Cellular Source-Specific Effects of Apolipoprotein (Apo) E4 on Dendrite Arborization and Dendritic Spine Development

Apolipoprotein (apo) E4 is the leading genetic risk factor for Alzheimer’s disease (AD), and it has a gene dose-dependent effect on the risk and age of onset of AD. Although apoE4 is primarily produced by astrocytes in the brain, neurons can also produce apoE4 under stress conditions. ApoE4 is known to inhibit neurite outgrowth and spine development in vitro and in vivo, but the potential influence of apoE4’s cellular source on dendritic arborization and spine development has not yet been investigated. In this study, we report impairments in dendritic arborization and a loss of spines, especially thin (learning) and mushroom (memory) spines, in the hippocampus and entorhinal cortex of 19–21-month-old female neuron-specific-enolase (NSE)-apoE4 and apoE4-knockin (KI) mice compared to their respective apoE3-expressing counterparts. In general, NSE-apoE4 mice had more severe and widespread deficits in dendritic arborization as well as spine density and morphology than apoE4-KI mice. The loss of dendritic spines, especially mushroom spines, occurred in NSE-apoE4 mice as early as 7–8 months of age. In contrast, glial fibrillary acidic protein (GFAP)-apoE4 mice, which express apoE4 solely in astrocytes, did not have impairments in their dendrite arborization or spine density and morphology compared to GFAP-apoE3 mice at both ages. These results indicate that the effects of apoE4 on dendrite arborization, spine density, and spine morphology depend critically on its cellular source, with neuronal apoE4 having more detrimental effects than astrocytic apoE4.     

Hilar GABAergic interneuron activity controls spatial learning and memory retrieval

Background

Although extensive research has demonstrated the importance of excitatory granule neurons in the dentate gyrus of the hippocampus in normal learning and memory and in the pathogenesis of amnesia in Alzheimer''s disease (AD), the role of hilar GABAergic inhibitory interneurons, which control the granule neuron activity, remains unclear.

Methodology and Principal Findings

We explored the function of hilar GABAergic interneurons in spatial learning and memory by inhibiting their activity through Cre-dependent viral expression of enhanced halorhodopsin (eNpHR3.0)—a light-driven chloride pump. Hilar GABAergic interneuron-specific expression of eNpHR3.0 was achieved by bilaterally injecting adeno-associated virus containing a double-floxed inverted open-reading frame encoding eNpHR3.0 into the hilus of the dentate gyrus of mice expressing Cre recombinase under the control of an enhancer specific for GABAergic interneurons. In vitro and in vivo illumination with a yellow laser elicited inhibition of hilar GABAergic interneurons and consequent activation of dentate granule neurons, without affecting pyramidal neurons in the CA3 and CA1 regions of the hippocampus. We found that optogenetic inhibition of hilar GABAergic interneuron activity impaired spatial learning and memory retrieval, without affecting memory retention, as determined in the Morris water maze test. Importantly, optogenetic inhibition of hilar GABAergic interneuron activity did not alter short-term working memory, motor coordination, or exploratory activity.

Conclusions and Significance

Our findings establish a critical role for hilar GABAergic interneuron activity in controlling spatial learning and memory retrieval and provide evidence for the potential contribution of GABAergic interneuron impairment to the pathogenesis of amnesia in AD.     

Isoform‐specific effects of apolipoprotein E on cognitive performance in targeted‐replacement mice overexpressing human APP

The ε4 allele of apolipoprotein E (apoE4) is the predominant genetic risk factor for late‐onset Alzheimer's disease (AD) and is also implicated in cognitive deficits associated with normal aging. The biological mechanisms by which APOE genotype affects cognitive processes or AD pathogenesis remain unclear, but interactions of apoE with amyloid β peptide (Aβ) are thought to play an important role in mediating apoE's isoform‐specific effects on brain function. Here, we investigated the potential isoform‐dependent effects of apoE on behavioral and cognitive performance in human apoE3 and apoE4 targeted‐replacement (TR) mice that also overexpress the human amyloid precursor protein (APP). Beginning at 6–7 months of age, female APP‐Yac/apoE3‐TR (‘poE3’) and APP‐Yac/apoE4‐TR (‘poE4’) mice were tested on a battery of tests to evaluate basic sensorimotor functioning, spatial working memory, spatial recognition, episodic‐like memory and attentional processing. Compared with apoE3 mice, a generalized reduction in locomotor activity was observed in apoE4 mice. Moderate, but significant, cognitive impairments were also detected in apoE4 mice in the novel object‐location preference task, the contextual fear conditioning test, and a two‐choice visual discrimination/detection test, however spontaneous alternation performance in the Y‐maze was spared. These results offer additional support for the negative impact of apoE4 on both memory and attention and further suggest that APP‐Yac/apoE‐TR mice provide a novel and useful model for investigating the role of apoE in mediating susceptibility to cognitive decline.     

Regional-Specific Effects of Ovarian Hormone Loss on Synaptic Plasticity in Adult Human APOE Targeted Replacement Mice

The human apolipoprotein ε4 allele (APOE4) has been implicated as one of the strongest genetic risk factors associated with Alzheimer’s disease (AD) and in influencing normal cognitive functioning. Previous studies have demonstrated that mice expressing human apoE4 display deficits in behavioral and neurophysiological outcomes compared to those with apoE3. Ovarian hormones have also been shown to be important in modulating synaptic processes underlying cognitive function, yet little is known about how their effects are influenced by apoE. In the current study, female adult human APOE targeted replacement (TR) mice were utilized to examine the effects of human APOE genotype and long-term ovarian hormone loss on synaptic plasticity in limbic regions by measuring dendritic spine density and electrophysiological function. No significant genotype differences were observed on any outcomes within intact mice. However, there was a significant main effect of genotype on total spine density in apical dendrites in the hippocampus, with post-hoc t-tests revealing a significant reduction in spine density in apoE3 ovariectomized (OVX) mice compared to sham operated mice. There was also a significant main effect of OVX on the magnitude of LTP, with post-hoc t-tests revealing a decrease in apoE3 OVX mice relative to sham. In contrast, apoE4 OVX mice showed increased synaptic activity relative to sham. In the lateral amygdala, there was a significant increase in total spine density in apoE4 OVX mice relative to sham. This increase in spine density was consistent with a significant increase in spontaneous excitatory activity in apoE4 OVX mice. These findings suggest that ovarian hormones differentially modulate synaptic integrity in an apoE-dependent manner within brain regions that are susceptible to neurophysiological dysfunction associated with AD.     

Isoform-specific effects of ApoE on neurite outgrowth in Olfactory Epithelium culture

Background

The apolipoprotein E4 (apoE4) genotype is a major risk factor for developing late-onset Alzheimer’s disease (AD). Inheritance of apoE4 is also associated with impairments in olfactory function in early stages of AD. In this project we examined the effects of the three common isoforms of human apoE (apoE2, apoE3, and apoE4) on neuronal differentiation and neurite outgrowth in explant cultures of mouse olfactory epithelium (OE).

Results

The OE cultures derived from apoE-deficient/knockout (KO) mice have significantly fewer neurons with shorter neurite outgrowth than cultures from wild-type (WT) mice. Treatment of the apoE KO culture with either purified human apoE2 or with human apoE3 significantly increased neurite outgrowth. In contrast, treatment with apoE4 did not have an effect on neurite outgrowth. The differential effects of human apoE isoforms on neurite outgrowth were abolished by blocking the low-density lipoprotein receptor-related protein (LRP) with lactoferrin and receptor-associated protein (RAP).

Conclusion

ApoE2 and apoE3 stimulate neurite outgrowth in OE cultures by interacting with the lipoprotein receptor, LRP. ApoE4, the isoform associated with AD, failed to promote neurite outgrowth, suggesting a potential mechanism whereby apoE4 may lead to olfactory dysfunction in AD patients.     

Differential Effects of apoE4 and Activation of ABCA1 on Brain and Plasma Lipoproteins

Apolipoprotein E4 (apoE4), the leading genetic risk factor for Alzheimer''s disease (AD), is less lipidated compared to the most common and AD-benign allele, apoE3. We have recently shown that i.p. injections of the ATP-binding cassette A1 (ABCA1) agonist peptide CS-6253 to apoE mice reverse the hypolipidation of apoE4 and the associated brain pathology and behavioral deficits. While in the brain apoE is the main cholesterol transporter, in the periphery apoE and apoA-I both serve as the major cholesterol transporters. We presently investigated the extent to which apoE genotype and CS-6253 treatment to apoE3 and apoE4-targeted replacement mice affects the plasma levels and lipid particle distribution of apoE, and those of plasma and brain apoA-I and apoJ. This revealed that plasma levels of apoE4 were lower and eluted faster following FPLC than plasma apoE3. Treatment with CS-6253 increased the levels of plasma apoE4 and rendered the elution profile of apoE4 similar to that of apoE3. Similarly, the levels of plasma apoA-I were lower in the apoE4 mice compared to apoE3 mice, and this effect was partially reversed by CS-6253. Conversely, the levels of apoA-I in the brain which were higher in the apoE4 mice, were unaffected by CS-6253. The plasma levels of apoJ were higher in apoE4 mice than apoE3 mice and this effect was abolished by CS-6253. Similar but less pronounced effects were obtained in the brain. In conclusion, these results suggest that apoE4 affects the levels of apoA-I and apoJ and that the anti-apoE4 beneficial effects of CS-6253 may be related to both central and peripheral mechanisms.     

Environmental enrichment stimulates neurogenesis in apolipoprotein E3 and neuronal apoptosis in apolipoprotein E4 transgenic mice

Neurodegeneration in Alzheimer's disease (AD) is associated with the activation of neurogenesis. The mechanisms underlying this crosstalk between neuronal death and birth and the extent to which it is affected by genetic risk factors of AD are not known. We employed transgenic mice expressing human apolipoprotein E4 (apoE4), the most prevalent genetic risk factor for AD, or expressing human apoE3 (an AD-benign allele), in order to examine the hypothesis that apoE4 tilts the balance between neurogenesis and neuronal cell death in favor of the latter. The results showed an isoform-specific increase in neurogenesis in the hippocampal dentate gyrus (DG) under standard conditions in apoE4-transgenic mice. Environmental stimulation, which increases neurogenesis in the DG of apoE3-transgenic and wild-type mice, had the opposite effect on the apoE4 mice, where it triggered apoptosis while decreasing hippocampal neurogenesis. These effects were specific to the DG and were not observed in the subventricular zone, where neurogenesis was unaffected by either the apoE genotype or the environmental conditions. These in vivo findings demonstrate a linkage between neuronal apoptosis and the impaired neuronal plasticity and cognition of apoE4-transgenic mice, and suggest that similar interactions between apoE4 and environmental factors might occur in AD.     

M1 Muscarinic Agonist Treatment Reverses Cognitive and Cholinergic Impairments of Apolipoprotein E-Deficient Mice

Abstract: Recent studies suggest that apolipoprotein E (apoE) plays a specific role in brain cholinergic function and that the E4 allele of apoE (apoE4), a major risk factor for Alzheimer's disease (AD), may predict the extent of cholinergic dysfunction and the efficacy of cholinergic therapy in this disease. Animal model studies relevant to this hypothesis revealed that apoE-deficient (knockout) mice have working memory impairments that are associated with distinct dysfunction of basal forebrain cholinergic neurons. Cholinergic replacement therapy utilizing M₁-selective muscarinic agonists has been proposed as effective treatment for AD patients. In the present study, we examined whether the memory deficits and brain cholinergic deficiency of apoE-deficient mice can be ameliorated by the M₁-selective agonist 1-methylpiperidine-4-spiro-(2'-methylthiazoline), [AF150(S)]. Treatment of apoE-deficient mice with AF150(S) for 3 weeks completely abolished their working memory impairments. Furthermore, this reversal of cognitive deficit was associated with a parallel increase of histochemically determined brain choline acetyltransferase and acetylcholinesterase levels and with the recovery of these cholinergic markers back to control levels. These findings show that apoE deficiency-related cognitive and cholinergic deficits can be ameliorated by M₁-selective muscarinic treatment. They also provide a novel model system for development and evaluation of therapeutic strategies directed specifically at the AD patients whose condition is attributed to the apoE genotype.     

Isoform-dependent effects of apoE on doublecortin-positive cells and microtubule-associated protein 2 immunoreactivity following 137Cs irradiation

Previously we found apoE isoform-dependent effects of ¹³⁷Cs irradiation on cognitive function of female mice 3 months following irradiation. Alterations in the number of immature neurons and in the levels of the dendritic marker microtubule-associated protein 2 (MAP-2) might contribute to the cognitive changes following irradiation. Therefore, in the present study we determined if, following ¹³⁷Cs irradiation, there are apoE isoform-dependent effects on loss of doublecortin-positive neuroprogenitor cells or MAP-2 immumonoreactivity. In the dentate gyrus, CA1 and CA3 regions of the hippocampus, enthorhinal and sensorimotor cortex, and central and basolateral nuclei of the amygdala of apoE3 female mice, MAP-2 immunoreactivity increased 3 months following ¹³⁷Cs irradiation. In addition, at 8 h following irradiation, the number of doublecortin-positive cells was higher in apoE3 than apoE2 or apoE4 mice. Together, these data indicate that brains of apoE3 mice respond differently to ¹³⁷Cs irradiation than those of apoE2 or apoE4 mice.     

Generation and characterization of two transgenic mouse lines expressing human ApoE2 in neurons and glial cells

Apolipoprotein E (apoE) isoforms are key determinants of susceptibility to late-onset Alzheimer's disease (AD). The epsilon 4 and epsilon 2 alleles have been associated with increased and decreased risk for AD, respectively. We have generated and characterized transgenic mice in which the human apoE2 gene is expressed under the control of the platelet-derived growth factor B-chain (PDGF-B) promoter, or the transferrin (TF) promoter. S1 nuclease analysis and immunoblotting showed that the PDGF-B apoE2 mice express apoE2 exclusively in the brain whereas the TF apoE2 mice express apoE2 in the liver and in the brain. In the TF apoE2 mouse line, apoE2 is also detected in the plasma. The PDGF-B apoE2 and the TF apoE2 transgenic mice were bred back to apoE(-)(/)(-) background. Immunohistochemical analysis showed that the PDGF apoE2 x apoE(-)(/)(-) and the TF apoE2 x apoE(-)(/)(-) mice express human apoE2 within the neocortex in hippocampal neurons and glial cells, respectively. ApoE(-)(/)(-) mice have been shown to develop age-dependent loss of synaptophysin. Immunoblotting of mouse brain extracts and immunohistochemical analysis of brain sections showed that apoE expression in both apoE2 x apoE(-)(/)(-) transgenic lines was associated with significant recovery of brain synaptophysin levels as compared to the levels of apoE(-)(/)(-) littermates of the same age. These apoE2-expressing mice, when bred back on amyloid precursor protein (APP) transgenic mice or other mouse lines featuring alterations in lipoprotein metabolism, may provide new mouse models for elucidating the role of apoE2 in lipid homeostasis in the brain and in the pathogenesis of AD.     

Passive avoidance learning and memory of 56Fe sham-irradiated and irradiated human apoE transgenic mice

Cranial irradiation is associated with long-term cognitive impairments, including deficits in hippocampus-dependent learning and memory. Not all people exposed to cranial radiation develop cognitive injury, suggesting the involvement of genetic risk factors. There may also be sex differences in susceptibility to develop radiation-induced cognitive injury. The three major human apolipoprotein E (apoE) isoforms are encoded by distinct alleles (epsilon2, epsilon3, and epsilon4). Compared with epsilon3, epsilon4 increases the risk of cognitive impairments following various challenges while epsilon2 provides relative protection. Women are at higher risk to develop Alzheimer's disease (AD) than men, particularly those carrying epsilon4. In previous experiments using male and female mice expressing human apoE-isoforms E2, E3 or E4 under the mouse apoE promoter, we showed that cranial irradiation with 137Cs (10 Gy) results in hippocampus-dependent cognitive impairments that are sex- and apoE-isoform dependent. 137Cs is a form of irradiation often used in the clinical setting. To investigate whether 56Fe irradiation also has sex- and apoE-isoform dependent effects on hippocampus-dependent cognitive function in human apoE mice, we sham-irradiated and irradiated 2-month old male and female human apoE mice at 3 Gy and assessed their performance in a passive avoidance learning and memory test three to five months later.     

Tfm-AR modulates the effects of ApoE4 on cognition

Female mice are more susceptible to apolipoprotein E (apoE4)-induced cognitive deficits than male mice. These deficits can be antagonized by stimulating androgen receptors (ARs). To determine the role of AR in the cognitive effects of apoE4, we backcrossed mutant mice with a naturally occurring defect in the AR [testicular feminization mutant ( tfm )] onto the Apoe −/− background to eliminate mouse apoE gene resulting in non-functional AR, and crossed the tfm / Apoe −/− female mice with apoE4 transgenic male mice. We behaviorally compared Apoe −/−, apoE4, tfm, and tfm /apoE4 male mice. Apoe −/−, apoE4, and tfm mice showed hippocampus-dependent novel location recognition but tfm /apoE4 mice did not. In contrast, all groups showed hippocampus-independent novel object recognition. Hippocampus-dependent learning and memory were also assessed in the water maze. In the water maze probe trial following the second day of hidden platform training, Apoe−/− and apoE4 mice showed spatial memory retention, but tfm and tfm /ApoE4 mice did not. In the water maze, probe trial following the third day of hidden platform training, Apoe−/− , apoE4, and tfm /Apoe −/− mice showed spatial memory retention, but tfm mice did not. These data support an important role for AR in protecting against the detrimental effects of apoE4 on hippocampus-dependent learning and memory.     

Alterations in brain leptin signalling in spite of unchanged CSF leptin levels in Alzheimer's disease

Several studies support the relation between leptin and Alzheimer's disease (AD). We show that leptin levels in CSF are unchanged as subjects progress to AD. However, in AD hippocampus, leptin signalling was decreased and leptin localization was shifted, being more abundant in reactive astrocytes and less in neurons. Similar translocation of leptin was found in brains from Tg2576 and apoE4 mice. Moreover, an enhancement of leptin receptors was found in hippocampus of young Tg2576 mice and in primary astrocytes and neurons treated with Aβ_1‐42. In contrast, old Tg2576 mice showed decreased leptin receptors levels. Similar findings to those seen in Tg2576 mice were found in apoE4, but not in apoE3 mice. These results suggest that leptin levels are intact, but leptin signalling is impaired in AD. Thus, Aβ accumulation and apoE4 genotype result in a transient enhancement of leptin signalling that might lead to a leptin resistance state over time.     

Cognitive Deficits and Disruption of Neurogenesis in a Mouse Model of Apolipoprotein E4 Domain Interaction

Apolipoprotein E4 (apoE4) allele is the major genetic risk factor for sporadic Alzheimer disease (AD) due to the higher prevalence and earlier onset of AD in apoE4 carriers. Accumulating data suggest that the interaction between the N- and the C-terminal domains in the protein may be the main pathologic feature of apoE4. To test this hypothesis, we used Arg-61 mice, a model of apoE4 domain interaction, by introducing the domain interaction feature of human apoE4 into native mouse apoE. We carried out hippocampus-dependent learning and memory tests and related cellular and molecular assays on 12- and 3-month-old Arg-61 and age-matched background C57BL/6J mice. Learning and memory task performance were impaired in Arg-61 mice at both old and young ages compared with C57BL/6J mice. Surprisingly, young Arg-61 mice had more mitotic doublecortin-positive cells in the subgranular zone; mRNA levels of brain-derived neurotrophic factor (BDNF) and TrkB were also higher in 3-month-old Arg-61 hippocampus compared with C57BL/6J mice. These early-age neurotrophic and neurogenic (proliferative) effects in the Arg-61 mouse may be an inadequate compensatory but eventually detrimental attempt by the system to “repair” itself. This is supported by the higher cleaved caspase-3 levels in the young animals that not only persisted, but increased in old age, and the lower levels of doublecortin at old age in the hippocampus of Arg-61 mice. These results are consistent with human apoE4-dependent cognitive and neuro-pathologic changes, supporting the principal role of domain interaction in the pathologic effect of apoE4. Domain interaction is, therefore, a viable therapeutic/prophylactic target for cognitive impairment and AD in apoE4 subjects.     

Increased tau phosphorylation in apolipoprotein E4 transgenic mice is associated with activation of extracellular signal-regulated kinase: modulation by zinc

Although apolipoprotein (apo) E4 is present in amyloid plaques and neurofibrillary tangles, its pathogenic role in Alzheimer's disease (AD) is unclear. Neuronal expression of apoE4 or apoE4 fragments in transgenic mice increases tau phosphorylation. To identify the kinase responsible for the increase, we studied transgenic mice expressing human apoE3 or apoE4 in neurons under the control of the neuron-specific enolase promoter. Brain levels of phosphorylated tau (p-tau) and phosphorylated (active) extracellular signal-regulated kinase (p-Erk) increased with age in both groups but were considerably higher in the apoE4 mice. Other candidate kinases, including glycogen synthase kinase 3beta and cyclin-dependent kinase-5 and its activators p25 and p35, were not significantly altered. The increases in p-Erk and p-tau were highest in the hippocampus, intermediate in the cortex, and lowest in the cerebellum. In the hippocampus, p-Erk and p-tau accumulated in the hilus and CA3 region of the dentate gyrus, where high levels of zinc are found along mossy fibers. In Neuro-2a cells stably expressing apoE3 or apoE4, treatment with ZnCl2 generated 2-fold more p-Erk and 3-fold more p-tau in the apoE4-expressing cells. Phosphorylation of Erk and tau was reduced by preincubation with the Erk pathway inhibitor U0126. Thus, increased tau phosphorylation in apoE4 transgenic mice was associated with Erk activation and could be modified by zinc, suggesting that apoE4 and zinc act in concert to contribute to the pathogenesis of AD.     

ApoE isoform‐dependent deficits in extinction of contextual fear conditioning

The three major human apoE isoforms (apoE2, apoE3 and apoE4) are encoded by distinct alleles (?2, ?3 and ?4). Compared with ?3, ?4 is associated with increased risk to develop Alzheimer's disease (AD), cognitive impairments in Parkinson's disease (PD), and other conditions. In contrast, a recent study indicated an increased susceptibility to the recurring and re‐experiencing symptom cluster of Post‐Traumatic Stress Disorder (PTSD), as well as related memory impairments, in patients carrying at least one ?2 allele. Contextual fear conditioning and extinction are used in human and animal models to study this symptom cluster. In this study, acquisition (day 1, training), consolidation (day 2, first day of re‐exposure) and extinction (days 2–5) of conditioned contextual fear in human apoE2, apoE3 and apoE4 targeted replacement and C57BL/6J wild‐type (WT) mice was investigated. Male and female apoE2 showed acquisition and retrieval of conditioned fear, but failed to exhibit extinction. In contrast, WT, apoE3 and apoE4 mice showed extinction. While apoE2 mice exhibited lower freezing in response to the context on day 2 than apoE3 and apoE4 mice, this cannot explain their extinction deficit as WT mice exhibited similar freezing levels as apoE2 mice on day 2 but still exhibited extinction. Elevating freezing through extended training preserved extinction in controls, but failed to ameliorate extinction deficits in apoE2 animals. These data along with clinical data showing an association of apoE2 with susceptibility to specific symptom clusters in PTSD supports an important role for apoE isoform in the extinction of conditioned fear.     

Effects of human apolipoprotein E isoforms on the amyloid beta-protein concentration and lipid composition in brain low-density membrane domains

Apolipoprotein E4 (apoE4) encoded by epsilon 4 allele is a strong genetic risk factor for Alzheimer's disease (AD). ApoE4 carriers have accelerated amyloid beta-protein (A beta) deposition in their brains, which may account for their unusual susceptibility to AD. We hypothesized that the accelerated A beta deposition in the brain of apoE4 carriers is mediated through cholesterol-enriched low-density membrane (LDM) domains. Thus, the concentrations of A beta and various lipids in LDM domains were quantified in the brains of homozygous apoE3 and apoE4 knock-in (KI) mice, and in the brains of those mice bred with beta-amyloid precursor protein (APP) transgenic mice (Tg2576). The A beta 40 and A beta 42 concentrations and the A beta 42 proportions in LDM domains did not differ between apoE3 and apoE4 KI mice up to 18 months of age. The A beta 40 concentration in the LDM domains was slightly, but significantly higher in apoE3/APP mice than in apoE4/APP mice. The lipid composition of LDM domains was modulated in an apoE isoform-specific manner, but its significance for A beta deposition remains unknown. These data show that the apoE isoform-specific effects on the A beta concentration in LDM domains do not occur in KI mouse models.     

Age- and sex-dependent effect of exogenous melatonin on expression pattern of melatonin receptor (MT1 and MT2) proteins in spleen of mice

Spleen is an important lymphoid organ which exerts immune activities throughout the life in mammals. In this study, we investigated the age- and sex-dependent effect of exogenous melatonin on expression pattern of MT1 and MT2 melatonin receptor proteins in spleen of laboratory Swiss albino mice in three different age-groups – 2, 4, and 8 months. The melatonin receptor expression patterns were studied by immunohistochemical localization and Western blot analysis. Immunohistochemical study showed reactivity of MT1 and MT2 melatonin receptors in spleen of both male and female mice. Exogenous melatonin significantly showed age- and sex-dependent expression pattern of MT1 receptor protein, while MT2 receptors showed only age-dependent differential expression patterns in both male and female mice. Therefore, this study may suggest that exogenous melatonin is modulating MT1 and MT2 receptor protein expression pattern in age- and sex-dependent manner in spleen of mice.     

Intraneuronal amyloid-beta plays a role in mediating the synergistic pathological effects of apoE4 and environmental stimulation

The allele E4 of apolipoprotein E4 (apoE4), which is the most prevalent genetic risk factor of Alzheimer's disease (AD), inhibits synaptogenesis and neurogenesis and stimulates apoptosis in brains of apoE4 transgenic mice that have been exposed to an enriched environment. In the present study, we investigated the hypothesis that the brain activity-dependent impairments in neuronal plasticity, induced by apoE4, are mediated via the amyloid cascade. Importantly, we found that exposure of mice transgenic for either apoE4, or the Alzheimer's disease benign allele apoE3, to an enriched environment elevates similarly the hippocampal levels of amyloid-beta peptide (Abeta) and apoE of these mice, but that the degree of aggregation and spatial distribution of Abeta in these mice are markedly affected by the apoE genotype. Accordingly, environmental stimulation triggered the formation of extracellular plaque-like Abeta deposits and the accumulation of intra-neuronal oligomerized Abeta specifically in brains of apoE4 mice. Further experiments revealed that hippocampal dentate gyrus neurons are particularly susceptible to apoE4 and environmental stimulation and that these neurons are specifically enriched in both oligomerized Abeta and apoE. These findings show that the impairments in neuroplasticity which are induced by apoE4 following environmental stimulation are associated with the accumulation of intraneuronal Abeta and suggest that oligomerized Abeta mediates the synergistic pathological effects of apoE4 and environmental stimulation.