Alzheimer's Aß42 and Aß40 form mixed oligomers with direct molecular interactions.

Formation of Aß oligomers and fibrils plays a central role in the pathogenesis of Alzheimer's disease. There are two major forms of Aß in the brain: Aß42 and Aß40. Aß42 is the major component of the amyloid plaques, but the overall abundance of Aß40 is several times that of Aß42. In vitro experiments show that Aß42 and Aß40 affect each other's aggregation. In mouse models of Alzheimer's disease, overexpression of Aß40 has been shown to reduce the plaque pathology, suggesting that Aß42 and Aß40 also interact in vivo. Here we address the question of whether Aß42 and Aß40 interact with each other in the formation of oligomers using electron paramagnetic resonance (EPR) spectroscopy. When the Aß42 oligomers were formed using only spin-labeled Aß42, the dipolar interaction between spin labels that are within 20 Å range broadened the EPR spectrum and reduced its amplitude. Oligomers formed with a mixture of spin-labeled Aß42 and wild-type Aß42 gave an EPR spectrum with higher amplitude due to weakened spin-spin interactions, suggesting molecular mixing of labeled and wild-type Aß42. When spin-labeled Aß42 and wild-type Aß40 were mixed to form oligomers, the resulting EPR spectrum also showed reduced amplitude, suggesting that wild-type Aß40 can also form oligomers with spin-labeled Aß42. Therefore, our results suggest that Aß42 and Aß40 form mixed oligomers with direct molecular interactions. Our results point to the importance of investigating Aß42-Aß40 interactions in the brain for a complete understanding of Alzheimer's pathogenesis and therapeutic interventions.

Small-angle X-ray scattering characterization of a [Formula: see text]-amyloid model in phantoms.

OBJECTIVE: We present a method to prepare an amyloid model at scalable quantities for phantom studies to evaluate small-angle x-ray scattering systems for amyloid detection. Two amyloid models were made from a plasma protein with and without heating. Both models mimic the [Formula: see text]-sheet structure of the [Formula: see text]-amyloid ([Formula: see text]) plaques in Alzheimer's disease. Amyloid detection is based on the distinct peaks in the scattering signature of the [Formula: see text]-sheet structure. We characterized the amyloid models using a spectral small-angle x-ray scattering (sSAXS) prototype with samples in a plastic syringe and within a cylindrical polymethyl methacrylate (PMMA) phantom. RESULTS: sSAXS data show that we can detect the scattering peaks characteristic of amyloid [Formula: see text]-sheet structure in both models around 6 and 13 [Formula: see text]. The [Formula: see text] model prepared without heating provides a stronger signal in the PMMA phantom. The methods described can be used to prepare models in sufficiently large quantities and used in samples with different packing density to assess the performance of [Formula: see text] quantification systems.

CRANAD-28: A Robust Fluorescent Compound for Visualization of Amyloid Beta Plaques.

CRANAD-28, a difluoroboron curcumin analogue, has been demonstrated in earlier reports to successfully label amyloid beta (Aß) plaques for imaging both ex vivo and in vivo. CRANAD-28's imaging brightness, ability to penetrate the blood brain barrier, and low toxicity make the compound a potentially potent imaging tool in Alzheimer's research. In this study, the Aß-labeling ability of CRANAD-28 was investigated in further detail using histological staining to assess different criteria, including stained Aß plaque brightness, Aß plaque size, and Aß plaque number count. The results of this study demonstrated CRANAD-28 to be superior across all criteria assessed. Furthermore, CRANAD-28 and IBA-1 antibody were used to label Aß-plaques and microglia respectively. Statistical analysis with Spearman regression revealed a statistically significant negative correlation between the size of labeled Aß plaques and surrounding microglia density. This finding provides interesting insight into Aß plaque and microglia dynamism in AD pathology and corroborates the findings of previous studies. In addition, we found that CRANAD-28 provided distinct spectral signatures for Aßs in the core and periphery of the plaques. Based on the study's results, CRANAD-28 could be considered as an alternative standard for imaging Aß-plaques in future research studies.

Bis(ethylmaltolato)oxidovanadium(iv) inhibited the pathogenesis of Alzheimer's disease in triple transgenic model mice.

Vanadium compounds have been reported to mimic the anti-diabetes effects of insulin on rodent models, but their effects on Alzheimer's disease (AD) have rarely been explored. In this paper, 9-month-old triple transgenic AD model mice (3×Tg-AD) received bis(ethylmaltolato)oxidovanadium(iv) (BEOV) at doses of 0.2 mmol L-1 (68.4 µg mL-1) and 1.0 mmol L-1 (342 µg mL-1) for 3 months. BEOV at both doses was found to improve contextual memory and spatial learning in AD mice. It also improved glucose metabolism and protected neuronal synapses in the AD brain, as evidenced respectively by 18F-labeled fluoro-deoxyglucose positron emission tomography (18F-FDG-PET) scanning and by transmission electron microscopy. Inhibitory effects of BEOV on ß-amyloid (Aß) plaques and neuronal impairment in the cortex and hippocampus of fluorescent AD mice were visualized three-dimensionally by applying optical clearing technology to brain slices before confocal laser scanning microscopy. Western blot analysis semi-quantitatively revealed the altered levels of Aß42 in the brains of wildtype, AD, and AD treated with 0.2 and 1.0 mmol L-1 BEOV mice (70.3%, 100%, 83.2% and 56.8% in the hippocampus; 82.4%, 100%, 66.9% and 42% in the cortex, respectively). The mechanism study showed that BEOV increased the expression of peroxisome proliferator-activated receptor γ (PPARγ) (140%, 100%, 142% and 160% in the hippocampus; 167%, 100%, 124% and 133% in the cortex) to inactivate the JAK2/STAT3/SOCS-1 pathway and to block the amyloidogenesis cascade, thus attenuating Aß-induced insulin resistance in AD models. BEOV also reduced protein tyrosine phosphatase 1B (PTP1B) expression (74.8%, 100%, 76.5% and 53.8% in the hippocampus; 71.8%, 100%, 94.2% and 81.8% in cortex) to promote insulin sensitivity and to stimulate the PI3K/Akt/GSK3ß pathway, subsequently reducing tau hyperphosphorylation (phosphorylated tau396 levels were 51.1%, 100%, 56.1% and 50.2% in the hippocampus; 22.2%, 100%, 36.1%, and 24% in the cortex). Our results suggested that BEOV reduced the pathological hallmarks of AD by targeting the pathways of PPARγ and PTP1B in 3×Tg AD mice.

Unobstructed Multiscale Imaging of Tissue Sections for Ultrastructural Pathology Analysis by Backscattered Electron Scanning Microscopy.

Ultrastructural analysis of healthy, diseased, or experimental tissues is essential in diagnostic and investigative pathology. Evaluation of large tissue areas with suborganelle resolution is challenging because biological structures ranging from several millimeters to nanometers in size need to be identified and imaged while maintaining context over multiple scales. Imaging with field emission scanning electron microscopes (FE-SEMs) is uniquely suited for this task. We describe an efficient workflow for the preparation and unobstructed multiscale imaging of tissue sections with backscattered electron scanning electron microscopy (BSE-SEM) for applications in ultrastructural pathology. We demonstrate that a diverse range of tissues, processed by conventional electron microscopy protocols and avoiding the use of mordanting agents, can be imaged on standard glass slides over multiple scales, from the histological to the ultrastructural level, without any visual obstructions. Our workflow takes advantage of the very large scan fields possible with modern FE-SEMs that allow for the acquisition of wide-field overview images which can be explored at the ultrastructural level by digitally zooming into the images. Examples from applications in pulmonary research and neuropathology demonstrate the versatility and efficiency of this method. This BSE-SEM-based multiscale imaging procedure promises to substantially simplify and accelerate ultrastructural tissue analysis in pathology.

Immunoelectron microscopy and mass spectrometry for classification of amyloid deposits.

Amyloidosis is a shared name for several rare, complex and serious diseases caused by extra-cellular deposits of different misfolded proteins. Accurate characterization of the amyloid protein is essential for patient care. Immunoelectron microscopy (IEM) and laser microdissection followed by tandem mass spectrometry (LMD-MS) are new gold standards for molecular subtyping. Both methods perform superiorly to immunohistochemistry, but their complementarities, strengths and weaknesses across amyloid subtypes and organ biopsy origin remain undefined. Therefore, we performed a retrospective study of 106 Congo Red positive biopsies from different involved organs; heart, kidney, lung, gut mucosa, skin and bone marrow. IEM, performed with gold-labelled antibodies against kappa light chains, lambda light chains, transthyretin and amyloid A, identified specific staining of amyloid fibrils in 91.6%; in six biopsies amyloid fibrils were not identified, and in two, the fibril subtype could not be established. LMD-MS identified amyloid protein signature in 98.1%, but in nine the amyloid protein could not be clearly identified. MS identified protein subtype in 89.6%. Corresponding specificities ranged at organ level from 94-100%. Concordance was 89.6-100% for different amyloid subtypes. Importantly, combined use of both methods increased the diagnostic classification to 100%. Some variety in performances at organ level was observed.

Dengzhan Shengmai capsules and their active component scutellarin prevent cognitive decline in APP/PS1 mice by accelerating Aß aggregation and reducing oligomers formation.

There is currently no effective treatment to prevent the progress of Alzheimer's disease (AD). The traditional Chinese herbs Dengzhan Shengmai (DZSM) capsules and their active component scutellarin possess multiple effects and are clinically used for the treatment of cerebrovascular diseases. Scutellarin has been reported to affect Aß aggregation. However, the effects of DZSM capsules on AD remain unknown. Through in vivo experiments, our study proved that the alleviating effects of DZSM capsules on cognitive deficits of AD mice were due to the role of scutellarin, which up-regulated low toxic amyloid plaques and down-regulated highly toxic soluble Aß42 and Aß40 levels in cortex. In vitro, we confirmed scutellarin's role in accelerating transforming Aß42 monomers into high-molecular-mass aggregates by biochemical assays, which supported the results observed in drug-treated APP/PS1 mice. In detail, the 1:10 ratio of scutellarin/Aß42 mixtures promoted production of large ß-sheet-rich fibrils whereas the 1:1 ratio promoted production of protofibrils. In addition, the binding between scutellarin and Aß monomers was quantified by microscale thermophoresis test and the apparent dissociation constant (Kd) was 1284.4 ±â€¯238.8 µM. What's more, binding regions between scutellarin and Aß fibrils were predicted by computational docking models and scutellarin might bind parallel to the long axis of Aß42 fibrils targeting hydrophobic grooves at residues 35-36 or 39. In conclusion, DZSM capsules protected against cognitive defects of AD through scutellarin-mediated acceleration of Aß aggregation into fibrils or protofibrils and reduction of soluble Aß oligomers, thus suggesting potential clinical applications of DZSM capsules and scutellarin in the treatment of AD.

Hidden Aggregation Hot-Spots on Human Apolipoprotein E: A Structural Study.

Human apolipoprotein E (apoE) is a major component of lipoprotein particles, and under physiological conditions, is involved in plasma cholesterol transport. Human apolipoprotein E found in three isoforms (E2; E3; E4) is a member of a family of apolipoproteins that under pathological conditions are detected in extracellular amyloid depositions in several amyloidoses. Interestingly, the lipid-free apoE form has been shown to be co-localized with the amyloidogenic Aß peptide in amyloid plaques in Alzheimer's disease, whereas in particular, the apoE4 isoform is a crucial risk factor for late-onset Alzheimer's disease. Evidence at the experimental level proves that apoE self-assembles into amyloid fibrilsin vitro, although the misfolding mechanism has not been clarified yet. Here, we explored the mechanistic insights of apoE misfolding by testing short apoE stretches predicted as amyloidogenic determinants by AMYLPRED, and we computationally investigated the dynamics of apoE and an apoE-Αß complex. Our in vitro biophysical results prove that apoE peptide-analogues may act as the driving force needed to trigger apoE aggregation and are supported by the computational apoE outcome. Additional computational work concerning the apoE-Αß complex also designates apoE amyloidogenic regions as important binding sites for oligomeric Αß; taking an important step forward in the field of Alzheimer's anti-aggregation drug development.

Senolytic therapy alleviates Aß-associated oligodendrocyte progenitor cell senescence and cognitive deficits in an Alzheimer's disease model.

Neuritic plaques, a pathological hallmark in Alzheimer's disease (AD) brains, comprise extracellular aggregates of amyloid-beta (Aß) peptide and degenerating neurites that accumulate autolysosomes. We found that, in the brains of patients with AD and in AD mouse models, Aß plaque-associated Olig2- and NG2-expressing oligodendrocyte progenitor cells (OPCs), but not astrocytes, microglia, or oligodendrocytes, exhibit a senescence-like phenotype characterized by the upregulation of p21/CDKN1A, p16/INK4/CDKN2A proteins, and senescence-associated ß-galactosidase activity. Molecular interrogation of the Aß plaque environment revealed elevated levels of transcripts encoding proteins involved in OPC function, replicative senescence, and inflammation. Direct exposure of cultured OPCs to aggregating Aß triggered cell senescence. Senolytic treatment of AD mice selectively removed senescent cells from the plaque environment, reduced neuroinflammation, lessened Aß load, and ameliorated cognitive deficits. Our findings suggest a role for Aß-induced OPC cell senescence in neuroinflammation and cognitive deficits in AD, and a potential therapeutic benefit of senolytic treatments.

Copper Redox Cycling Inhibits Aß Fibre Formation and Promotes Fibre Fragmentation, while Generating a Dityrosine Aß Dimer.

Oxidative stress and the formation of plaques which contain amyloid-ß (Aß) peptides are two key hallmarks of Alzheimer's disease (AD). Dityrosine is found in the plaques of AD patients and Aß dimers have been linked to neurotoxicity. Here we investigate the formation of Aß dityrosine dimers promoted by Cu2+/+ Fenton reactions. Using fluorescence measurements and UV absorbance, we show that dityrosine can be formed aerobically when Aß is incubated with Cu2+ and hydrogen-peroxide, or in a Cu2+ and ascorbate redox mixture. The dityrosine cross-linking can occur for both monomeric and fibrillar forms of Aß. We show that oxidative modification of Aß impedes the ability for Aß monomer to form fibres, as indicated by the amyloid specific dye Thioflavin T (ThT). Transmission electron microscopy (TEM) indicates the limited amyloid assemblies that form have a marked reduction in fibre length for Aß(1-40). Importantly, the addition of Cu2+ and a reductant to preformed Aß(1-40) fibers causes their widespread fragmentation, reducing median fibre lengths from 800 nm to 150 nm upon oxidation. The processes of covalent cross-linking of Aß fibres, dimer formation, and fibre fragmentation within plaques are likely to have a significant impact on Aß clearance and neurotoxicity.

When can we believe what we see?

Abigail Sawyer and Joseph Martin investigate how microscopy is evolving in order for cells and processes to be better visualized in their native environments.

Diffuse Amyloid-ß Plaques, Neurofibrillary Tangles, and the Impact of APOE in Elderly Persons' Brains Lacking Neuritic Amyloid Plaques.

Data from a large autopsy series were analyzed to address questions pertinent to primary age-related tauopathy (PART) and Alzheimer's disease (AD): what factors are associated with increased severity of neurofibrillary degeneration in brains that lack neuritic amyloid plaques?; is there an association between Apolipoprotein E (APOE) alleles and PART pathologic severity independent of amyloid-ß (Aß) deposits?; and, how do the stains used to detect plaques and tangles impact the experimental results? Neuropathologic data were evaluated from elderly research volunteers whose brain autopsies were performed at University of Kentucky Alzheimer's Disease Center (UK-ADC; N = 145 subjects). All of the included subjects' brains lacked neuritic amyloid plaques according to the CERAD diagnostic criteria and the average final MMSE score before death was 26.8±4.6 stdev. The study incorporated evaluation of tissue with both silver histochemical stains and immunohistochemical stains to compare results; the immunohistochemical stains (Aß and phospho-tau) were scanned and quantified using digital pathologic methods. Immunohistochemical stains provided important advantages over histochemical stains due to sensitivity and detectability via digital methods. When AD-type pathology was in its presumed earliest phases, neocortical parenchymal Aß deposits were associated with increased medial temporal lobe neurofibrillary tangles. The observation supports the NIA-AA consensus recommendation for neuropathologic diagnoses, because even these "diffuse" Aß deposits signal that AD pathobiologic mechanisms are occurring. Further, the data were most compatible with the hypothesis that the APOEɛ4 allele exerts its effect(s) via driving Aß deposition, i.e., an "upstream" influence, rather than being associated directly with Aß- independent PART pathology.

Stereological Quantification of Plaques and Tangles in Neocortex from Alzheimer's Disease Patients.

We estimated by stereological methods the neocortical volume occupied by plaques and tangles in females dying with severe Alzheimer's disease (AD), age-matched female subjects with severe vascular dementia (VaD), and normal control brains. Stereological investigations include a uniform sampling of the tissue in the whole of neocortex and its subdivisions. Resultant volume estimates provide information about the overall burden of these two pathological changes and their volume fractions and allow for correlational studies between the pathological changes and factors such as the total neocortical neuronal cell numbers, dementia test scores, and age. We estimated the volume of plaques and tangles in the entire neocortex and frontal-, temporal-, parietal-, and occipital cortex in nine female AD brains, four female patients dying with VaD, and six neurologically normal female control brains using point-counting in uniform samples of neocortex. The volume occupied by plaques comprised approximately 1% of neocortex, while the neocortical tangles made up approximately 0.1% of neocortex of AD patients but were scarcely present in the other study groups. The individual tangle and plaque volumes did not correlate to the ultimate dementia score of the AD subjects, despite correlating with reduced neocortical volume. In neocortex of AD patients, the burden of plaques and tangles is much higher than that in patients with severe vascular dementia or normal older women but only occupy a small fraction of the neocortical volume.

Doublecortin expression in CD8+ T-cells and microglia at sites of amyloid-ß plaques: A potential role in shaping plaque pathology?

INTRODUCTION: One characteristic of Alzheimer's disease is the formation of amyloid-ß plaques, which are typically linked to neuroinflammation and surrounded by inflammatory cells such as microglia and infiltrating immune cells. METHODS: Here, we describe nonneurogenic doublecortin (DCX) positive cells, DCX being generally used as a marker for young immature neurons, at sites of amyloid-ß plaques in various transgenic amyloid mouse models and in human brains with plaque pathology. RESULTS: The plaque-associated DCX+ cells were not of neurogenic identity, instead most of them showed coexpression with markers for microglia (ionized calcium-binding adapter molecule 1) and for phagocytosis (CD68 and TREM2). Another subpopulation of plaque-associated DCX+ cells was negative for ionized calcium-binding adapter molecule 1 but was highly positive for the pan-leukocyte marker CD45. These hematopoietic cells were identified as CD3-and CD8-positive and CD4-negative T-cells. DISCUSSION: Peculiarly, the DCX+/ionized calcium-binding adapter molecule 1+ microglia and DCX+/CD8+ T-cells were closely attached, suggesting that these two cell types are tightly interacting and that this interaction might shape plaque pathology.

Electron microscopic and confocal laser microscopy analysis of amyloid plaques in chronic wasting disease transmitted to transgenic mice.

We report here on the ultrastructure of amyloid plaques in chronic wasting disease (CWD) transmitted to Tg20 transgenic mice overexpressing prion protein (PrPc). We identified three main types of amyloid deposits in mCWD: large amyloid deposits, unicentric plaques similar to kuru plaques in human prion diseases and multicentric plaques reminiscent of plaques typical of GSS. The most unique type of plaques were large subpial amyloid deposits. They were composed of large areas of amyloid fibrils but did not form "star-like" appearances of unicentric plaques. All types of plaques were totally devoid of dystrophic neuritic elements. However, numerous microglial cells invaded them. The plaques observed by confocal laser microscope were of the same types as those analyzed by electron microscopy. Neuronal processes surrounding the plaques did not show typical features of neuroaxonal dystrophy.

Visualization of neuritic plaques in Alzheimer's disease by polarization-sensitive optical coherence microscopy.

One major hallmark of Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA) is the deposition of extracellular senile plaques and vessel wall deposits composed of amyloid-beta (Aß). In AD, degeneration of neurons is preceded by the formation of Aß plaques, which show different morphological forms. Most of them are birefringent owing to the parallel arrangement of amyloid fibrils. Here, we present polarization sensitive optical coherence microscopy (PS-OCM) for imaging mature neuritic Aß plaques based on their birefringent properties. Formalin-fixed, post-mortem brain samples of advanced stage AD patients were investigated. In several cortical brain regions, neuritic Aß plaques were successfully visualized in tomographic and three-dimensional (3D) images. Cortical grey matter appeared polarization preserving, whereas neuritic plaques caused increased phase retardation. Consistent with the results from PS-OCM imaging, the 3D structure of senile Aß plaques was computationally modelled for different illumination settings and plaque sizes. Furthermore, the birefringent properties of cortical and meningeal vessel walls in CAA were investigated in selected samples. Significantly increased birefringence was found in smaller vessels. Overall, these results provide evidence that PS-OCM is able to assess amyloidosis based on intrinsic birefringent properties.

Amyloid plaque structure and cell surface interactions of ß-amyloid fibrils revealed by electron tomography.

The deposition of amyloid fibrils as plaques is a key feature of several neurodegenerative diseases including in particular Alzheimer's. This disease is characterized, if not provoked, by amyloid aggregates formed from Aß peptide that deposit inside the brain or are toxic to neuronal cells. We here used scanning transmission electron microscopy (STEM) to determine the fibril network structure and interactions of Aß fibrils within a cell culture model of Alzheimer's disease. STEM images taken from the formed Aß amyloid deposits revealed three main types of fibril network structures, termed amorphous meshwork, fibril bundle and amyloid star. All three were infiltrated by different types of lipid inclusions from small-sized exosome-like structures (50-100 nm diameter) to large-sized extracellular vesicles (up to 300 nm). The fibrils also presented strong interactions with the surrounding cells such that fibril bundles extended into tubular invaginations of the plasma membrane. Amyloid formation in the cell model was previously found to have an intracellular origin and we show here that it functionally destroys the integrity of the intracellular membranes as it leads to lysosomal leakage. These data provide a mechanistic link to explain why intracellular fibril formation is toxic to the cell.

Non-Fibrillar Oligomeric Amyloid-ß within Synapses.

Alzheimer's disease (AD) is characterized by memory loss, insidious cognitive decline, profound neurodegeneration, and the extracellular accumulation of amyloid-ß (Aß) peptide in senile plaques and intracellular accumulation of tau in neurofibrillary tangles. Loss and dysfunction of synapses are believed to underlie the devastating cognitive decline in AD. A large amount of evidence suggests that oligomeric forms of Aß associated with senile plaques are toxic to synapses, but the precise sub-synaptic localization of Aß and which forms are synaptotoxic remain unknown. Here, we characterize the sub-synaptic localization of Aß oligomers using three high-resolution imaging techniques, stochastic optical reconstruction microscopy, immunogold electron microscopy, and Förster resonance energy transfer in a plaque-bearing mouse model of AD. With all three techniques, we observe oligomeric Aß inside synaptic terminals. Further, we tested a panel of Aß antibodies using the relatively high-throughput array tomography technique to determine which forms are present in synapses. Our results show that different oligomeric Aß species are present in synapses and highlight the potential of array tomography for rapid testing of aggregation state specific Aß antibodies in brain tissue.

Increase of Positive Net Charge and Conformational Rigidity Enhances the Efficacy of d-Enantiomeric Peptides Designed to Eliminate Cytotoxic Aß Species.

Alzheimer's disease (AD) is a neurodegenerative disorder and the most common type of dementia. Until now, there is no curative therapy available. Previously, we selected the amyloid-beta (Aß) targeting peptide D3 consisting of 12 d-enantiomeric amino acid residues by mirror image phage display as a potential drug candidate for the treatment of AD. In the current approach, we investigated the optimization potential of linear D3 with free C-terminus (D3COOH) by chemical modifications. First, the impact of the net charge was investigated and second, cyclization was introduced which is a well-known tool for the optimization of peptides for enhanced target affinity. Following this strategy, three D3 derivatives in addition to D3COOH were designed: C-terminally amidated linear D3 (D3CONH2), cyclic D3 (cD3), and cyclic D3 with an additional arginine residue (cD3r) to maintain the net charge of linear D3CONH2. These four compounds were compared to each other according to their binding affinities to Aß(1-42), their efficacy to eliminate cytotoxic oligomers, and consequently their potency to neutralize Aß(1-42) oligomer induced neurotoxicity. D3CONH2 and cD3r versions with equally increased net charge showed superior properties over D3COOH and cD3, respectively. The cyclic versions showed superior properties compared to their linear version with equal net charge, suggesting cD3r to be the most efficient compound among these four. Indeed, treatment of the transgenic AD mouse model Tg-SwDI with cD3r significantly enhanced spatial memory and cognition of these animals as revealed by water maze performance. Therefore, charge increase and cyclization imply suitable modification steps for an optimization approach of the Aß targeting compound D3.

Human ApoE ɛ2 Promotes Regulatory Mechanisms of Bioenergetic and Synaptic Function in Female Brain: A Focus on V-type H+-ATPase.

Humans possess three major isoforms of the apolipoprotein E (ApoE) gene encoded by three alleles: ApoE ɛ2 (ApoE2), ApoE ɛ3 (ApoE3), and ApoE ɛ4 (ApoE4). It is established that the three ApoE isoforms confer differential susceptibility to Alzheimer's disease (AD); however, an in-depth molecular understanding of the underlying mechanisms is currently unavailable. In this study, we examined the cortical proteome differences among the three ApoE isoforms using 6-month-old female, human ApoE2, ApoE3, and ApoE4 gene-targeted replacement mice and two-dimensional proteomic analyses. The results reveal that the three ApoE brains differ primarily in two areas: cellular bioenergetics and synaptic transmission. Of particular significance, we show for the first time that the three ApoE brains differentially express a key component of the catalytic domain of the V-type H+-ATPase (Atp6v), a proton pump that mediates the concentration of neurotransmitters into synaptic vesicles and thus is crucial in synaptic transmission. Specifically, our data demonstrate that ApoE2 brain exhibits significantly higher levels of the B subunit of Atp6v (Atp6v1B2) when compared to both ApoE3 and ApoE4 brains, with ApoE4 brain exhibiting the lowest expression. Our additional analyses show that Atp6v1B2 is significantly impacted by aging and AD pathology and the data suggest that Atp6v1B2 deficiency could be involved in the progressive loss of synaptic integrity during early development of AD. Collectively, our findings indicate that human ApoE isoforms differentially modulate regulatory mechanisms of bioenergetic and synaptic function in female brain. A more efficient and robust status in both areas-in which Atp6v may play a role-could serve as a potential mechanism contributing to the neuroprotective and cognition-favoring properties associated with the ApoE2 genotype.