9%to 120.7±8.4%(P＜0.05);chronic 100 nM apoE4-induced[Ca²⁺]_i elevation was also suppressed from 172.5±6.4%to 131.0±7.1%(P＜0.01); chronic 100 nM apoE4-induced reduction of calcium response to NMDA was reversed from 200.8±9.7%to 267.8±8.9%(P＜0.01) respectively in apoE4 alone group and in co-application group. On the contrary,both acute and chronic application of apoE3,even at 300 nM concentration,did not induce any significant change in intracellular calcium level and NMDA-induced calcium response.These data indicated that apoE4,not apoE3,exerts isoform-specific and detrimental effects on neuronal calcium signaling at physiologically relevant concentration by activating,at least partly, NMDA receptor pathway,which suggests that apoE4-induced calcium overloading may be one of crucial events in the early stage of AD and blocking NMDA receptors by designed specific antagonist should be a potential strategy in the treatment of AD.In conclusion,the present study,by using electrophysiological technologies,i.e.field potential recording and whole-cell patch clamp technique,and confocal fluorescent calcium imaging, observed the effects of apoE isoforms(apoE4 and apoE3) on rat hippocampal LTP in vivo, NMDA-induced currents in both hippocampal and cortical neurons,and intracellular resting calcium level and NMDA-induced calcium response in cultured primary rat cortical neurons.Additionally, the roles of NMDA receptors in apoE4-induced LTP suppression and alteration of calcium signaling were also examined.The results reveal that apoE exerts isoform-specific effects on hippocampal LTP and NMDA-induced currents,as well as intracellular calcium level,with apoE4 posing a detrimental effect.Therefore,the present study demonstrates for the first time that apoE4-induced impairment of LTP induction in hippocampal CA1 region is resulted from the pathological activation of NMDARs and resultant intracellular calcium overload,which would be helpful not only in the elucidating the neurotoxic mechanisms of apoE4 but also in the preventing against apoE4-induced deficit of cognition in AD,even in the treatment of AD in the future.