Synaptic loss is accompanied by an increase in synaptic area in the dentate gyrus of aged human apolipoprotein E4 transgenic mice

To investigate the relationship between the three isoforms of apolipoprotein E (E2, E3 and E4) and the integrity of the synaptic circuitry in the dentate gyrus of the hippocampus, we have estimated the synapse per neuron ratio and mean apposition zone area per synapse at the electron microscope level in the dentate gyrus of apolipoprotein E knockout and human apolipoprotein E transgenic mice aged six to 24months. During ageing, only in human apolipoprotein E4 mice was there a decrease in synapse per neuron ratio, accompanied by an increase in synaptic size. When these mice were compared with human apolipoprotein E2, apolipoprotein E knockout and wild-type mice at old age, they displayed the lowest synapse per neuron ratio, but similar apposition zone area. In contrast, as in our previous study, aged apolipoprotein E knockout mice did not show any sign of synaptic degeneration.The functional consequences of such morphological changes remain to be determined. However, if such age-related loss of synapses occurred in the brain of Alzheimer apolipoprotein E4 patients, they might be additive to pathological processes and could contribute to greater cognitive impairment.     

Impaired synaptic plasticity and learning in aged amyloid precursor protein transgenic mice

We investigated synaptic communication and plasticity in hippocampal slices from mice overexpressing mutated 695-amino-acid human amyloid precursor protein (APP695SWE), which show behavioral and histopathological abnormalities simulating Alzheimer's disease. Although aged APP transgenic mice exhibit normal fast synaptic transmission and short term plasticity, they are severely impaired in in-vitro and in-vivo long-term potentiation (LTP) in both the CA1 and dentate gyrus regions of the hippocampus. The LTP deficit was correlated with impaired performance in a spatial working memory task in aged transgenics. These deficits are accompanied by minimal or no loss of presynaptic or postsynaptic elementary structural elements in the hippocampus, suggesting that impairments in functional synaptic plasticity may underlie some of the cognitive deficits in these mice and, possibly, in Alzheimer's patients.     

Selective working memory impairments following intradentate injection of colchicine: attenuation of the behavioral but not the neuropathological effects by gangliosides GM1 and AGF2

Bilateral injection of 3.5 micrograms of colchicine into the dentate gyrus produced specific learning and memory impairments together with a selective pattern of neuropathology. Animals injected with colchicine exhibited a significant impairment in their ability to perform the working memory, but not the reference memory, component of a multiple component T-maze task. These deficits were transient and over time all animals were able to reaquire the task to preoperative levels of performance. Histological analyses revealed that intradentate injection of colchicine produced 1) a significant decrease in the width of both the superior and inferior blades of the dentate gyrus reflecting the extensive loss of granule cells, 2) a related decrease in the size of the dentate molecular layer, and 3) a decrease in the number of cholinergic neurons in the medial septum. The second phase of the experiment demonstrated that gangliosides GM1 and AGF2 did not prevent the initial impairments in working memory performance induced by colchicine but rather accelerated the rate at which it recovered. The gangliosides did not decrease the extent of neuronal damage; there was no sparing of granule cells in the dentate gyrus or cholinergic neurons in the medial septum. These data further support a role for the hippocampus in working memory processes and they also indicate that gangliosides GM1 and AGF2 might be useful for treating the behavioral deficits induced by hippocampal damage.     

Apolipoprotein E genotype influences spatial distribution of cerebral microbleeds

In cerebral amyloid angiopathy patients, microbleeds often cluster, mostly occipital, and are associated with apolipoprotein E (APOE) genotype. Microbleeds also frequently occur in the asymptomatic, general population. In this population, we investigated spatial distribution of microbleeds and whether this is influenced by APOE genotype. In 292 persons with microbleeds, we labeled microbleeds on baseline and follow-up magnetic resonance images. We calculated distance between incident and prevalent microbleeds within and between persons and performed lobar segmentation on the magnetic resonance images. Subsequently, we investigated proximity and lobar distribution in strata of APOE genotype. Microbleeds occurred closer within persons than between persons (−42.2 mm, 95% confidence interval, −44.6 to −39.9; p < 0.001). Microbleeds within APOE ε2 and ε4 carriers occurred closer than those in persons with ε3ε3 genotype (−11.9 mm, 95% confidence interval, −24.4 to 0.6; p = 0.06). Persons with ε2 and ε4 alleles had a larger proportion of microbleeds in the occipital lobe than persons with ε3ε3 genotype. Similar to cerebral amyloid angiopathy patients, microbleeds in the general population cluster and the distribution is affected by APOE genotype.     

Dissecting spatial knowledge from spatial choice by hippocampal NMDA receptor deletion

Hippocampal NMDA receptors (NMDARs) and NMDAR-dependent synaptic plasticity are widely considered crucial substrates of long-term spatial memory, although their precise role remains uncertain. Here we show that Grin1(ΔDGCA1) mice, lacking GluN1 and hence NMDARs in all dentate gyrus and dorsal CA1 principal cells, acquired the spatial reference memory water maze task as well as controls, despite impairments on the spatial reference memory radial maze task. When we ran a spatial discrimination water maze task using two visually identical beacons, Grin1(ΔDGCA1) mice were impaired at using spatial information to inhibit selecting the decoy beacon, despite knowing the platform's actual spatial location. This failure could suffice to impair radial maze performance despite spatial memory itself being normal. Thus, these hippocampal NMDARs are not essential for encoding or storing long-term, associative spatial memories. Instead, we demonstrate an important function of the hippocampus in using spatial knowledge to select between alternative responses that arise from competing or overlapping memories.     

Impact of aging on hippocampal function: plasticity,network dynamics,and cognition

Aging is associated with specific impairments of learning and memory, some of which are similar to those caused by hippocampal damage. Studies of the effects of aging on hippocampal anatomy, physiology, plasticity, and network dynamics may lead to a better understanding of age-related cognitive deficits. Anatomical and electrophysiological studies indicate that the hippocampus of the aged rat sustains a loss of synapses in the dentate gyrus, a loss of functional synapses in area CA1, a decrease in the NMDA-receptor-mediated response at perforant path synapses onto dentate gyrus granule cells, and an alteration of Ca(2+) regulation in area CA1. These changes may contribute to the observed age-related impairments of synaptic plasticity, which include deficits in the induction and maintenance of long-term potentiation (LTP) and lower thresholds for depotentiation and long-term depression (LTD). This shift in the balance of LTP and LTD could, in turn, impair the encoding of memories and enhance the erasure of memories, and therefore contribute to cognitive deficits experienced by many aged mammals. Altered synaptic plasticity may also change the dynamic interactions among cells in hippocampal networks, causing deficits in the storage and retrieval of information about the spatial organization of the environment. Further studies of the aged hippocampus will not only lead to treatments for age-related cognitive impairments, but may also clarify the mechanisms of learning in adult mammals.     

Evolution of human apolipoprotein E (APOE) isoforms: Gene structure,protein function and interaction with dietary factors

Apolipoprotein E (APOE) is a member of the vertebrate protein family of exchangeable apolipoproteins that is characterized by amphipathic α-helices encoded by multiple nucleotide tandem repeats. Its equivalent in flying insects − apolipophorin-III − shares structural and functional commonalities with APOE, suggesting the possibility of an evolutionary relationship between the proteins. In contrast to all other known species, human APOE is functionally polymorphic and possesses three major allelic variants (ε4, ε3 and ε2). The present review examines the current knowledge on APOE gene structure, phylogeny and APOE protein topology as well as its human isoforms. The ε4 allele is associated with an increased age-related disease risk but is also the ancestral form. Despite increased mortality in the elderly, ε4 has not become extinct and is the second-most common allele worldwide after ε3. APOE ε4, moreover, shows a non-random geographical distribution, and similarly, the ε2 allele is not homogenously distributed among ethnic populations. This likely suggests the existence of selective forces that are driving the evolution of human APOE isoforms, which may include differential interactions with dietary factors. To that effect, micronutrients such as vitamin D and carotenoids or dietary macronutrient composition are elucidated with respect to APOE evolution.     

Medial temporal lobe dysfunction during encoding and retrieval of episodic memory in non-demented APOE ε4 carriers

Presence of the apolipoprotein E (APOE) ε4 allele is linked to an increased risk to develop Alzheimer's dementia (AD). However, there are controversial data concerning the impact of the APOE genotype on cognitive functioning and brain activity in healthy subjects. We used event-related functional magnetic resonance imaging (fMRI) to investigate the effects of APOE genotype on spatial contextual memory encoding and retrieval success in healthy older adults. Eighteen subjects (eight APOE4 heterozygotes (ε4+) and 10 non-carriers (ε4−), mean age 60.0±5.0 years) were included in the present analysis. Behaviorally, ε4+ subjects performed significantly worse than ε4− subjects in item memory and spatial context retrieval. fMRI data revealed that ε4+ subjects, compared to ε4-subjects, predominantly showed an increase of neural activity specific to encoding of items and their spatial context in prefrontal, temporal and parietal regions. In contrast, ε4+ subjects showed activity decreases in the right amygdala during successful item recognition and in the prefrontal cortex bilaterally during spatial context retrieval when compared to ε4− subjects. While the activity increases during encoding may reflect compensatory activity in the attempt to maintain normal performance, the decreases during retrieval indicate incipient neural decline in ε4+ subjects. These data highlight that preclinical ApoE-related changes in neural activity are not unidirectional but dissociate depending on the memory phase, i.e., encoding or retrieval.     

Treadmill running improves long-term potentiation (LTP) defects in streptozotocin-induced diabetes at dentate gyrus in rats

ObjectivesIt has been demonstrated that exercise has neuroprotective effects in the central nervous system (CNS), especially in hippocampus. Previous studies have indicated that diabetes mellitus affects synaptic plasticity in the hippocampus leading to impairments in learning and memory. The aim of this study was to evaluate the effects of treadmill running on synaptic plasticity at dentate gyrus (DG) of streptozotocin-induced diabetic rats.Study designExperimental groups were the control, the diabetes and the diabetes-exercise groups. Long-term potentiation (LTP) in perforant path-DG synapses was assessed (by 400 Hz tetanization) in order to investigate the effect of exercise on synaptic plasticity. Field excitatory post-synaptic potential (fEPSP) slope and population spike (PS) amplitude were measured.ResultsWith respect to the control group, fEPSP were significantly decreased in the diabetes group. However, there were no differences between responses of the diabetes-exercise group and the control.ConclusionThe present results suggest that LTP induction in the dentate gyrus is affected under diabetic conditions and that treadmill running prevents these effects. The data suggest that treadmill running protect against diabetes-induced decrease of learning ability and memory function of the hippocampus.     

Age-related spatial learning impairment is unrelated to spinophilin immunoreactive spine number and protein levels in rat hippocampus

Age-related impairments in hippocampus-dependent learning and memory tasks are not associated with a loss of hippocampal neurons, but may be related to alterations in synaptic integrity. Here we used stereological techniques to estimate spine number in hippocampal subfields using immunostaining for the spine-associated protein, spinophilin, as a marker. Quantification of the immunoreactive profiles was performed using the optical disector/fractionator technique. Aging was associated with a modest increase in spine number in the molecular layer of the dentate gyrus and CA1 stratum lacunosum-moleculare. By comparison, spinophilin protein levels in the hippocampus, measured by Western blot analysis, failed to differ as a function of age. Neither the morphological nor the protein level data were correlated with spatial learning ability across individual aged rats. The results extend current evidence on synaptic integrity in the aged brain, indicating that a substantial loss of dendritic spines and spinophilin protein in the hippocampus are unlikely to contribute to age-related impairment in spatial learning.     

Progressive loss of synaptic integrity in human apolipoprotein E4 targeted replacement mice and attenuation by apolipoprotein E2

Inheritance of the APOE4 allele is a well established genetic risk factor linked to the development of late onset Alzheimer's disease. As the major lipid transport protein in the central nervous system, apolipoprotein (apo) E plays an important role in the assembly and maintenance of synaptic connections. Our previous work showed that 7 month old human apoE4 targeted replacement (TR) mice displayed significant synaptic deficits in the principal neurons of the lateral amygdala, a region that is critical for memory formation and also one of the primary regions affected in Alzheimer's disease, compared to apoE3 TR mice. In the current study, we determined how age and varying APOE genotype affect synaptic integrity of amygdala neurons by comparing electrophysiological and morphometric properties in C57BL6, apoE knockout, and human apoE3, E4 and E2/4 TR mice at 1 month and 7 months. The apoE4 TR mice exhibited the lowest level of excitatory synaptic activity and dendritic arbor compared to other cohorts at both ages, and became progressively worse by 7 months. In contrast, the apoE3 TR mice exhibited the highest synaptic activity and dendritic arbor of all cohorts at both ages. C57BL6 mice displayed virtually identical synaptic activity to apoE3 TR mice at 1 month; however this activity decreased by 7 months. ApoE knockout mice exhibited a similar synaptic activity profile with apoE4 TR mice at 7 months. Consistent with previous reports that APOE2 confers protection, the apoE4-dependent deficits in excitatory activity were significantly attenuated in apoE2/4 TR mice at both ages. These findings demonstrate that expression of human apoE4 contributes to functional deficits in the amygdala very early in development and may be responsible for altering neuronal circuitry that eventually leads to cognitive and affective disorders later in life.     

Alteration of CREB phosphorylation and spatial memory deficits in aged 129T2/Sv mice

Phosphorylation of cAMP-response element binding protein (CREB) is required for hippocampus-dependent long-term memory formation. The present study was designed to determine whether spatial memory deficits in aged mice were associated with alteration of hippocampal CREB phosphorylation. We examined the temporal pattern of CREB activation in 5–6 and 23–24-month-old 129T2/Sv mice trained on a spatial reference memory task in the water maze. Phosphorylated CREB (pCREB), total CREB (t-CREB) and c-Fos immunoreactivity (ir) were measured at four time points after the end of training. In young mice, pCREB-ir was significantly increased 15 and 60 min after training in the CA1 region and dentate gyrus. In aged mice sacrificed 15 min after training, pCREB-ir in these structures was reduced whereas t-CREB-ir remained unchanged compared to respective young-adults. An age-related reduction of c-Fos-ir also occurred selectively in hippocampal CA1 region. Since reduced pCREB-ir in CA1 from the 15 min-aged group strongly correlated with individual learning performance, we suggest that altered CREB phosphorylation in CA1 may account for spatial memory impairments during normal aging.     

Role of apolipoprotein E and estrogen in mossy fiber sprouting in hippocampal slice cultures.

A role for apolipoprotein E is implicated in regeneration of synaptic circuitry after neural injury. The in vitro mouse organotypic hippocampal slice culture system shows Timm's stained mossy fiber sprouting into the dentate gyrus molecular layer in response to deafferentation of the entorhinal cortex. We show that cultures derived from apolipoprotein E knockout mice are defective in this sprouting response; specifically, they show no sprouting in the dorsal region of the dentate gyrus, yet retain sprouting in the ventral region. Dorsal but not ventral sprouting in cultures from C57B1/6J mice is increased 75% by treatment with 100 pM 17beta-estradiol; this response is blocked by both progesterone and tamoxifen. These results show that neuronal sprouting is increased by estrogen in the same region where sprouting is dependent on apolipoprotein E. Sprouting may be stimulated by estrogen through its up-regulation of apolipoprotein E expression leading to increased recycling of membrane lipids for use by sprouting neurons. Estrogen and apolipoprotein E may therefore interact in their modulation of both Alzheimer's disease risk and recovery from CNS injury.     

Reduced levels of human apoE4 protein in an animal model of cognitive impairment

The APOE4 allele is the most common genetic determinant for Alzheimer's disease (AD) in the developed world. APOE genotype specific differences in brain apolipoprotein E protein levels have been observed in numerous studies since the discovery of APOE4's link to AD. Since the human apoE4 targeted replacement mice display characteristics of cognitive impairment we sought to determine if reduced levels of apoE might provide one explanation for this impairment. We developed a novel mass spectrometry method to measure apoE protein levels in plasma. Additionally, we developed an ELISA that replicates the mass spectrometry data and enables the rapid quantitation of apoE in plasma, brain and cerebrospinal fluid. We detected a significant decrease in plasma, brain and cerebrospinal fluid apoE levels in the apoE4 mice compared to apoE2 and E3 mice. We also measured a small (∼19%) decrease in brain apoE levels from aged, non-demented APOE4 carriers. Our findings suggest that a fraction of APOE4-linked AD may be due to insufficient levels of functional apoE required to maintain neuronal health.     

Gene dose of apolipoprotein E and age-related hearing loss

Next to outer hair cell dysfunction, age-related hearing loss may be explained by apolipoprotein E (APOE) genotype. In the Leiden 85-plus Study, a population-based study, the participants were 85 years old. We measured hearing loss by pure-tone audiometry in 435 participants in relation to APOE. Results demonstrated that those with the APOE-ε4/ε4 genotype had the highest levels of hearing loss (n = 6; 56.1 dB), those with the APOE-ε3/ε4 or ε2/ε4 genotype (n = 89) had intermediate levels of hearing loss (51.0 dB), and those without the APOE-ε4 allele (n = 340) had the lowest levels of hearing loss (48.9 dB), p for trend = 0.02. Eighty percent of participants had hearing loss of 35 dB and more, that is, hearing impairment. The APOE-ε4 allele was associated with a 2.0-fold increased risk of hearing impairment (confidence interval [CI 95%], 1.0-4.0), compared with those without the APOE-ε4 allele. The risk for hearing impairment in subjects with the APOE-ε4 allele remained similar after adjustment for cardiovascular disease, stroke, and cognitive impairment. Our results suggest that the APOE-ε4 allele contributes to age-related hearing loss.     

Sortilin receptor 1 predicts longitudinal cognitive change

The gene encoding sortilin receptor 1 (SORL1) has been associated with Alzheimer's disease risk. We examined 15 SORL1 variants and single nucleotide polymorphism (SNP) set risk scores in relation to longitudinal verbal, spatial, memory, and perceptual speed performance, testing for age trends and sex-specific effects. Altogether, 1609 individuals from 3 population-based Swedish twin studies were assessed up to 5 times across 16 years. Controlling for apolipoprotein E genotype (APOE), multiple simple and sex-moderated associations were observed for spatial, episodic memory, and verbal trajectories (p = 1.25E-03 to p = 4.83E-02). Five variants (rs11600875, rs753780, rs7105365, rs11820794, rs2070045) were associated across domains. Notably, in those homozygous for the rs2070045 risk allele, men demonstrated initially favorable performance but accelerating declines, and women showed overall lower performance. SNP set risk scores predicted spatial (Card Rotations, p = 5.92E-03) and episodic memory trajectories (Thurstone Picture Memory, p = 3.34E-02), where higher risk scores benefited men's versus women's performance up to age 75 but with accelerating declines. SORL1 is associated with cognitive aging, and might contribute differentially to change in men and women.     

Breaches of the pial basement membrane are associated with defective dentate gyrus development in mouse models of congenital muscular dystrophies

A subset of congenital muscular dystrophies (CMDs) has central nervous system manifestations. There are good mouse models for these CMDs that include POMGnT1 knockout, POMT2 knockout and Large^myd mice with all exhibiting defects in dentate gyrus. It is not known how the abnormal dentate gyrus is formed during the development. In this study, we conducted a detailed morphological examination of the dentate gyrus in adult and newborn POMGnT1 knockout, POMT2 knockout, and Large^myd mice by immunofluorescence staining and electron microscopic analyses. We observed that the pial basement membrane overlying the dentate gyrus was disrupted and there was ectopia of granule cell precursors through the breached pial basement membrane. Besides these, the knockout dentate gyrus exhibited reactive gliosis in these mouse models. Thus, breaches in the pial basement membrane are associated with defective dentate gyrus development in mouse models of congenital muscular dystrophies.     

Hippocampal neuron and synaptophysin-positive bouton number in aging C57BL/6 mice

A loss of hippocampal neurons and synapses had been considered a hallmark of normal aging and, furthermore, to be a substrate of age-related learning and memory deficits. Recent stereological studies in humans have shown that only a relatively minor neuron loss occurs with aging and that this loss is restricted to specific brain regions, including hippocampal subregions. Here, we investigate these age-related changes in C57BL/6J mice, one of the most commonly used laboratory mouse strains. Twenty-five mice (groups at 2, 14, and 28–31 months of age) were assessed for Morris water-maze performance, and modern stereological techniques were used to estimate total neuron and synaptophysin-positive bouton number in hippocampal subregions at the light microscopic level. Results revealed that performance in the water maze was largely maintained with aging. No age-related decline was observed in number of dentate gyrus granule cells or CA1 pyramidal cells. In addition, no age-related change in number of synaptophysin-positive boutons was observed in the molecular layer of the dentate gyrus or CA1 region of hippocampus. We observed a significant correlation between dentate gyrus synaptophysin-positive bouton number and water-maze performance. These results demonstrate that C57BL/6J mice do not exhibit major age-related deficits in spatial learning or hippocampal structure, providing a baseline for further study of mouse brain aging.