Alzheimer's Disease Brain Tissue Research Articles

Gene Expression Profiling in the APP/PS1KI Mouse Model of Familial Alzheimer's Disease.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder characterized by early intraneuronal amyloid-β (Aβ) accumulation, extracellular deposition of Aβ peptides, and intracellular hyperphosphorylated tau aggregates. These lesions cause dendritic and synaptic alterations and induce an inflammatory response in the diseased brain. Although the neuropathological characteristics of AD have been known for decades, the molecular mechanisms causing the disease are still under investigation. Studying gene expression changes in postmortem AD brain tissue can yield new insights into the molecular disease mechanisms. To that end, one can employ transgenic AD mouse models and the next-generation sequencing technology. In this study, a whole-brain transcriptome analysis was carried out using the well-characterized APP/PS1KI mouse model for AD. These mice display a robust phenotype reflected by working memory deficits at 6 months of age, a significant neuron loss in a variety of brain areas including the CA1 region of the hippocampus and a severe amyloid pathology. Based on deep sequencing, differentially expressed genes (DEGs) between 6-month-old WT or PS1KI and APP/PS1KI were identified and verified by qRT-PCR. Compared to WT mice, 250 DEGs were found in APP/PS1KI mice, while 186 DEGs could be found compared to PS1KI control mice. Most of the DEGs were upregulated in APP/PS1KI mice and belong to either inflammation-associated pathways or lysosomal activation, which is likely due to the robust intraneuronal accumulation of Aβ in this mouse model. Our comprehensive brain transcriptome study further highlights APP/PS1KI mice as a valuable model for AD, covering molecular inflammatory and immune responses.

Identification of neurotoxic cytokines by profiling Alzheimer's disease tissues and neuron culture viability screening.

Alzheimer’s disease (AD) therapeutics based on the amyloid hypothesis have shown minimal efficacy in patients, suggesting that the activity of amyloid beta (Aβ) represents only one aspect of AD pathogenesis. Since neuroinflammation is thought to play an important role in AD, we hypothesized that cytokines may play a direct role in promoting neuronal death. Here, we profiled cytokine expression in a small cohort of human AD and control brain tissues. We identified AD-associated cytokines using partial least squares regression to correlate cytokine expression with quantified pathologic disease state and then used neuron cultures to test whether cytokines up-regulated in AD tissues could affect neuronal viability. This analysis identified cytokines that were associated with the pathological severity. Of the top correlates, only TNF-α reduced viability in neuron culture when applied alone. VEGF also reduced viability when applied together with Aβ, which was surprising because VEGF has been viewed as a neuro-protective protein. We found that this synthetic pro-death effect of VEGF in the context of Aβ was commensurate with VEGFR-dependent changes in multiple signaling pathways that govern cell fate. Our findings suggest that profiling of tissues combined with a culture-based screening approach can successfully identify new mechanisms driving neuronal death.

Synthesis and Preliminary Evaluation of Phenyl 4-123I-Iodophenylcarbamate for Visualization of Cholinesterases Associated with Alzheimer Disease Pathology.

Acetylcholinesterase and butyrylcholinesterase accumulate with brain β-amyloid (Aβ) plaques in Alzheimer disease (AD). The overall activity of acetylcholinesterase is found to decline in AD, whereas butyrylcholinesterase has been found to either increase or remain the same. Although some cognitively normal older adults also have Aβ plaques within the brain, cholinesterase-associated plaques are generally less abundant in such individuals. Thus, brain imaging of cholinesterase activity associated with Aβ plaques has the potential to distinguish AD from cognitively normal older adults, with or without Aβ accumulation, during life. Current Aβ imaging agents are not able to provide this distinction. To address this unmet need, synthesis and evaluation of a cholinesterase-binding ligand, phenyl 4-(123)I-iodophenylcarbamate ((123)I-PIP), is described. Phenyl 4-iodophenylcarbamate was synthesized and evaluated for binding potency toward acetylcholinesterase and butyrylcholinesterase using enzyme kinetic analysis. This compound was subsequently rapidly radiolabeled with (123)I and purified by high-performance liquid chromatography. Autoradiographic analyses were performed with (123)I-PIP using postmortem orbitofrontal cortex from cognitively normal and AD human brains. Comparisons were made with an Aβ imaging agent, 2-(4'-dimethylaminophenyl)-6-(123)I-iodo-imidazo[1,2-a]pyridine ((123)I-IMPY), in adjacent brain sections. Tissues were also stained for Aβ and cholinesterase activity to visualize Aβ plaque load for comparison with radioligand uptake. Synthesized and purified PIP exhibited binding to cholinesterases. (123)I was successfully incorporated into this ligand. (123)I-PIP autoradiography with human tissue revealed accumulation of radioactivity only in AD brain tissues in which Aβ plaques had cholinesterase activity. (123)I-IMPY accumulated in brain tissues with Aβ plaques from both AD and cognitively normal individuals. Radiolabeled ligands specific for cholinesterases have potential for use in neuroimaging AD plaques during life. The compound herein described, (123)I-PIP, can detect cholinesterases associated with Aβ plaques and can distinguish AD brain tissues from those of cognitively normal older adults with Aβ plaques. Imaging cholinesterase activity associated with Aβ plaques in the living brain may contribute to the definitive diagnosis of AD during life.

Preclinical properties and human in vivo assessment of 123I-ABC577 as a novel SPECT agent for imaging amyloid-β.

Non-invasive imaging of amyloid-β in the brain, a hallmark of Alzheimer's disease, may support earlier and more accurate diagnosis of the disease. In this study, we assessed the novel single photon emission computed tomography tracer (123)I-ABC577 as a potential imaging biomarker for amyloid-β in the brain. The radio-iodinated imidazopyridine derivative (123)I-ABC577 was designed as a candidate for a novel amyloid-β imaging agent. The binding affinity of (123)I-ABC577 for amyloid-β was evaluated by saturation binding assay and in vitro autoradiography using post-mortem Alzheimer's disease brain tissue. Biodistribution experiments using normal rats were performed to evaluate the biokinetics of (123)I-ABC577. Furthermore, to validate (123)I-ABC577 as a biomarker for Alzheimer's disease, we performed a clinical study to compare the brain uptake of (123)I-ABC577 in three patients with Alzheimer's disease and three healthy control subjects. (123)I-ABC577 binding was quantified by use of the standardized uptake value ratio, which was calculated for the cortex using the cerebellum as a reference region. Standardized uptake value ratio images were visually scored as positive or negative. As a result, (123)I-ABC577 showed high binding affinity for amyloid-β and desirable pharmacokinetics in the preclinical studies. In the clinical study, (123)I-ABC577 was an effective marker for discriminating patients with Alzheimer's disease from healthy control subjects based on visual images or the ratio of cortical-to-cerebellar binding. In patients with Alzheimer's disease, (123)I-ABC577 demonstrated clear retention in cortical regions known to accumulate amyloid, such as the frontal cortex, temporal cortex, and posterior cingulate. In contrast, less, more diffuse, and non-specific uptake without localization to these key regions was observed in healthy controls. At 150 min after injection, the cortical standardized uptake value ratio increased by ∼ 60% in patients with Alzheimer's disease relative to healthy control subjects. Both healthy control subjects and patients with Alzheimer's disease showed minimal (123)I-ABC577 retention in the white matter. These observations indicate that (123)I-ABC577 may be a useful single photon emission computed tomography imaging maker to identify amyloid-β in the human brain. The availability of an amyloid-β tracer for single photon emission computed tomography might increase the accessibility of diagnostic imaging for Alzheimer's disease.

Oligomeric α-synuclein and β-amyloid variants as potential biomarkers for Parkinson's and Alzheimer's diseases.

Oligomeric forms of α-synuclein and β-amyloid are toxic protein variants that are thought to contribute to the onset and progression of Parkinson's disease (PD) and Alzheimer's disease (AD), respectively. The detection of toxic variants in human cerebrospinal fluid (CSF) and blood has great promise for facilitating early and accurate diagnoses of these devastating diseases. Two hurdles that have impeded the use of these protein variants as biomarkers are the availability of reagents that can bind the different variants and a sensitive assay to detect their very low concentrations. We previously isolated antibody-based reagents that selectively bind two different oligomeric variants of α-synuclein and two of β-amyloid, and developed a phage-based capture enzyme-linked immunosorbent assay (ELISA) with subfemtomolar sensitivity to quantify their presence. Here, we used these reagents to show that these oligomeric α-synuclein variants are preferentially present in PD brain tissue, CSF and serum, and that the oligomeric β-amyloid variants are preferentially present in AD brain tissue, CSF, and serum. Some AD samples also had α-synuclein pathology and some PD samples also had β-amyloid pathology, and, very intriguingly, these PD cases also had a history of dementia. Detection of different oligomeric α-synuclein and β-amyloid species is an effective method for identifying tissue, CSF and sera from PD and AD samples, respectively, and samples that also contained early stages of other protein pathologies, indicating their potential value as blood-based biomarkers for neurodegenerative diseases.

α1B-Adrenoceptor signaling molecule expression in Alzheimer’s disease brain tissue / Alzeimer hastalığı olan beyin dokusunda α1B-Adrenoseptör Sinyal Molekülü Ekspresyonu

AbstractObjective: Gαh (tissue transglutaminase; TGII), known as the α1B-adrenoceptor signal mediator, is a bifunctional enzyme possessing transglutaminase and guanosine triphosphatase activities. The microtubule-associated protein tau plays a role in formation of neurofibrillary tangles in Alzheimer’s disease (AD) brain. Tau is also known as a substrate of transglutaminase. We aimed to better understand the correlation between α1B-adrenoceptor signaling and AD.Methods: We examined not only the cross-linking ability of TGII for tau, but also the expression level of tau as well as α1B-adrenoceptor signaling molecules, TGII and phospholipase C-δ1, in the human brain.Results: When the tau protein was assayed as a transglutaminase substrate of TGII, tau proteins formed cross-linked products. However, phospholipase C-δ1 inhibited transglutaminase activity in TGII to cross-link with tau in vitro. The amount of expressed mRNA in AD brain tissue was elevated 2~10 fold for tau and 3~20 fold for TGII. Consistent with these observations, the densities of expressed proteins in AD brain tissue also increased 9 fold for tau and 15 fold for TGII. Moreover, phospholipase C-δ1, which is a negative regulator for transglutaminase activity of TGII, also increased 2~25 fold for mRNA as well as 8 fold for protein in AD brain tissue. In contrast, expressed mRNA and protein activity for α1B-adrenoceptor were almost the same between AD and normal brains.Conclusion: These results suggest that the α1B-adrenoceptor signal mediator, TGII and tau, as a substrate of TGII might have a pathophysiological role in AD, but this correlation may be independent of the α1B-adrenoceptor signaling system, including α1B-adrenoceptor and phospholipase C-δ1.

CFH Variants Affect Structural and Functional Brain Changes and Genetic Risk of Alzheimer's Disease.

The immune response is highly active in Alzheimer's disease (AD). Identification of genetic risk contributed by immune genes to AD may provide essential insight for the prognosis, diagnosis, and treatment of this neurodegenerative disease. In this study, we performed a genetic screening for AD-related top immune genes identified in Europeans in a Chinese cohort, followed by a multiple-stage study focusing on Complement Factor H (CFH) gene. Effects of the risk SNPs on AD-related neuroimaging endophenotypes were evaluated through magnetic resonance imaging scan, and the effects on AD cerebrospinal fluid biomarkers (CSF) and CFH expression changes were measured in aged and AD brain tissues and AD cellular models. Our results showed that the AD-associated top immune genes reported in Europeans (CR1, CD33, CLU, and TREML2) have weak effects in Chinese, whereas CFH showed strong effects. In particular, rs1061170 (P(meta)=5.0 × 10(-4)) and rs800292 (P(meta)=1.3 × 10(-5)) showed robust associations with AD, which were confirmed in multiple world-wide sample sets (4317 cases and 16 795 controls). Rs1061170 (P=2.5 × 10(-3)) and rs800292 (P=4.7 × 10(-4)) risk-allele carriers have an increased entorhinal thickness in their young age and a higher atrophy rate as the disease progresses. Rs800292 risk-allele carriers have higher CSF tau and Aβ levels and severe cognitive decline. CFH expression level, which was affected by the risk-alleles, was increased in AD brains and cellular models. These comprehensive analyses suggested that CFH is an important immune factor in AD and affects multiple pathological changes in early life and during disease progress.

Dickkopf-related protein 3 is a potential Aβ-associated protein in Alzheimer's Disease.

Amyloid-β (Aβ) is the most prominent protein in Alzheimer's disease (AD) senile plaques. In addition, Aβ interacts with a variety of Aβ-associated proteins (AAPs), some of which can form complexes with Aβ and influence its clearance, aggregation or toxicity. Identification of novel AAPs may shed new light on the pathophysiology of AD and the metabolic fate of Aβ. In this study, we aimed to identify new AAPs by searching for proteins that may form soluble complexes with Aβ in CSF, using a proteomics approach. We identified the secreted Wnt pathway protein Dickkopf-related protein 3 (Dkk-3) as a potential Aβ-associated protein. Using immunohistochemistry on human AD brain tissue, we observed that (i) Dkk-3 co-localizes with Aβ in the brain, both in diffuse and classic plaques. (ii) Dkk-3 is expressed in neurons and in blood vessel walls in the brain and (iii) is secreted by leptomeningeal smooth muscle cells in vitro. Finally, measurements using ELISA revealed that (iv) Dkk-3 protein is abundantly present in both cerebrospinal fluid and serum, but its levels are similar in non-demented controls and patients with AD, Lewy body dementia, and frontotemporal dementia. Our study demonstrates that Dkk-3 is a hitherto unidentified Aβ-associated protein which, given its relatively high cerebral concentrations and co-localization with Aβ, is potentially involved in AD pathology. In this study, we propose that Dickkopf-related protein-3 (Dkk-3) might be a novel Amyloid-β (Aβ) associated protein. We demonstrate that Dkk-3 is expressed in the brain, especially in vessel walls, and co-localizes with Aβ in senile plaques. Furthermore, Dkk-3 levels in cerebrospinal fluid strongly correlate with Aβ40 levels, but were not suitable to discriminate non-demented controls and patients with dementia.

Visualization of regional tau deposits using 3H-THK5117 in Alzheimer brain tissue

Introduction The accumulation of neurofibrillary tangles, composed of aggregated hyperphosphorylated tau protein, starts spreading early in specific regions in the course of Alzheimer’s disease (AD), correlating with the progression of memory dysfunction. The non-invasive imaging of tau could therefore facilitate the early diagnosis of AD, differentiate it from other dementing disorders and allow evaluation of tau immunization therapy outcomes. In this study we characterized the in vitro binding properties of THK5117, a tentative radiotracer for positron emission tomography (PET) imaging of tau brain deposits.ResultsSaturation and competition binding studies of 3H-THK5117 in post-mortem AD brain tissue showed the presence of multiple binding sites. THK5117 binding was significantly higher in hippocampal (p < 0.001) and temporal (p < 0.01) tissue homogenates in AD compared to controls. Autoradiography studies with 3H-THK5117 was performed on large frozen brain sections from three AD cases who had been followed clinically and earlier undergone in vivo18F-FDG PET investigations. The three AD cases showed distinct differences in regional THK5117 binding that were also observed in tau immunohistopathology as well as in clinical presentation. A negative correlation between in vivo18F-FDG PET and in vitro3H-THK5117 autoradiography was observed in two of the three AD cases.ConclusionsThis study supports that new tau PET tracers will provide further understanding on the role of tau pathology in the diversity of the clinical presentation in AD.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-015-0220-4) contains supplementary material, which is available to authorized users.

Microglial internalization and degradation of pathological tau is enhanced by an anti-tau monoclonal antibody.

Microglia have been shown to contribute to the clearance of brain amyloid β peptides (Aβ), the major component of amyloid plaques, in Alzheimer’s disease (AD). However, it is not known whether microglia play a similar role in the clearance of tau, the major component of neurofibrillary tangles (NFTs). We now report that murine microglia rapidly internalize and degrade hyperphosphorylated pathological tau isolated from AD brain tissue in a time-dependent manner in vitro. We further demonstrate that microglia readily degrade human tau species released from AD brain sections and eliminate NFTs from brain sections of P301S tauopathy mice. The anti-tau monoclonal antibody MC1 enhances microglia-mediated tau degradation in an Fc-dependent manner. Our data identify a potential role for microglia in the degradation and clearance of pathological tau species in brain and provide a mechanism explaining the potential therapeutic actions of passively administered anti-tau monoclonal antibodies.

Cellular Models of Aggregation-dependent Template-directed Proteolysis to Characterize Tau Aggregation Inhibitors for Treatment of Alzheimer Disease

Alzheimer disease (AD) is a degenerative tauopathy characterized by aggregation of Tau protein through the repeat domain to form intraneuronal paired helical filaments (PHFs). We report two cell models in which we control the inherent toxicity of the core Tau fragment. These models demonstrate the properties of prion-like recruitment of full-length Tau into an aggregation pathway in which template-directed, endogenous truncation propagates aggregation through the core Tau binding domain. We use these in combination with dissolution of native PHFs to quantify the activity of Tau aggregation inhibitors (TAIs). We report the synthesis of novel stable crystalline leucomethylthioninium salts (LMTX®), which overcome the pharmacokinetic limitations of methylthioninium chloride. LMTX®, as either a dihydromesylate or a dihydrobromide salt, retains TAI activity in vitro and disrupts PHFs isolated from AD brain tissues at 0.16 μM. The Ki value for intracellular TAI activity, which we have been able to determine for the first time, is 0.12 μM. These values are close to the steady state trough brain concentration of methylthioninium ion (0.18 μM) that is required to arrest progression of AD on clinical and imaging end points and the minimum brain concentration (0.13 μM) required to reverse behavioral deficits and pathology in Tau transgenic mice.

Tau phosphosignatures discriminate Alzheimer Disease from other tauopathies

Neurofibrillary tangles (NFTs) comprised of highly phosphorylated tau are a core pathological feature of Alzheimer's disease (AD) and several other neurodegenerative diseases collectively termed tauopathies, which include progressive supranuclear palsy, corticobasal degeneration and certain frontotemporal dementias. There are six isoforms of human tau in brain and over 40 serine (S), threonine (T), and tyrosine (Y) tau phosphorylation sites have been identified. Phosphorylation of tau at specific S/T/Y sites is hypothesized to cause both functional deficits as well as gain‐of‐function toxicity that ultimately lead to NFT formation, synapse loss and cognitive dysfunction. Despite being clinically distinct diseases it remains unclear whether site‐specific tau phosphorylation differs between AD and other tauopathies. To address this question we employed immobilized metal affinity chromatography followed by liquid chromatography‐tandem mass spectrometry to identify and quantify tau phosphopeptides from individual AD, tauopathy and age‐matched control postmortem human brain tissues (n= 7 per group). In total 77 tau phosphopeptides representing 32 S/T/Y sites were analyzed by weighted correlation network analysis to delineate tau phosphosites co‐enriched in a disease specific manner. Our findings suggest that the mechanisms underlying site‐specific tau phosphorylation differ between AD and other tauopathies.

Potassium channel Kv1.3 is highly expressed by microglia in human Alzheimer's disease.

Recent genetic studies suggest a central role for innate immunity in Alzheimer's disease (AD) pathogenesis, wherein microglia orchestrate neuroinflammation. Kv1.3, a voltage-gated potassium channel of therapeutic relevance in autoimmunity, is upregulated by activated microglia and mediates amyloid-mediated microglial priming and reactive oxygen species production in vitro. We hypothesized that Kv1.3 channel expression is increased in human AD brain tissue. In a blinded postmortem immunohistochemical semi-quantitative analysis performed on ten AD patients and ten non-disease controls, we observed a significantly higher Kv1.3 staining intensity (p = 0.03) and Kv1.3-positive cell density (p = 0.03) in the frontal cortex of AD brains, compared to controls. This paralleled an increased number of Iba1-positive microglia in AD brains. Kv1.3-positive cells had microglial morphology and were associated with amyloid-β plaques. In immunofluorescence studies, Kv1.3 channels co-localized primarily with Iba1 but not with astrocyte marker GFAP, confirming that elevated Kv1.3 expression is limited to microglia. Higher Kv1.3 expression in AD brains was also confirmed by western blot analysis. Our findings support that Kv1.3 channels are biologically relevant and microglia-specific targets in human AD.

Isolation and characterization of antibody fragments selective for toxic oligomeric tau

Oligomeric tau species are important in the onset and progression of Alzheimer's disease (AD), as they are neurotoxic and can propagate tau-tangle pathology. Therefore, reagents that selectively recognize different key morphologies of tau are needed to help define the role of tau in AD and related diseases. We utilized a biopanning protocol that combines the binding diversity of phage-displayed antibody libraries with the powerful imaging capability of atomic force microscopy to isolate single-chain antibody fragments (scFvs) that selectively bind toxic oligomeric tau. We isolated 3 different antibody fragments that bind oligomeric but not monomeric or fibrillar tau. The scFvs differentiate brain tissue homogenates of both 3×TG and tau-AD mice from wild-type mice, detecting oligomeric tau at much earlier ages than when neurofibrillary tangles are typically detected. The scFvs also distinguish human postmortem AD brain tissue from cognitively normal postmortem human brain tissue, demonstrating the potential of this approach for developing biomarkers for early detection and progression of AD.

Increased acetyl and total histone levels in post-mortem Alzheimer's disease brain

Histone acetylation is an epigenetic modification that plays a critical role in chromatin remodelling and transcriptional regulation. There is increasing evidence that epigenetic modifications may become compromised in aging and increase susceptibility to the development of neurodegenerative disorders such as Alzheimer's disease. Immunohistochemical labelling of free-floating sections from the inferior temporal gyrus (Alzheimer's disease, n=14; control, n=17) and paraffin-embedded tissue microarrays containing tissue from the middle temporal gyrus (Alzheimer's disease, n=29; control, n=28) demonstrated that acetyl histone H3 and acetyl histone H4 levels, as well as total histone H3 and total histone H4 protein levels, were significantly increased in post-mortem Alzheimer's disease brain tissue compared to age- and sex-matched neurologically normal control brain tissue. Changes in acetyl histone levels were proportional to changes in total histone levels. The increase in acetyl histone H3 and H4 was observed in Neuronal N immunopositive pyramidal neurons in Alzheimer's disease brain. Using immunolabelling, histone markers correlated significantly with the level of glial fibrillary acidic protein and HLA-DP, -DQ and -DR immunopositive cells and with the pathological hallmarks of Alzheimer's disease (hyperphosphorylated tau load and β-amyloid plaques). Given that histone acetylation changes were correlated with changes in total histone protein, it was important to evaluate if protein degradation pathways may be compromised in Alzheimer's disease. Consequently, significant positive correlations were also found between ubiquitin load and histone modifications. The relationship between histone acetylation and ubiquitin levels was further investigated in an in vitro model of SK-N-SH cells treated with the proteasome inhibitor Mg132 and the histone deacetylase inhibitor valproic acid. In this model, compromised protein degradation caused by Mg132 lead to elevated histone labelling. In addition, valproic acid worked synergistically with Mg132 in elevating ubiquitin load and causing cell death. These findings highlight important pathological relationships linking a compromise in protein turnover with the histone changes observed in Alzheimer's disease post-mortem human brain.

Regulation of synaptic plasticity and cognition by SUMO in normal physiology and Alzheimer's disease.

Learning and memory and the underlying cellular correlate, long-term synaptic plasticity, involve regulation by posttranslational modifications (PTMs). Here we demonstrate that conjugation with the small ubiquitin-like modifier (SUMO) is a novel PTM required for normal synaptic and cognitive functioning. Acute inhibition of SUMOylation impairs long-term potentiation (LTP) and hippocampal-dependent learning. Since Alzheimer's disease (AD) prominently features both synaptic and PTM dysregulation, we investigated SUMOylation under pathology induced by amyloid-β (Aβ), a primary neurotoxic molecule implicated in AD. We observed that SUMOylation is dysregulated in both human AD brain tissue and the Tg2576 transgenic AD mouse model. While neuronal activation normally induced upregulation of SUMOylation, this effect was impaired by Aβ42 oligomers. However, supplementing SUMOylation via transduction of its conjugating enzyme, Ubc9, rescued Aβ-induced deficits in LTP and hippocampal-dependent learning and memory. Our data establish SUMO as a novel regulator of LTP and hippocampal-dependent cognition and additionally implicate SUMOylation impairments in AD pathogenesis.

Synthetic Aβ peptides acquire prion-like properties in the brain.

In transmission studies with Alzheimer's disease (AD) animal models, the formation of Aβ plaques is proposed to be initiated by seeding the inoculated amyloid β (Aβ) peptides in the brain. Like the misfolded scrapie prion protein (PrPSc) in prion diseases, Aβ in AD shows a certain degree of resistance to protease digestion while the biochemical basis for protease resistance of Aβ remains poorly understood. Using in vitro assays, histoblotting, and electron microscopy, we characterize the biochemical and morphological features of synthetic Aβ peptides and Aβ isolated from AD brain tissues. Consistent with previous observations, monomeric and oligomeric Aβ species extracted from AD brains are insoluble in detergent buffers and resistant to digestions with proteinase K (PK). Histoblotting of AD brain tissue sections exhibits an increased Aβ immunoreactivity after digestion with PK. In contrast, synthetic Aβ40 and Aβ42 are soluble in detergent buffers and fully digested by PK. Electron microscopy of Aβ40 and Aβ42 synthetic peptides shows that both species of Aβ form mature fibrils. Those generated from Aβ40 are longer but less numerous than those made of Aβ42. When spiked into human brain homogenates, both Aβ40 and Aβ42 acquire insolubility in detergent and resistance to PK. Our study favors the hypothesis that the human brain may contain cofactor(s) that confers the synthetic Aβ peptides PrPSc-like physicochemical properties.

Micellar extraction possesses a new advantage for the analysis of Alzheimer's disease brain proteome.

Integral membrane proteins (MPs), such as transporters, receptors, and ion channels, are of great interest because of their participation in various vital cellular functions including cell-cell interactions, ion transport, and signal transduction. However, studies of MPs are complicated because of their hydrophobic nature, heterogeneity, and low abundance. Cloud-point extraction (CPE) with the non-ionic surfactant Triton X-114 was performed to simultaneously extract and phase separate hydrophobic and hydrophilic proteins from Alzheimer's disease (AD) and unaffected control brain tissue. Quantitative proteomics analysis of temporal neocortex samples of AD patients and controls was performed using a shotgun approach based on stable isotope dimethyl labeling (DML) quantification technique followed by nanoLC-MS/MS analysis. A total of 1096 unique proteins were identified and quantified, with 40.3% (211/524) predicted as integral MPs with at least one transmembrane domain (TMD) found in the detergent phase, and 10% (80/798) in the detergent-depleted phase. Among these, 62 proteins were shown to be significantly altered (p-value <0.05), in AD versus control samples. In the detergent fraction, we found 10 hydrophobic transmembrane proteins containing up to 14 putative TMDs that were significantly up- or down-regulated in AD compared with control brains. Changes in four of these proteins, alpha-enolase (ENOA), lysosome-associated membrane glycoprotein 1 (LAMP1), 14-3-3 protein gamma (1433G), and sarcoplasmic/endoplasmic reticulum calcium ATPase2 (AT2A2) were validated by immunoblotting. Our results emphasize that separating hydrophobic MPs in CPE contributes to an increased understanding of the underlying molecular mechanisms in AD. Such knowledge can become useful for the development of novel disease biomarkers.

Abnormal cortical lysosomal β-hexosaminidase and β-galactosidase activity at post-synaptic sites during Alzheimer's disease progression

A critical role of endosomal–lysosomal system alteration in neurodegeneration is supported by several studies. Dysfunction of the lysosomal compartment is a common feature also in Alzheimer's disease. Altered expression of lysosomal glycohydrolases has been demonstrated not only in the brain and peripheral tissues of Alzheimer's disease patients, but also in presymptomatic subjects before degenerative phenomenon becomes evident. Moreover, the presence of glycohydrolases associated to the plasma membrane have been widely demonstrated and their alteration in pathological conditions has been documented. In particular, lipid microdomains-associated glycohydrolases can be functional to the maintenance of the proper glycosphingolipids pattern, especially at cell surface level, where they are crucial for the function of cell types such as neurons. In this study we investigated the localization of β-hexosaminidase and β-galactosidase glycohydrolases, both involved in step by step degradation of the GM1 to GM3 gangliosides, in lipid microdomains from the cortex of both an early and advanced TgCRND8 mouse model of Alzheimer's disease. Throughout immunoprecipitation experiments of purified cortical lipid microdomains, we demonstrated for the first time that β-hexosaminidase and β-galactosidase are associated with post-synaptic vesicles and that their activities are increased at both the early and the advanced stage of Alzheimer's disease. The early increase of lipid microdomain-associated β-hexosaminidase and β-galactosidase activities could have relevant implications for the pathophysiology of the disease since their possible pharmacological manipulation could shed light on new reliable targets and biological markers of Alzheimer's disease.

The Ca2+ sensor S100A1 modulates neuroinflammation, histopathology and Akt activity in the PSAPP Alzheimer's disease mouse model

The contribution of the Ca2+ sensor S100A1 to in vivo Alzheimer's disease (AD) pathobiology has not been elucidated although S100A1 regulates numerous cellular processes linked to AD. This study uses genetic ablation to ascertain the effects of S100A1 on neuroinflammation, beta-amyloid (Aβ) plaque deposition and Akt activity in the PSAPP AD mouse model. PSAPP/S100A1−/− mice exhibited decreases in astrocytosis (GFAP burden), microgliosis (Iba1 burden) and plaque load/number when compared to PSAPP/S100A1+/+ mice at six and twelve months of age. The presence of detectable S100A1 staining in human AD specimens is consistent with a detrimental gain of S100A1 function in AD. S100A1 ablation also reduced plaque associated and increased non-plaque associated PO4-Akt and PO4-GSK3β staining. S100A1·Akt complexes were undetectable in PC12 cells and AD brain tissue suggesting that S100A1 indirectly modulates Akt activity. In contrast, S100A1·RyR (ryanodine receptor) complexes were present in human/mouse AD brain and exhibited Ca2+-dependent formation in neuronal cells. This is the first direct demonstration of an S100A1· target protein complex in tissue/cells and identifies the RyR as a primary S100A1 target protein in the brain. Collectively, these data suggest that S100A1 inhibition may be a novel strategy for normalizing aberrant Ca2+ signaling in AD.