Attenuated amplitude of circadian and sleep-dependent modulation of electroencephalographic sleep spindle characteristics in elderly human subjects

The low density lipoprotein receptor-related protein (LRP) gene is a candidate gene for Alzheimer's disease (AD) due to its role as a receptor for apolipoprotein E (apoE), a major genetic risk factor for late-onset familial and sporadic AD. Recently, several studies have reported a correlation between a polymorphism (C766T) in exon 3 of LRP and AD. We examined this polymorphism in a Caucasian population of 225 neuropathologically confirmed cases with AD and 187 elderly cases without any AD neuropathological changes. We found that the exon 3 LRP C/C genotype was slightly but not significantly higher in the AD group when compared to the control group. A meta-analysis of previous studies revealed only a weak correlation of this polymorphism with AD (odds ratio 1.34, [95% CI 1.16-1.54], P < 0.0001). These data indicate that the polymorphism in exon 3 of LRP is only a minor risk factor for AD and that another locus on chromosome 12 is likely responsible for the associations observed in other studies.     

Effects of high cholesterol diet on gliosis in apolipoprotein E knockout mice. Implications for Alzheimer's disease and stroke

Hypercholesterolemia has been suggested as a risk factor for Alzheimer's disease (AD). A genetic risk factor for AD is the E4 allele of apolipoprotein E (apoE). ApoE is the major lipoprotein transporter in the brain, and is mainly produced by glial cells. The present study is focussed on analysing the effects of high cholesterol (HC) diet, duration 9 months, on glial activation in the brain, both in wild type (WT) mice and in mice with a null mutation in the apoE gene (knock-out, KO) mice. The activation of astrocytes and microglia was analysed after immunohistochemical labelling of glial fibrillary acidic protein (GFAP), and F4/80, respectively. In addition, the expression of the antioxidant enzyme NAD(P)H:quinone oxidoreductase (NQO1) was analysed. There was a marked stimulation of astrocyte and microglial activation as well as induced expression of NQO1 in the hippocampus and cerebral cortex upon HC diet. Furthermore, there was significant astrocyte activation in the apoE KO mice, as compared to the WT mice, on ND. The long time exposure to HC diet combined with apoE deficiency resulted in a synergistic effect on the expression of NQO1 in the brain.     

Apolipoprotein E Isoforms Increase Intracellular Ca2+ Differentially Through a ω-Agatoxin IVa-Sensitive Ca2+-Channel

Apolipoprotein E (apoE) is the major apolipoprotein in the brain and is known for its important role in plasticity and neurodegeneration. We show that apoE dose-dependently increases intracellular free Ca²⁺ in rat hippocampal astrocytes and neurons. This effect varies with isoforms in the order E4>E3>E2. It is insensitive to blockade of action potentials by tetrodotoxin or inhibition of binding of apoE by heparinase, by the LRP ligand lactoferrin and by low density lipoprotein. ApoE evoked Ca²⁺-increases are blocked in zero [Ca]o and by the Ca-channel antagonists nickel and ω-Agatoxin-IVa but not by nifedipine and ω-Conotoxin-GVIa, demonstrating an isoform-specific activation of P/Q type Ca²⁺-channels. This novel mechanism is discussed with respect to Alzheimer's disease, that is linked for most cases to the apoE ε-allelic variation (ε4 > ε3 > ε2).     

Estradiol replacement increases the low-density lipoprotein receptor related protein (LRP) in the mouse brain

Numerous epidemiology studies have shown protective effects of hormone therapy (HT) on chronic neurological diseases. We have proposed that some of the neuroprotective effects of estrogen are mediated by apolipoprotein E (apoE). Polymorphisms of receptors for apoE modify the risk for dementia. To our knowledge, no reports exist showing CNS effects of estrogen replacement on members of the low-density lipoprotein receptor family. The current study focused on the effect of estradiol-17beta (E2) replacement on protein expression of two members of the receptor family, the low-density lipoprotein receptor (LDL-r) and low-density lipoprotein receptor related protein (LRP) in ovariectomized mice. Five days of E2 replacement significantly increased LRP expression in the hippocampus, olfactory bulb and neocortex but not in cerebellum. In contrast, E2 treatment decreased LDL-r protein expression in olfactory bulb. HT modification of both apoE and LRP could have wide-spread effects on cellular function given LRP's manifold signaling functions.     

PKC modulation of transmitter release by SNAP-25 at sensory-to-motor synapses in aplysia

Activation of phosphokinase C (PKC) can increase transmitter release at sensory-motor neuron synapses in Aplysia, but the target of PKC phosphorylation has not been determined. One putative target of PKC at synapses is the synaptosomal-associated protein of 25 kDa (SNAP-25), a member of the SNARE protein complex implicated in synaptic vesicle docking and fusion. To determine whether PKC regulated transmitter release through phosphorylation of SNAP-25, we cloned Aplysia SNAP-25 and expressed enhanced green fluorescent protein (EGFP)-coupled SNAP-25 constructs mutated at the PKC phosphorylation site Ser198 in Aplysia sensory neurons. We found several distinct effects of expression of EGFP-SNAP-25 constructs. First, the rates of synaptic depression were slowed when cells contained SNAP-25 with phosphomimetic residues Glu or Asp. Second, PDBu-mediated increases in transmitter release at na?ve synapses were blocked in cells expressing nonphosphorylated-state SNAP-25. Finally, expression of EGFP-coupled SNAP-25 but not uncoupled SNAP-25 inhibited 5-HT-mediated reversal of depression and the ability of EGFP-coupled SNAP-25 to inhibit the reversal of depression was affected by changes at Ser198. These results suggest SNAP-25 and phosphorylation of SNAP-25 by PKC can regulate transmitter release at Aplysia sensory-motor neuron synapses by a number of distinct processes.     

ApoE genotype does not affect plasma tPA and PAI-1 antigen levels

The presence of one or two apoliprotein E4 (apoE4) alleles constitutes a major risk factor for Alzheimer's disease (AD) and coronary heart disease (CHD). Numerous observations have suggested that misregulation of proteases may be instrumental in both diseases. Tissue-type plasminogen activator (tPA) has been recently demonstrated to play a key role in neuronal plasticity and in experimental neurodegeneration. One receptor for the ApoE protein is the LRP/α2 macroglobulin receptor, which also binds to and endocytoses tPA and plasminogen activator inhibitor I (PAI-1). Here we tested whether the apoE genotype has an influence on the plasma levels of these proteins. We demonstrate that there is no difference in plasma levels of tPA- and PAI-1-antigens between middled-aged individuals with one apoE4 allele and those having none. This suggests that the impact of apoE4 on Alzheimer's disease is not the result of altered clearance of tPA or PAI-1 by the LRP receptor. Am. J. Med. Genet. 74:172–175, 1997. © 1997 Wiley-Liss, Inc.     

Increased cholesterol in Abeta-positive nerve terminals from Alzheimer's disease cortex

Synapse loss in Alzheimer's disease (AD) is poorly understood but evidence suggests it is a key pathological event. In order to precisely detect stable synaptic changes, we have developed methods for flow cytometry analysis of synaptosomes prepared from cryopreserved AD samples, and have previously shown that amyloid-beta (Abeta) accumulates in surviving presynaptic terminals in AD cortex. In the present experiments we have examined amyloid-containing terminals in more detail, first dual labeling synaptosomes from AD cortex for Abeta and a series of markers, and then using quadrant analysis to compare amyloid-positive and amyloid-negative terminals. Amyloid-positive synaptosomes were larger in size than amyloid-negatives (p<0.007), and significant increases were observed in mean fluorescence for the lipid raft markers cholesterol (27%; p<0.0005) and GM1 ganglioside (24%; p<0.005). SNAP-25 immunofluorescence was increased by 31% (p<0.0001) in amyloid-bearing terminals, consistent with a sprouting response to amyloid accumulation. These results suggest that Abeta accumulation in synaptic terminals may underly dysfunction prior to or independent of extracellular amyloid deposition.     

Localization of SNARE proteins and secretory organelle proteins in astrocytes in vitro and in situ

Astrocytes are capable of regulated release of messenger molecules. Astrocytes cultured from new born rodent brain express a variety of classical presynaptic proteins. We investigated the question whether the capability to express synaptic proteins in culture was a feature only of immature astrocytes, and whether these proteins were also expressed by astrocytes in situ. Experiments were performed with transgenic mice expressing the enhanced green fluorescent protein under the control of the human glial fibrillary acidic protein promoter. Using double fluorescence and astrocytes cultured from 1 to 16 day-old animals we show that the astrocytic expression of synaptic proteins in culture is invariant of the age of donor animals. Culturing can induce the astrocytic expression of specific synaptic proteins such as SV2, synaptophysin and SNAP-25. Astrocytes in brain sections of 1-16 day-old animals revealed a punctuate immunofluorescence for secretory carrier membrane protein (SCAMP), SNAP-23, synaptobrevin II, and cellubrevin, to a minor extent for SNAP-25 and synaptophysin, and none for SV2. Our results demonstrate that cultured astrocytes express synaptic proteins not present in situ. Nevertheless, astrocytic organelles in situ are equipped with molecules that could be involved in regulated exocytosis of messenger substances.     

ApoAI deficiency results in marked reductions in plasma cholesterol but no alterations in amyloid-beta pathology in a mouse model of Alzheimer's disease-like cerebral amyloidosis

Epidemiological studies suggest links between cholesterol metabolism and Alzheimer's disease (AD), with hypercholesterolemia associated with increased AD risk, and use of cholesterol-lowering drugs associated with decreased risk. Animal models using cholesterol-modifying dietary or pharmacological interventions demonstrate similar findings. Proposed mechanisms include effects of cholesterol on the metabolism of amyloid-beta (Abeta), the protein that deposits in AD brain. To investigate the effect of genetic alterations in plasma cholesterol on Abeta pathology, we crossed the PDAPP transgenic mouse model of AD-like cerebral amyloidosis to apolipoprotein AI-null mice that have markedly reduced plasma cholesterol levels due to a virtual absence of high density lipoproteins, the primary lipoprotein in mice. Interestingly and in contrast to models using non-physiological high fat diets or cholesterol-lowering drugs to modify plasma cholesterol, we observed no differences in Abeta pathology in PDAPP mice of the various apoAI genotypes despite robust differences in plasma cholesterol levels between the groups. Absence of apoAI also resulted in reductions in brain but not cerebrospinal fluid cholesterol, but had no effect on brain apolipoprotein E levels. These and other data suggest that it is perhaps the level of brain apolipoprotein E, not cholesterol per se, that plays a primary role in brain Abeta metabolism.     

Analysis of SNAP-25 immunoreactivity in hippocampal inhibitory neurons during development in culture and in situ

Synaptosomal associated protein of 25 kDa (SNAP-25) is a component of the soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor (SNARE) complex which plays a central role in synaptic vesicle exocytosis. We have previously demonstrated that adult rat hippocampal GABAergic synapses, both in culture and in brain, are virtually devoid of SNAP-25 immunoreactivity and are less sensitive to the action of botulinum toxin type A, which cleaves this SNARE protein [Neuron 41 (2004) 599]. In the present study, we extend our findings to the adult mouse hippocampus and we also provide demonstration that hippocampal inhibitory synapses lacking SNAP-25 labeling belong to parvalbumin-, calretinin- and cholecystokinin-positive interneurons. A partial colocalization between SNAP-25 and glutamic acid decarboxylase is instead detectable in developing mouse hippocampus at P0 and, at a lesser extent, at P5. In rat embryonic hippocampal cultures at early developmental stages, SNAP-25 immunoreactivity is detectable in a percentage of GABAergic neurons, which progressively reduces with time in culture. Consistent with the presence of the substrate, botulinum toxin type A is partially effective in inhibiting synaptic vesicle recycling in immature GABAergic neurons. Since SNAP-25, beside its role as a SNARE protein, is involved in additional processes, such as neurite outgrowth and regulation of calcium dynamics, the presence of higher levels of the protein at specific stages of neuronal differentiation may have implications for the construction and for the functional properties of brain circuits.     

Synaptic protein deficits are associated with dementia irrespective of extreme old age

Recent evidence shows that despite high incidence of dementia in the very old, they exhibit significantly lower levels of Alzheimer's disease (AD) neuropathology relative to younger persons with dementia. The levels and distributions of some synaptic proteins have been found to be associated with dementia severity, even in the oldest-old, but the molecular and functional nature of these deficits have not been studied in detail. The objective of this study was to assess the relationship of dementia with gene and protein expression of a panel of synaptic markers associated with different synaptic functions in young-, middle-, and oldest-old individuals. The protein and messenger RNA (mRNA) levels of 7 synaptic markers (complexin-1, complexin-2, synaptophysin, synaptobrevin, syntaxin, synaptosomal-associated protein 25 (SNAP-25), and septin-5) were compared in the brains of nondemented and demented individuals ranging from 70 to 103 years of age. One hundred eleven brains were selected to have either no significant neuropathology or only AD-associated pathology (neuritic plaques [NPs] and neurofibrillary tangles [NFTs]). The cohort was then stratified into tertiles as young-old (70-81 years old), middle-old (82-88), and oldest-old (89-103). The brains of persons with dementia evidenced significantly lower levels of gene and protein expression of synaptic markers regardless of age. Importantly, dementia was associated with reductions in all measured synaptic markers irrespective of their role(s) in synaptic function. Although other dementia-associated hallmarks of AD neuropathology (neuritic plaques and neurofibrillary tangles) become less prominent with increasing age, synaptic marker abnormalities in dementia remain constant with increasing age and may represent an independent substrate of dementia spanning all ages.     

Altered neurotransmission in the lateral amygdala in aged human apoE4 targeted replacement mice

The human APOE4 allele is associated with an early age of onset and increased risk of Alzheimer's disease (AD). Apolipoprotein E is secreted as part of a high-density lipoprotein-like particle by glial cells in the brain for the primary purpose of transport of lipophilic compounds involved in the maintenance of synapses. Previous studies examining synaptic integrity in the amygdala of human apoE targeted replacement (TR) mice showed a decrease in spontaneous excitatory synaptic activity, dendritic arbor, and spine density associated with apoE4 compared with apoE3 and apoE2 in adult male mice. In the present study, we assessed how APOE genotype affects synaptic integrity of amygdala neurons by comparing electrophysiological and morphometric properties in human apoE3, E4, and E2/4 TR mice at the age of 18–20 months. In contrast to adult mice, we found that aged apoE4 TR mice exhibited the highest level of excitatory synaptic activity compared with other cohorts. Additionally, apoE4 mice had significantly greater spontaneous inhibitory activity than all other cohorts. Taken together, there was a significant interaction between genotypes when comparing inhibition relative to excitation; there was a simple main effect of frequency type with an imbalance toward inhibition in apoE4 mice but not in apoE3 or apoE2/4 mice. These results suggest that apoE isoforms differentially influence synaptic transmission throughout the life span, where aging coupled with apoE4 expression, results in an imbalance in maintaining integrity of synaptic transmission.     

Snapin: a SNARE-associated protein implicated in synaptic transmission

Synaptic vesicle docking and fusion are mediated by the assembly of a stable SNARE core complex of proteins, which include the synaptic vesicle membrane protein VAMP/synaptobrevin and the plasmalemmal proteins syntaxin and SNAP-25. We have now identified another SNAP-25-binding protein, called Snapin. Snapin was enriched in neurons and exclusively located on synaptic vesicle membranes. It associated with the SNARE complex through direct interaction with SNAP-25. Binding of recombinant Snapin-CT to SNAP-25 blocked the association of the SNARE complex with synaptotagmin. Introduction of Snapin-CT and peptides containing the SNAP-25 binding sequence into presynaptic superior cervical ganglion neurons in culture reversibly inhibited synaptic transmission. These results suggest that Snapin is an important component of the neurotransmitter release process through its modulation of the sequential interactions between the SNAREs and synaptotagmin.     

Protein kinase C modulates NMDA receptor trafficking and gating

Regulation of neuronal N-methyl-D-aspartate receptors (NMDARs) by protein kinases is critical in synaptic transmission. However, the molecular mechanisms underlying protein kinase C (PKC) potentiation of NMDARs are uncertain. Here we demonstrate that PKC increases NMDA channel opening rate and delivers new NMDA channels to the plasma membrane through regulated exocytosis. PKC induced a rapid delivery of functional NMDARs to the cell surface and increased surface NR1 immunofluorescence in Xenopus oocytes expressing NMDARs. PKC potentiation was inhibited by botulinum neurotoxin A and a dominant negative mutant of soluble NSF-associated protein (SNAP-25), suggesting that receptor trafficking occurs via SNARE-dependent exocytosis. In neurons, PKC induced a rapid delivery of functional NMDARs, assessed by electrophysiology, and an increase in NMDAR clusters on the surface of dendrites and dendritic spines, as indicated by immunofluorescence. Thus, PKC regulates NMDAR channel gating and trafficking in recombinant systems and in neurons, mechanisms that may be relevant to synaptic plasticity.     

Apolipoprotein E, cholesterol transport and synthesis in sporadic Alzheimer's disease

The discovery that the apolipoprotein E4 (apoE4) allele is genetically linked to both sporadic and familial late onset Alzheimer's disease (AD) raises the possibility that a dysfunction of the lipid transport system could seriously affect lipid homeostasis in the brain of AD subjects. The presence of the E4 allele has been associated with lower levels of apoE in both serum and brain tissues of normal and AD subjects. In an attempt to reverse the apoE deficit in AD, we identified and characterized several apoE inducer agents using a low throughput-screening assay. The most promising of these compounds is called probucol. Administration of probucol, an old cholesterol lowering drug, in mild to moderate sporadic AD led to significant increases in CSF apoE levels and a decrease of CSF beta amyloid 1-42 without significant modifications of CSF tau concentration or CSF lipid peroxides levels. These results are consistent with recent reports suggesting that the long term use of cholesterol lowering drugs that block 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) activity in the fourth and fifth decade of life may help reduce the risk of developing AD at later age. These results indicate that, in addition to lipid transport mediated by apoE, cholesterol homeostasis in the brain is markedly altered in response to changes in the HMGR pathway; suggesting a possible explanation for the potentially beneficial effect of statins in common AD.     

Genetic association between low-density lipoprotein receptor-related protein gene polymorphisms and Alzheimer's disease in Chinese Han population

Alzheimer's disease (AD) is the most common neurodegenerative disorders in the elderly. Low-density lipoprotein receptor-related protein (LRP), as a receptor of apolipoprotein E (APOE), APP, and alpha2 macroglobulin (alpha2-M), keeps the balance between degeneration and production of beta-amyloid protein (Abeta) clearance. Its gene had been defined as a candidate gene for AD, but the results were not universal. Total 496 AD patients and 478 controls were recruited in Chinese Han population and real-time PCR was used to detect the polymorphism of LRP C766T. Multiple logistic regression, Chi-square test and survival analysis were performed to explore the association. The distribution of LRP genotypes and alleles was significantly different between cases and controls, and T allele could reduce the risk for developing AD (OR of CT genotype: 0.57; 95% CI: 0.38-0.85, rho=0.003; OR of T allele: 0.57; 95% CI: 0.39-0.83, rho=0.003). TT genotype carriers had 5 years later for developing AD compared with CC genotype carriers, but survival analysis did not conform this (LRP TT vs. CT and CC log rank chi(2)=2.71, rho=0.26). The distribution of LRP C766T genotypes and alleles was different among different severity stratified by MMSE yet (rho=0.26). Our data suggested that the polymorphism of LRP C766T was strongly associated with AD and T allele might be a protective factor for AD in Chinese Han population.     

Snap-25 is polarized to axons and abundant along the axolemma: an immunogold study of intact neurons

SNAP-25, synaptosomal associated protein of 25 kDa, is reported to be a t-SNARE (target receptor associated with the presynaptic plasma membrane) involved in the docking and fusion of synaptic vesicles. We present here the first ultrastructural localization of SNAP-25 in intact neurons by pre-embedding EM immunocytochemistry in rat brains, hippocampal slice cultures, and PC12 cells. In differentiated neurons, SNAP-25 labeling was clearly membrane-associated. The labeling was most prominent in the plasma membrane of axons and excluded from the plasma membranes of soma and dendrites. Furthermore, SNAP-25 did not appear to be restricted to the synaptic junctions. SNAP-25 labeling was seen in the cytoplasm of the soma and large dendrites, mostly associated with the Golgi complexes. There were also some SNAP-25 labeled tubulo-vesicular structures in the cytoplasm of the soma and the axons, but rarely in the smaller dendrites. In PC12 cells, after 5–10 minutes of high potassium (75 mM) stimulation in the presence of HRP, SNAP-25 labeling appeared, additionally, on HRP-filled early endosomes. After a longer (20–30 minutes) HRP incubation, most of the later stage endosomes and lysosomes were loaded with HRP but they were negative for SNAP-25. These results suggest that SNAP-25 is sorted out of these late endosomal compartments, and that the bulk of the SNAP-25 protein is probably recycled back to the axolemma from the early endosomes. In contrast, in those samples which were incubated with HRP for longer periods, there were still some SNAP-25–positive vesicular structures which were HRP-negative. These structures most likely represent anterograde vesicles that carry newly synthesized SNAP-25 from the soma to the axolemma by axonal transport. SNAP-25 appears to be sorted at the Golgi complex to reach the axolemma specifically. Its widespread distribution all along the axolemma does not support the view of SNAP-25 as a t-SNARE limited for synaptic exocytosis.     

LRP1 modulates the microglial immune response via regulation of JNK and NF-κB signaling pathways

Background

Neuroinflammation is characterized by microglial activation and the increased levels of cytokines and chemokines in the central nervous system (CNS). Recent evidence has implicated both beneficial and toxic roles of microglia when over-activated upon nerve injury or in neurodegenerative diseases, including Alzheimer’s disease (AD). The low-density lipoprotein receptor-related protein 1 (LRP1) is a major receptor for apolipoprotein E (apoE) and amyloid-β (Aβ), which play critical roles in AD pathogenesis. LRP1 regulates inflammatory responses in peripheral tissues by modulating the release of inflammatory cytokines and phagocytosis. However, the roles of LRP1 in brain innate immunity and neuroinflammation remain unclear.

Methods

In this study, we determined whether LRP1 modulates microglial activation by knocking down Lrp1 in mouse primary microglia. LRP1-related functions in microglia were also assessed in the presence of LRP1 antagonist, the receptor-associated protein (RAP). The effects on the production of inflammatory cytokines were measured by quantitative real-time PCR (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). Potential involvement of specific signaling pathways in LRP1-regulated functions including mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) were assessed using specific inhibitors.

Results

We found that knocking down of Lrp1 in mouse primary microglia led to the activation of both c-Jun N-terminal kinase (JNK) and NF-κB pathways with corresponding enhanced sensitivity to lipopolysaccharide (LPS) in the production of pro-inflammatory cytokines. Similar effects were observed when microglia were treated with LRP1 antagonist RAP. In addition, treatment with pro-inflammatory stimuli suppressed Lrp1 expression in microglia. Interestingly, NF-κB inhibitor not only suppressed the production of cytokines induced by the knockdown of Lrp1 but also restored the down-regulated expression of Lrp1 by LPS.

Conclusions

Our study uncovers that LRP1 suppresses microglial activation by modulating JNK and NF-κB signaling pathways. Given that dysregulation of LRP1 has been associated with AD pathogenesis, our work reveals a critical regulatory mechanism of microglial activation by LRP1 that could be associated with other AD-related pathways thus further nominating LRP1 as a potential disease-modifying target for the treatment of AD.