A Novel Triple Repeat Mutant Tau Transgenic Model That Mimics Aspects of Pick’s Disease and Fronto-Temporal Tauopathies

Tauopathies are a group of disorders leading to cognitive and behavioral impairment in the aging population. While four-repeat (4R) Tau is more abundant in corticobasal degeneration, progressive supranuclear palsy, and Alzheimer’s disease, three-repeat (3R) Tau is the most abundant splice, in Pick''s disease. A number of transgenic models expressing wild-type and mutant forms of the 4R Tau have been developed. However, few models of three-repeat Tau are available. A transgenic mouse model expressing three-repeat Tau was developed bearing the mutations associated with familial forms of Pick''s disease (L266V and G272V mutations). Two lines expressing high (Line 13) and low (Line 2) levels of the three-repeat mutant Tau were analyzed. By Western blot, using antibodies specific to three-repeat Tau, Line 13 expressed 5-times more Tau than Line 2. The Tau expressed by these mice was most abundant in the frontal-temporal cortex and limbic system and was phosphorylated at residues detected by the PHF-1, AT8, CP9 and CP13 antibodies. The higher-expressing mice displayed hyperactivity, memory deficits in the water maze and alterations in the round beam. The behavioral deficits started at 6-8 months of age and were associated with a progressive increase in the accumulation of 3R Tau. By immunocytochemistry, mice from Line 13 displayed extensive accumulation of 3R Tau in neuronal cells bodies in the pyramidal neurons of the neocortex, CA1-3 regions, and dentate gyrus of the hippocampus. Aggregates in the granular cells had a globus appearance and mimic Pick’s-like inclusions. There were abundant dystrophic neurites, astrogliosis and synapto-dendritic damage in the neocortex and hippocampus of the higher expresser line. The hippocampal lesions were moderately argyrophilic and Thioflavin-S negative. By electron microscopy, discrete straight filament aggregates were detected in some neurons in the hippocampus. This model holds promise for better understanding the natural history and progression of 3R tauopathies and their relationship with mitochondrial alterations and might be suitable for therapeutical testing.     

Apolipoprotein E4 Causes Age- and Sex-Dependent Impairments of Hilar GABAergic Interneurons and Learning and Memory Deficits in Mice

Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimer''s disease (AD). ApoE4 has sex-dependent effects, whereby the risk of developing AD is higher in apoE4-expressing females than males. However, the mechanism underlying the sex difference, in relation to apoE4, is unknown. Previous findings indicate that apoE4 causes age-dependent impairments of hilar GABAergic interneurons in female mice, leading to learning and memory deficits. Here, we investigate whether the detrimental effects of apoE4 on hilar GABAergic interneurons are sex-dependent using apoE knock-in (KI) mice across different ages. We found that in female apoE-KI mice, there was an age-dependent depletion of hilar GABAergic interneurons, whereby GAD67- or somatostatin-positive–but not NPY- or parvalbumin-positive–interneuron loss was exacerbated by apoE4. Loss of these neuronal populations was correlated with the severity of spatial learning deficits at 16 months of age in female apoE4-KI mice; however, this effect was not observed in female apoE3-KI mice. In contrast, we found an increase in the numbers of hilar GABAergic interneurons with advancing age in male apoE-KI mice, regardless of apoE genotype. Moreover, male apoE-KI mice showed a consistent ratio of hilar inhibitory GABAergic interneurons to excitatory mossy cells approximating 1.5 that is independent of apoE genotype and age, whereas female apoE-KI mice exhibited an age-dependent decrease in this ratio, which was exacerbated by apoE4. Interestingly, there are no apoE genotype effects on GABAergic interneurons in the CA1 and CA3 subregions of the hippocampus as well as the entorhinal and auditory cortexes. These findings suggest that the sex-dependent effects of apoE4 on developing AD is in part attributable to inherent sex-based differences in the numbers of hilar GABAergic interneurons, which is further modulated by apoE genotype.     

Mass Spectrometry Quantification Revealed Accumulation of C-Terminal Fragment of Apolipoprotein E in the Alzheimer's Frontal Cortex

Polymorphic variation in the apolipoprotein E (apoE) gene is the major genetic susceptibility factor for late-onset Alzheimer''s disease (AD) and likely contributes to neuropathology through various pathways. It is also recognized that apoE undergoes proteolytic cleavage in the brain and the resultant apoE fragments likely have a variety of bioactive properties that regulate neuronal signaling and may promote neurodegeneration. ApoE fragmentation in the human brain has been intensively studied using different immunochemical methods, but has never been analyzed in a quantitative manner to establish preferably accumulated fragments. Here we report quantification using multiple reaction monitoring mass spectrometry (MRM MS) with ¹⁵N-labeled full-length apoE4 as an internal standard. Measurements were performed on frontal cortex from control and severe AD donors. Our data point to a preferable accumulation of C-terminal apoE fragment in the insoluble fraction of tissue homogenate in the severe AD group versus the control group. Further insight into the biological consequences of this accumulation may lead to a better understanding of the basic mechanism of AD pathology.     

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.     

微管相关蛋白tau在不同年龄大鼠海马内的定位分布特点

目的 检测微管相关蛋白tau和Ser396/404位点磷酸化tau在胚胎期大鼠和出生后直至成熟期大鼠海马内的表达及变化规律,浅析与神经细胞分裂和分化的关系.方法 用免疫组织化学SABC法显示孕18d、出生后1d、1w、2w、2m的大鼠脑冠状切面总tau（R134d）、Ser396/404位点磷酸化tau（PHF-1）的表达.结果 ①胚胎期大鼠海马CA区的锥体细胞数量明显多于出生后,随着脑的发育,锥体细胞层的神经细胞数量逐渐下降;②孕18d大鼠海马内有丰富的总tau和PHF-1 tau的表达,并且海马内各区的阳性物质表达均匀,生后1d和1w两种物质表达仍然很强,阳性产物主要分布在海马CA1和CA2区以锥体细胞轴突为主要成分的始层,在锥体细胞树突集中的分子层和胞核内也有少量分布,而在生后2w和2m大鼠海马内各区均未见密集的阳性产物表达.结论 不同年龄大鼠海马内总tau和PHF-1 tau的表达和分布变化可能与神经系统发育过程中细胞的分裂和分化有关.     

Apolipoprotein E localization in rat hepatocytes by immunogold labeling of cryothin sections

The distribution of apolipoprotein (apo) E in rat hepatocytes was investigated with an affinity-purified polyclonal antibody raised against apoE isolated from hepatogeneous very low density lipoproteins (VLDL). The distribution of this antibody was visualized with colloidal gold complexed to anti-rabbit IgG. By epipolarization microscopy, apoE was found uniformly along the basolateral surfaces of all hepatic parenchymal cells, showing a striking intensity along the sinusoidal front. Punctate deposits of colloidal gold appeared to be randomly distributed within all hepatocytes. Widely scattered Kupffer cells also stained for apoE. Electron microscopic examination of immunogold-labeled cryothin sections showed that hepatocytic microvilli projecting into the space of Disse consistently contained clusters of immunogold. The gold particles were variably associated with evident lipoprotein particles, raising the possibility that apoE alone may bind to receptors or other macromolecules at the surface of hepatocytes. Endosomes near the sinusoidal front and multivesicular bodies in the Golgi/biliary area labeled intensely for apoE, consistent with a high content of apoE associated with triglyceride-rich lipoprotein remnants contained within these organelles. Some but not all nascent VLDL particles within putative forming Golgi secretory vesicles were labeled, but many other Golgi vesicles and cisternae that lacked evident VLDL particles were also labeled. These results suggest that at least some apoE associates with nascent VLDL in forming Golgi secretory vesicles. Unexpectedly, the matrix of all hepatocytic peroxisomes was heavily labeled. Immunoblots with the affinity-purified anti-rat apoE IgG against proteins from highly purified peroxisomes isolated from rat hepatocytes revealed a protein with an apparent molecular mass of 34.5 kDa, similar to that of rat apoE in rat blood plasma. In addition, gold was sometimes found in the area either adjacent to peroxisomes or between multivesicular bodies and the bile canaliculus not evidently associated with a membranous compartment. These observations suggest that apoE may participate in interorganellar cholesterol transport within hepatocytes.     

Increased Levels of Apolipoprotein D in Cerebrospinal Fluid and Hippocampus of Alzheimer's Patients

Abstract: Apolipoprotein D (apoD) is a member of the lipocalin family of proteins. Most members of this family are transporters of small hydrophobic ligands, although in the case of apoD, neither its physiological function(s) nor its putative ligand(s) have been unequivocally identified. In humans, apoD is expressed in several tissues, including the CNS, and its synthesis is greatly increased during regeneration of rat peripheral nerves. As apoD may have an important function in the nervous system and, particularly, in nerve regeneration, we measured immunoreactive apoD levels in the hippocampus and in CSF of patients with either Alzheimer's disease (AD) or other neuropathologies. In parallel, we determined the concentrations of apolipoprotein E (apoE), another apolipoprotein also implicated in nerve regeneration and in the etiology of AD. Levels of apoD but not apoE were increased in the hippocampus of AD patients compared with controls. ApoD concentrations, as determined by radioimmunoassay, were significantly increased in the CSF of AD patients (4.23 ± 1.58 µg/ml) and patients with other pathologies (3.29 ± 1.35 µg/ml) compared with those in the CSF of normal subjects (1.15 ± 0.71 µg/ml). Although the differences were smaller than for apoD, the mean apoE concentrations in the CSF of both groups of patients were also significantly higher than those of controls. In AD patients, apoD, but not apoE, levels in CSF and hippocampus increased as a function of inheritance of the ε4 apoE allele. This study therefore demonstrates that increased apoD levels in the hippocampus and in CSF are a marker of neuropathology, including that associated with AD, and are independent of apoE concentrations.     

褪黑素对异丙肾上腺素诱导大鼠tau蛋白过度磷酸化的预防作用

异常过度磷酸化的微管相关蛋白tau是阿尔茨海默病(Alzheimer's disease,AD)患者大脑中神经原纤维缠结的主要组成部分.迄今为止,尚无有效的措施阻止tau蛋白的过度磷酸化.为探讨褪黑素(melatonin,Mel)对AD样tau蛋白过度磷酸化的预防作用,我们以β受体激动剂异丙肾上腺素(isoproterenol,IP)来复制AD样tau蛋白过度磷酸化的动物模型,在大鼠双侧海马注射IP前,以褪黑素作为保护组药物,于腹腔连续注射5 d.应用磷酸化位点特异性抗体(PHF-1和Tau-1)作免疫印迹和免疫组织化学检测tau蛋白的磷酸化水平,并用非磷酸化依赖的总tau蛋白抗体(111e)进行标准化.免疫印迹结果显示在注射IP 48 h后,tau蛋白在PHF-1表位的免疫反应显著增强,在Tau-1表位显著减弱,表明tau蛋白在Ser396/Ser404(PHF-1)和Ser199/Ser202(Tau-1)位点有过度磷酸化.免疫组织化学染色结果与免疫印迹结果相似,主要检测到在大鼠海马CA3区的神经纤维有tau蛋白过度磷酸化.褪黑素预处理大鼠可有效地阻止IP诱导tau蛋白在Tau-1和PHF-1位点的过度磷酸化.上述结果提示褪黑素可预防大鼠脑组织中由异丙肾上腺素引起的AD样tau蛋白的过度磷酸化.     

Structural mapping techniques distinguish the surfaces of fibrillar 1N3R and 1N4R human tau

The rigid core of intracellular tau filaments from Alzheimer''s disease (AD), Pick''s disease (PiD), and Corticobasal disease (CBD) brains has been shown to differ in their cryo-EM atomic structure. Despite providing critical information on the intimate arrangement of a fraction of htau molecule within the fibrillar scaffold, the cryo-EM studies neither yield a complete picture of tau fibrillar assemblies structure nor contribute insights into the surfaces that define their interactions with numerous cellular components. Here, using proteomic approaches such as proteolysis and molecular covalent painting, we mapped the exposed amino acid stretches at the surface and those constituting the fibrillar core of in vitro-assembled fibrils of human htau containing one N-terminal domain and three (1N3R) or four (1N4R) C-terminal microtubule-binding repeat domains as a result of alternative splicing. Using limited proteolysis, we identified the proteolytic fragments composing the molecular “bar-code” for each type of fibril. Our results are in agreement with structural data reported for filamentous tau from AD, PiD, and CBD cases predigested with the protease pronase. Finally, we report two amino acid stretches, exposed to the solvent in 1N4R not in 1N3R htau, which distinguish the surfaces of these two kinds of fibrils. Our findings open new perspectives for the design of highly specific ligands with diagnostic and therapeutic potential.     

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.     

APPSWE转基因小鼠发育过程中海马CA1区神经细胞凋亡研究

目的探讨APPSWE转基因小鼠发育过程中海马CA1区神经细胞凋亡规律。方法取不同发育时间（P0、P7、P14、P30、P00、P180）APPSWE转基因模型鼠与同时问点对照鼠,Nissl染色观察海马结构和锥体细胞形态,免疫组织化学方法观察海马细胞内Caspase-3表达变化,RT—PCR检测Caspase-3 mRNA表达变化。结果随着小鼠的生长发育,P14时间点以后,模型组CA1区神经元Caspase-3阳性细胞密度比对照组高,RT—PCR检测结果与Caspase-3免疫组织化学结果基本一致。结论APPSWE转基因小鼠发育中的海马神经细胞过度凋亡可能与阿茨海默病的发生、发展具有联系。     

Comparative analysis of an improved thioflavin-s stain, Gallyas silver stain, and immunohistochemistry for neurofibrillary tangle demonstration on the same sections.

An improved thioflavin-S stain, Gallyas silver stain, and two immunostainings were quantitatively compared for demonstration of neurofibrillary tangles (NFTs) on the same sections. Sections of hippocampal formation from seven cases of Alzheimer's disease (AD) were immunofluorescently stained with a commercially available polyclonal NFT antibody or a PHF-1 monoclonal antibody, followed by an improved thioflavin-S stain, and finally by Gallyas silver staining. The thioflavin-S method was improved by using a combination quenching method that removes background autofluorescence without remarkable tissue damage and by post-treatment with concentrated phosphate buffer, which minimizes photobleaching. PHF-1 or NFT immunostaining is much less sensitive than the improved thioflavin-S staining and Gallyas silver staining, particularly in the transentorhinal region. Moreover PHF-1 immunoreactivity varied greatly among AD individuals. Thioflavin-S staining and Gallyas silver staining show almost the same sensitivity in NFT demonstration, but only the former depends on the secondary protein structure of NFTs. This study suggests that the improved thioflavin-S staining is a simple, sensitive, and consistent method for demonstration of neurofibrillary pathology.     

Mechanisms of neuronal death in brain aging and alzheimer's disease: Role of endocrine-mediated calcium dyshomeostasis

This paper reviews evidence that brain aging and Alzheimer's disease (AD) are somehow closely related and that the hippocampus (CA1) is highly vulnerable to cell loss under both conditions. In addition, two current lines of evidence on the mechanisms of hippocampal cell loss with aging are considered, including studies of neuronal calcium dysregulation and studies of cumulative glucocorticoid (GC) neurotoxicity. Moreover, recent electro-physiological studies have shown that excess glucocorticoid activation of hippocampal neurons increases the influx of calcium through voltage-activated calcium channels. Second messenger systems may mediate the steroid modulation of calcium channels. Therefore, it is hypothesized that excess glucocorticoid activation and neuronal calcium dysregulation may be two phases of a single process that increases the susceptibility of neurons to neurodegeneration during aging and Alzheimer's disease. © 1992 John Wiley & Sons, Inc.     

Specific Disruption of Hippocampal Mossy Fiber Synapses in a Mouse Model of Familial Alzheimer's Disease

The earliest stages of Alzheimer''s disease (AD) are characterized by deficits in memory and cognition indicating hippocampal pathology. While it is now recognized that synapse dysfunction precedes the hallmark pathological findings of AD, it is unclear if specific hippocampal synapses are particularly vulnerable. Since the mossy fiber (MF) synapse between dentate gyrus (DG) and CA3 regions underlies critical functions disrupted in AD, we utilized serial block-face electron microscopy (SBEM) to analyze MF microcircuitry in a mouse model of familial Alzheimer''s disease (FAD). FAD mutant MF terminal complexes were severely disrupted compared to control – they were smaller, contacted fewer postsynaptic spines and had greater numbers of presynaptic filopodial processes. Multi-headed CA3 dendritic spines in the FAD mutant condition were reduced in complexity and had significantly smaller sites of synaptic contact. Significantly, there was no change in the volume of classical dendritic spines at neighboring inputs to CA3 neurons suggesting input-specific defects in the early course of AD related pathology. These data indicate a specific vulnerability of the DG-CA3 network in AD pathogenesis and demonstrate the utility of SBEM to assess circuit specific alterations in mouse models of human disease.     

Comparison of Medial Temporal Measures between Binswanger's Disease and Alzheimer's Disease

Binswanger''s disease (BD) is a common cause of vascular dementia in elderly patients; however, few studies have investigated the medial temporal lobe (MTL) atrophy in BD, and the differences in the atrophic patterns between BD and Alzheimer''s disease (AD) remain largely unknown. Such knowledge is essential for understanding the pathologic basis of dementia. In this study, we collected structural magnetic resonance imaging (MRI) data from 16 normal controls, 14 patients with AD and 14 patients with BD. The volumes of the hippocampus and amygdala, and morphologic parameters (volume, surface area, cortical thickness and mean curvature) of the entorhinal cortex (ERC) and perirhinal cortex (PRC) were calculated using an automated approach. Volume reduction of the hippocampus, amygdala and ERC, and disturbance of the PRC curvature was found in both AD and BD patients compared with the controls (p<0.05, uncorrected). There were no significant differences among all the structural measures between the AD and BD patients. Finally, partial correlation analyses revealed that cognitive decline could be attributed to ERC thinning in AD and volume reduction of PRC in BD. We conclude that AD and BD exhibit similar atrophy patterns in the medial temporal cortices and deep gray matter but have distinct pathologic bases for cognitive impairments. Although atrophy of the MTL structures is a sensitive biomarker for AD, it is not superior for discrimination between AD and BD.     

Ceruloplasmin immunoreactivity in neurodegenerative disorders

Ceruloplasmin (CP) is a 132kd cuproprotein which, together with transferrin, provides the majority of anti-oxidant capacity in serum. Increased iron deposition and lipid peroxidation in the basal ganglia of subjects with hereditary CP deficiency suggest that CP may serve as an anti-oxidant in the brain as well. The present study compared CP immunoreactivity in brain specimens from normal controls and subjects with neurodegenerative disorders (Alzheimer's disease [AD], Parkinson's disease [PD], progressive supranuclear palsy [PSP], and Huntington's disease [HD]) (n = 5 per group). The relative intensity of neuronal CP staining and the numbers of CP-stained neurons per 25x microscope field were determined in hippocampus (CA1, subiculum, and parahippocampal gyrus), parietal cortex, frontal cortex, substantia nigra, and caudate. CP was detected in both neurons and astrocytes in all specimens, and in senile plaques and occasional neurofibrillary tangles in AD brain. Neuronal CP staining intensity tended to increase in most AD brain regions, but was statistically significant vs controls only in the CA1 region of hippocampus (p = .016). Neuronal CP staining in brain specimens from other neurodegenerative disorders showed a slight but nonsignificant increase vs controls. The numbers of CP-stained neurons per field did not differ between the various neurodegenerative disorders and controls. These results suggest that a modest increase in neuronal CP content is present in the AD brain, and lesser elevations in neuronal CP occur in the other neurodegenerative disorders in this study. Though CP functions as both an acute phase protein and an anti-oxidant in peripheral tissues, whether it does so in the brain remains to be determined.     

Autoradiographic Imaging of [3H]Phorbol 12,13-Dibutyrate Binding to Protein Kinase C in Alzheimer''s Disease

Quantitative autoradiography was used to examine the distribution of [3H]phorbol 12,13-dibutyrate ([3H]PDBu) binding to protein kinase C in the middle frontal and temporal cortices and the hippocampal region of nine control and nine elderly subjects with Alzheimer's disease (AD). AD patients had a clinical diagnosis of the disease that was confirmed neuropathologically by the presence of numerous plaques in the hippocampus and cerebral cortex. Choline acetyltransferase (ChAT) activity was significantly reduced in the middle frontal and temporal cortex and in the hippocampus of AD subjects, with the deficit being greater than 60% of control values. Quantitative autoradiographic analysis of [3H]PDBu binding to protein kinase C revealed a heterogeneous pattern in control brain, being particularly high in superficial layers of the cortex and CA1 of the hippocampus. There were no significant differences between control and AD sections in all areas examined within the middle frontal cortex; e.g., layers I-II control, 491 +/- 46 versus AD, 537 +/- 39 pmol/g of tissue; middle temporal cortex, e.g., layers I-II control, 565 +/- 68 versus AD, 465 +/- 72 pmol/g of tissue; and hippocampal formation, e.g., CA1 control, 511 +/- 28 versus AD, 498 +/- 25 pmol/g of tissue. In a parallel study, [3H]PDBu binding to homogenate preparations of control and AD brain confirmed that there was no significant difference in [3H]PDBu binding in either the particulate or the cytosolic fraction. We have demonstrated in a well-defined population of AD patients that [3H]PDBu binding to protein kinase C remains preserved in brain regions that are severely affected by the neuropathological and neurochemical correlates of AD.     

Intra- and extracellular Abeta and PHF in clinically evaluated cases of Alzheimer's disease

Temporal cortical sections from postmortem brains of individuals without any dementing condition and with different degrees of severity of Alzheimer's disease (AD) evaluated by the Clinical Dementia Rating scale (CDR 0-CDR 3) were analyzed using immunohistochemical procedures. To demonstrate the amyloid-beta-peptide (Abeta) deposition and the neurofibrillary pathology, two monoclonal antibodies were used, a human CERAD Abeta (10D5) antibody raised against the N-terminal region of the Abeta-peptide, and an antibody raised against paired helical filaments (PHF-1). The neuron cell bodies and the glial cells were also recognized by two polyclonal antibodies raised, respectively, against the protein gene peptide (PGP 9.5) and glial fibrillary acidic protein (GFAP). Directly related to severity of AD, progressive deposits of Abeta-peptide were found within cortical pyramidal-like neurons and forming senile plaques. Ultrastructurally, Abeta-peptide deposits were related to neuronal intracytoplasmic organelles, such as the ER, the mitochondria, the Nissl bodies and lipofuscin. We have also found that the intracellular deposition of the Abeta peptide is a neuropathological finding prior to the appearance of PHF-immunoreactive structures. We suggest that the intracellular Abeta deposition in cortical pyramidal neurons is a first neurodegenerative event in AD development and that it is involved in cell dysfunction, neuronal death, and plaque formation.     

Apolipoprotein E levels in cerebrospinal fluid and the effects of ABCA1 polymorphisms

Background

Animal studies suggest that brain apolipoprotein E (apoE) levels influence amyloid-β (Aβ) deposition and thus risk for Alzheimer's disease (AD). We have previously demonstrated that deletion of the ATP-binding cassette A1 transporter (ABCA1) in mice causes dramatic reductions in brain and cerebrospinal fluid (CSF) apoE levels and lipidation. To examine whether polymorphisms in ABCA1 affect CSF apoE levels in humans, we measured apoE in CSF taken from 168 subjects who were 43 to 91 years old and were either cognitively normal or who had mild AD. We then genotyped the subjects for ten previously identified ABCA1 single nucleotide polymorphisms (SNPs).

Results

In all subjects, the mean CSF apoE level was 9.09 μg/ml with a standard deviation of 2.70 μg/ml. Levels of apoE in CSF samples taken from the same individual two weeks apart were strongly correlated (r² = 0.93, p < 0.01). In contrast, CSF apoE levels in different individuals varied widely (coefficient of variation = 46%). CSF apoE levels did not vary according to AD status, APOE genotype, gender or race. Average apoE levels increased with age by ~0.5 μg/ml per 10 years (r² = 0.05, p = 0.003). We found no significant associations between CSF apoE levels and the ten ABCA1 SNPs we genotyped. Moreover, in a separate sample of 1225 AD cases and 1431 controls, we found no association between the ABCA1 SNP rs2230806 and AD as has been previously reported.

Conclusion

We found that CSF apoE levels vary widely between individuals, but are stable within individuals over a two-week interval. AD status, APOE genotype, gender and race do not affect CSF apoE levels, but average CSF apoE levels increase with age. Given the lack of association between CSF apoE levels and genotypes for the ABCA1 SNPs we examined, either these SNPs do not affect ABCA1 function or if they do, they do not have strong effects in the CNS. Finally, we find no evidence for an association between the ABCA1 SNP rs2230806 and AD in a large sample set.     

ApoE4 delays dendritic spine formation during neuron development and accelerates loss of mature spines in?vitro

The ε4 allele of the gene that encodes apolipoprotein E (APOE4) is the greatest genetic risk factor for Alzheimer''s disease (AD), while APOE2 reduces AD risk, compared to APOE3. The mechanism(s) underlying the effects of APOE on AD pathology remains unclear. In vivo, dendritic spine density is lower in APOE4-targeted replacement (APOE-TR) mice compared with APOE2- and APOE3-TR mice. To investigate whether this apoE4-induced decrease in spine density results from alterations in the formation or the loss of dendritic spines, the effects of neuron age and apoE isoform on the total number and subclasses of spines were examined in long-term wild-type neurons co-cultured with glia from APOE2-, APOE3- and APOE4-TR mice. Dendritic spine density and maturation were evaluated by immunocytochemistry via the presence of drebrin (an actin-binding protein) with GluN1 (NMDA receptor subunit) and GluA2 (AMPA receptor subunit) clusters. ApoE isoform effects were analyzed via a method previously established that identifies phases of spine formation (day-in-vitro, DIV10–18), maintenance (DIV18–21) and loss (DIV21–26). In the formation phase, apoE4 delayed total spine formation. During the maintenance phase, the density of GluN1+GluA2 spines did not change with apoE2, while the density of these spines decreased with apoE4 compared to apoE3, primarily due to the loss of GluA2 in spines. During the loss phase, total spine density was lower in neurons with apoE4 compared to apoE3. Thus, apoE4 delays total spine formation and may induce early synaptic dysfunction via impaired regulation of GluA2 in spines.