Alzheimer's Disease Brain Tissue Research Articles

Identification of diffusion, kurtosis, and propagator MRI markers of Alzheimer’s disease pathology in post-mortem human tissue

Abstract Alzheimer’s disease (AD) is an irreversible degenerative brain disease affecting 6.7 million Americans and while the hallmark AD pathologies of plaques and tangles follow a stereotyped progression during the course of the disease, clinical markers for early diagnosis are lacking and approximately 20% of all AD cases are ultimately misdiagnosed. Conventional clinical MRI is capable of reporting severe brain atrophy, but fails to recognize earlier biomarkers associated with more subtle cellular and molecular changes. Microstructural Magnetic Resonance Imaging (MRI) techniques are promising to address this challenge and may sensitively detect and distinguish tissue degeneration, tauopathies, and beta amyloid plaques to improve accuracy of diagnosis and enable early detection. The objective of this study was to identify and compare the most promising microstructural markers of AD pathology over a range of diffusion and relaxometry-based MRI techniques from conventional to advanced. To accomplish this, we performed MRI microscopy of post-mortem human temporal lobe specimens (n = 14) at high resolution and image quality and evaluated the relative influence of metrics across multiple microstructural MRI frameworks using principal component analysis (PCA). We performed additional correlation analysis between metrics identified by PCA and clinical neuropathology scores of Braak stage and plaque and tangle load. Hippocampal diffusion and restriction metrics contributed most to the first principal component, and the correlation with Braak score was positive for diffusivity and negative for restriction metrics. Additionally, the MAP-MRI propagator anisotropy (PA) metric of microscale anisotropy was strongly and negatively correlated with AD pathology while the conventional fractional anisotropy (FA) metric showed little or no correspondence and there was not a strong association between FA and PA by PCA. Entorhinal cortex findings were minimal except for reported increases in restriction due to plaque content. Taken together, our findings suggest that microstructural MRI metrics of restriction and diffusion are most prominent and may reflect degenerative processes in AD brain tissue and that microscale anisotropy may be more advantageous than conventional FA for the detection of subtle and earlier cellular changes in AD.

In Search for Low-Molecular-Weight Ligands of Human Serum Albumin That Affect Its Affinity for Monomeric Amyloid β Peptide.

An imbalance between production and excretion of amyloid β peptide (Aβ) in the brain tissues of Alzheimer's disease (AD) patients leads to Aβ accumulation and the formation of noxious Aβ oligomers/plaques. A promising approach to AD prevention is the reduction of free Aβ levels by directed enhancement of Aβ binding to its natural depot, human serum albumin (HSA). We previously demonstrated the ability of specific low-molecular-weight ligands (LMWLs) in HSA to improve its affinity for Aβ. Here we develop this approach through a bioinformatic search for the clinically approved AD-related LMWLs in HSA, followed by classification of the candidates according to the predicted location of their binding sites on the HSA surface, ranking of the candidates, and selective experimental validation of their impact on HSA affinity for Aβ. The top 100 candidate LMWLs were classified into five clusters. The specific representatives of the different clusters exhibit dramatically different behavior, with 3- to 13-fold changes in equilibrium dissociation constants for the HSA-Aβ40 interaction: prednisone favors HSA-Aβ interaction, mefenamic acid shows the opposite effect, and levothyroxine exhibits bidirectional effects. Overall, the LMWLs in HSA chosen here provide a basis for drug repurposing for AD prevention, and for the search of medications promoting AD progression.

Interaction of exogenous acetylcholinesterase and butyrylcholinesterase with amyloid-β plaques in human brain tissue

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are associated with amyloid-β (Aβ) plaques and exhibit altered biochemical properties in human Alzheimer's disease (AD), as well as in the transgenic 5XFAD mouse model of AD amyloidosis. In the brains of the 5XFAD mouse model devoid of BChE enzyme (5XFAD/BChE-KO), incubation of tissue sections with exogenous BChE purified from human plasma (pl-BChE) leads to its association with Aβ plaques and its biochemical properties are comparable to those reported for endogenous BChE associated with plaques in both human AD and in 5XFAD mouse brain tissue. We sought to determine whether these observations in 5XFAD/BChE-KO mice also apply to human brain tissues. To do so, endogenous ChE activity in human AD brain tissue sections was quenched with 50 % aqueous acetonitrile (MeCNaq) leaving the tissue suitable for further studies. Quenched sections were then incubated with recombinant AChE (r-AChE) or pl-BChE and stained for each enzymes' activity. Exogenous r-AChE or pl-BChE became associated with Aβ plaques, and when bound, had properties that were comparable to the endogenous ChE enzymes associated with plaques in AD brain tissues without acetonitrile treatment. These findings in human AD brain tissue extend previous observations in the 5XFAD/BChE-KO mouse model and demonstrate that exogenously applied r-AChE and pl-BChE have high affinity for Aβ plaques in human brain tissues. This association alters the biochemical properties of these enzymes, most likely due a conformational change. If incorporation of AChE and BChE in Aβ plaques facilitates AD pathogenesis, blocking this association could lead to disease-modifying approaches to AD. This work provides a method to study the mechanism of AChE and BChE interaction with Aβ plaque pathology in post-mortem human brain tissue.

Sensitive detection of pathological seeds of α-synuclein, tau and prion protein on solid surfaces.

Prions or prion-like aggregates such as those composed of PrP, α-synuclein, and tau are key features of proteinopathies such as prion, Parkinson's and Alzheimer's diseases, respectively. Their presence on solid surfaces may be biohazardous under some circumstances. PrP prions bound to solids are detectable by ultrasensitive real-time quaking-induced conversion (RT-QuIC) assays if the solids can be immersed in assay wells or the prions transferred to pads. Here we show that prion-like seeds can remain detectable on steel wires for at least a year, or even after enzymatic cleaning and sterilization. We also show that contamination of larger objects with pathological seeds of α-synuclein, tau, and PrP can be detected by simply assaying a sampling medium that has been transiently applied to the surface. Human α-synuclein seeds in dementia with Lewy bodies brain tissue were detected by α-synuclein RT-QuIC after drying of tissue dilutions with concentrations as low as 10-6 onto stainless steel. Tau RT-QuIC detected tau seeding activity on steel exposed to Alzheimer's disease brain tissue diluted as much as a billion fold. Prion RT-QuIC assays detected seeding activity on plates exposed to brain dilutions as extreme as 10-5-10-8 from prion-affected humans, sheep, cattle and cervids. Sampling medium collected from surgical instruments used in necropsies of sporadic Creutzfeldt-Jakob disease-infected transgenic mice was positive down to 10-6 dilution. Sensitivity for prion detection was not sacrificed by omitting the recombinant PrP substrate from the sampling medium during its application to a surface and subsequent storage as long as the substrate was added prior to performing the assay reaction. Our findings demonstrate practical prototypic surface RT-QuIC protocols for the highly sensitive detection of pathologic seeds of α-synuclein, tau, and PrP on solid objects.

ZCCHC17 knockdown phenocopies Alzheimer's disease-related loss of synaptic proteins and hyperexcitability.

ZCCHC17 is a master regulator of synaptic gene expression and has recently been shown to play a role in splicing of neuronal mRNA. We previously showed that ZCCHC17 protein declines in Alzheimer's disease (AD) brain tissue before there is significant gliosis and neuronal loss, that ZCCHC17 loss partially replicates observed splicing abnormalities in AD brain tissue, and that maintenance of ZCCHC17 levels is predicted to support cognitive resilience in AD. Here, we assessed the functional consequences of reduced ZCCHC17 expression in primary cortical neuronal cultures using siRNA knockdown. Consistent with its previously identified role in synaptic gene expression, loss of ZCCHC17 led to loss of synaptic protein expression. Patch recording of neurons shows that ZCCHC17 loss significantly disrupted the excitation/inhibition balance of neurotransmission, and favored excitatory-dominant synaptic activity as measured by an increase in spontaneous excitatory post synaptic currents and action potential firing rate, and a decrease in spontaneous inhibitory post synaptic currents. These findings are consistent with the hyperexcitable phenotype seen in AD animal models and in patients. We are the first to assess the functional consequences of ZCCHC17 knockdown in neurons and conclude that ZCCHC17 loss partially phenocopies AD-related loss of synaptic proteins and hyperexcitability.

Thiophene-Based Ligands for Specific Assignment of Distinct Aβ Pathologies in Alzheimer's Disease.

Aggregated species of amyloid-β (Aβ) are one of the pathological hallmarks in Alzheimer's disease (AD), and ligands that selectively target different Aβ deposits are of great interest. In this study, fluorescent thiophene-based ligands have been used to illustrate the features of different types of Aβ deposits found in AD brain tissue. A dual-staining protocol based on two ligands, HS-276 and LL-1, with different photophysical and binding properties, was developed and applied on brain tissue sections from patients affected by sporadic AD or familial AD associated with the PSEN1 A431E mutation. When binding to Aβ deposits, the ligands could easily be distinguished for their different fluorescence, and distinct staining patterns were revealed for these two types of AD. In sporadic AD, HS-276 consistently labeled all immunopositive Aβ plaques, whereas LL-1 mainly stained cored and neuritic Aβ deposits. In the PSEN1 A431E cases, each ligand was binding to specific types of Aβ plaques. The ligand-labeled Aβ deposits were localized in distinct cortical layers, and a laminar staining pattern could be seen. Biochemical characterization of the Aβ aggregates in the individual layers also showed that the variation of ligand binding properties was associated with certain Aβ peptide signatures. For the PSEN1 A431E cases, it was concluded that LL-1 was binding to cotton wool plaques, whereas HS-276 mainly stained diffuse Aβ deposits. Overall, our findings showed that a combination of ligands was essential to identify distinct aggregated Aβ species associated with different forms of AD.

Integrating multi-omics data to reveal the effect of genetic variant rs6430538 on Alzheimer's disease risk.

Growing evidence highlights a potential genetic overlap between Alzheimer's disease (AD) and Parkinson's disease (PD); however, the role of the PD risk variant rs6430538 in AD remains unclear. In Stage 1, we investigated the risk associated with the rs6430538 C allele in seven large-scale AD genome-wide association study (GWAS) cohorts. In Stage 2, we performed expression quantitative trait loci (eQTL) analysis to calculate the cis-regulated effect of rs6430538 on TMEM163 in both AD and neuropathologically normal samples. Stage 3 involved evaluating the differential expression of TMEM163 in 4 brain tissues from AD cases and controls. Finally, in Stage 4, we conducted a transcriptome-wide association study (TWAS) to identify any association between TMEM163 expression and AD. The results showed that genetic variant rs6430538 C allele might increase the risk of AD. eQTL analysis revealed that rs6430538 up-regulated TMEM163 expression in AD brain tissue, but down-regulated its expression in normal samples. Interestingly, TMEM163 showed differential expression in entorhinal cortex (EC) and temporal cortex (TCX). Furthermore, the TWAS analysis indicated strong associations between TMEM163 and AD in various tissues. In summary, our findings suggest that rs6430538 may influence AD by regulating TMEM163 expression. These discoveries may open up new opportunities for therapeutic strategies targeting AD.

APOE4/4 is linked to damaging lipid droplets in Alzheimer’s disease microglia

Several genetic risk factors for Alzheimer’s disease implicate genes involved in lipid metabolism and many of these lipid genes are highly expressed in glial cells1. However, the relationship between lipid metabolism in glia and Alzheimer’s disease pathology remains poorly understood. Through single-nucleus RNA sequencing of brain tissue in Alzheimer’s disease, we have identified a microglial state defined by the expression of the lipid droplet-associated enzyme ACSL1 with ACSL1-positive microglia being most abundant in patients with Alzheimer’s disease having the APOE4/4 genotype. In human induced pluripotent stem cell-derived microglia, fibrillar Aβ induces ACSL1 expression, triglyceride synthesis and lipid droplet accumulation in an APOE-dependent manner. Additionally, conditioned media from lipid droplet-containing microglia lead to Tau phosphorylation and neurotoxicity in an APOE-dependent manner. Our findings suggest a link between genetic risk factors for Alzheimer’s disease with microglial lipid droplet accumulation and neurotoxic microglia-derived factors, potentially providing therapeutic strategies for Alzheimer’s disease.

A NIR iridium(III) complex-based reversible probe for assessing AD progression and screening anti-AD drugs through visualization of the “Cu2+-H2S seesaw” phenomenon in Alzheimer’s disease

The progression of Alzheimer’s disease (AD) is accompanied by the increase of copper ions (Cu2+) concentration and decrease of hydrogen sulfide (H2S) level in brain tissue, thus, reversible detection of Cu2+ and H2S is regarded as an important strategy for the diagnosis and intervention of AD. However, reliable tools for reversible tracking of Cu2+ and H2S and demonstrate their dynamic changes in AD are still lacking. Herein, this work developed a novel NIR Ir(III) complex-based probe, named NIR-Ir-DA, for reversible tracking of Cu2+ and H2S in AD cells and brain tissues. The newly prepared NIR luminescence probe exhibited fast response, high sensitivity and selectivity, good biocompatibility, which makes it to be used for reversible sensing of Cu2+ and H2S in multiple cycles in living cells and assessing AD progression in vitro and in vivo. Furthermore, using NIR-Ir-DA as a probe, we screened the theaflavin-3,3′-digallate (TF3) from polyphenols with an outstanding ability to reduce neuroinflammation, which would be greatly beneficial for AD therapy. These results indicated that NIR-Ir-DA could be a useful tool for understanding the role of “Cu2+-H2S seesaw” phenomenon in amyloid-β deposition, as well as for assessing AD progression and screening anti-AD drugs.

Adjunctive silymarin supplementation and its effects on disease severity, oxidative stress, and inflammation in patients with Alzheimer’s disease

ABSTRACT Background Brain tissue in Alzheimer's patients is exposed to oxidative stress. Silymarin is an adjunct drug that has anti-inflammatory and antioxidant properties. Objective This study aimed to evaluate the effect of silymarin on biomarkers of oxidative stress, inflammation, and disease severity in Alzheimer's patients. Methods This randomized, single-blind clinical trial study was performed on 33 patients with Alzheimer's disease (AD) whose disease was confirmed by DSM-5 criteria and by brain imaging. Patients in the case group received three 250 mg silymarin capsules daily (each containing 150 mg silymarin), as an adjunctive medication in addition to the routine medication regimen. In the placebo group (control), patients received the same amount of placebo. All patients underwent Mini Mental State Exam (MMSE) and a panel of blood tests including malondialdehyde, neopterin, catalase, paraoxonase-1, total oxidative status, and total antioxidant capacity to reevaluate the changes pre/postintervention at the end of the trimester. Results The catalase and MDA serum levels after the adjunctive silymarin treatment decreased significantly (Catalasebefore silymarin = 9.29 ± 7.02 vs Catalaseafter silymarin = 5.32 ± 2.97, p = 0.007 and MDAbefore silymarin = 4.29 ± 1.90 vs MDAafter silymarin = 1.66 ± 0.84, p < 0.001) while MMSE increased notably (MMSEbefore silymarin = 10.39 ± 6.42 vs MMSEafter silymarin = 13.37 ± 6.81, p < 0.001). Conclusion Silymarin can be effective as an adjunct drug and a powerful antioxidant in reducing oxidative stress and improving the course of AD.

Perineuronal net deglycosylation associates with tauopathy-induced gliosis and neurodegeneration.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by clinical symptoms of memory and cognitive deficiencies. Postmortem evaluation of AD brain tissue shows proteinopathy that closely associate with the progression of this dementing disorder, including the accumulation of extracellular beta amyloid (Aβ) and intracellular hyperphosphorylated tau (pTau) with neurofibrillary tangles (NFTs). Current therapies targeting Aβ have limited clinical efficacy and life-threatening side effects and highlight the need for alternative treatments targeting pTau and other pathophysiologic mechanisms driving AD pathogenesis. The brain's extracellular matrices (ECM), particularly perineuronal nets (PNNs), play a crucial role in brain functioning and neurocircuit stability, and reorganization of these unique PNN matrices has been associated with the progression of AD and accumulation of pTau in humans. We hypothesize that AD-associated changes in PNNs may in part be driven by the accumulation of pTau within the brain. In this work, we investigated whether the presence of pTau influenced PNN structural integrity and PNN chondroitin sulfate-glycosaminoglycan (CS-GAG) compositional changes in two transgenic mouse models expressing tauopathy-related AD pathology, PS19 (P301S) and Tau4RTg2652 mice. We show that PS19 mice exhibit an age-dependent loss of hippocampal PNN CS-GAGs, but not the underlying aggrecan core protein structures, in association with pTau accumulation, gliosis, and neurodegeneration. The loss of PNN CS-GAGs were linked to shifts in CS-GAG sulfation patterns to favor the neuroregenerative isomer, 2S6S-CS. Conversely, Tau4RTg2652 mice exhibit stable PNN structures and normal CS-GAG isomer composition despite robust pTau accumulation, suggesting a critical interaction between neuronal PNN glycan integrity and neighboring glial cell activation. Overall, our findings provide insights into the complex relationship between PNN CS-GAGs, pTau pathology, gliosis, and neurodegeneration in mouse models of tauopathy, and offer new therapeutic insights and targets for AD treatment.

Proteomic analysis identifies HSP90AA1, PTK2B, and ANXA2 in the human entorhinal cortex in Alzheimer's disease: Potential role in synaptic homeostasis and Aβ pathology through microglial and astroglial cells.

Alzheimer's disease (AD), the most prevalent neurodegenerative disorder worldwide, is clinically characterized by cognitive deficits. Neuropathologically, AD brains accumulate deposits of amyloid-β (Aβ) and tau proteins. Furthermore, these misfolded proteins can propagate from cell to cell in a prion-like manner and induce native proteins to become pathological. The entorhinal cortex (EC) is among the earliest areas affected by tau accumulation along with volume reduction and neurodegeneration. Neuron-glia interactions have recently come into focus; however, the role of microglia and astroglia in the pathogenesis of AD remains unclear. Proteomic approaches allow the determination of changes in the proteome to better understand the pathology underlying AD. Bioinformatic analysis of proteomic data was performed to compare ECs from AD and non-AD human brain tissue. To validate the proteomic results, western blot, immunofluorescence, and confocal studies were carried out. The findings revealed that the most disturbed signaling pathway was synaptogenesis. Because of their involvement in synapse function, relationship with Aβ and tau proteins and interactions in the pathway analysis, three proteins were selected for in-depth study: HSP90AA1, PTK2B, and ANXA2. All these proteins showed colocalization with neurons and/or astroglia and microglia and with pathological Aβ and tau proteins. In particular, ANXA2, which is overexpressed in AD, colocalized with amoeboid microglial cells and Aβ plaques surrounded by astrocytes. Taken together, the evidence suggests that unbalanced expression of HSP90AA1, PTK2B, and ANXA2 may play a significant role in synaptic homeostasis and Aβ pathology through microglial and astroglial cells in the human EC in AD.

Advancements in high-resolution 3D microscopy analysis of endosomal morphology in postmortem Alzheimer's disease brains.

Abnormal endo-lysosomal morphology is an early cytopathological feature of Alzheimer's disease (AD) and genome-wide association studies (GWAS) have implicated genes involved in the endo-lysosomal network (ELN) as conferring increased risk for developing sporadic, late-onset AD (LOAD). Characterization of ELN pathology and the underlying pathophysiology is a promising area of translational AD research and drug development. However, rigorous study of ELN vesicles in AD and aged control brains poses a unique constellation of methodological challenges due in part to the small size of these structures and subsequent requirements for high-resolution imaging. Here we provide a detailed protocol for high-resolution 3D morphological quantification of neuronal endosomes in postmortem AD brain tissue, using immunofluorescent staining, confocal imaging with image deconvolution, and Imaris software analysis pipelines. To demonstrate these methods, we present neuronal endosome morphology data from 23 sporadic LOAD donors and one aged non-AD control donor. The techniques described here were developed across a range of AD neuropathology to best optimize these methods for future studies with large cohorts. Application of these methods in research cohorts will help advance understanding of ELN dysfunction and cytopathology in sporadic AD.

Determination of Alzheimer's disease based on morphology and atrophy using machine learning combined with automated segmentation.

To evaluate the degree of cerebral atrophy for Alzheimer's disease (AD), voxel-based morphometry has been performed with magnetic resonance imaging. Detailed morphological changes in a specific tissue area having the most evidence of atrophy were not considered by the machine-learning technique. To develop a machine-learning system that can capture morphology features for determination of atrophy of brain tissue in early-stage AD and classification of healthy participants or patients. Three-dimensional T1-weighted (3D-T1W) data were obtained from AD Neuroimaging Initiative (200 healthy controls and 200 patients with early-stage AD). Automated segmentation of 3D-T1W data was performed. Deep learning (DL) and support vector machine (SVM) were trained using 66-segmented volume values as input and AD diagnosis as output. DL was performed using 66 volume values or gray matter (GM) and white matter (WM) volume values. SVM learning was performed using 66 volume values and six regions with high variable importance. 3D convolutional neural network (3D-CNN) was trained using the segmented images. Accuracy and area under curve (AUC) were obtained. Variable importance was evaluated from logistic regression analysis. DL for GM and WM volume values, accuracy 0.6; SVM for all volume values, accuracy 0.82 and AUC 0.81; DL for all volume values, accuracy 0.82 and AUC 0.8; 3D-CNN using segmental images of the whole brain, accuracy 0.5 and AUC 0.51. SVM using volume values of six regions, accuracy 0.82; image-based 3D-CNN, highest accuracy 0.69. Our results show that atrophic features are more considerable than morphological features in the early detection of AD.

Synthesis and in vitro evaluation of novel indanone derivatives targeting β-amyloid

ObjectiveThe aim of this research is to synthesize and evaluate β-amyloid (Aβ) binding probes for early diagnosis of Alzheimer's disease (AD). MethodsWe designed and synthesized two new fluorine tag Indanone derivatives (compounds a and b), and then based on the platform of ultra-high-performance liquid chromatography-mass spectrometry for its affinity with β-amyloid (Aβ) protein determination, and use of compounds a and b solution as dyes for AD brain tissue staining in vitro. To evaluate the lipid solubility and stability of the drug, the lipid-water partition coefficient was determined using n-octanol as the lipid phase, and the stability of compound b was tested in vitro to mimic the in vivo environment. ResultsBoth compounds a and b exhibit high affinity for binding to Aβ. The Kd values of compound a for Aβ1-40 and Aβ1-42 were found to be 467.40 ± 63.26, and 227.57 ± 19.08 nmol/L, respectively. Similarly, the Kd values of compound b for Aβ1-40 and Aβ1-42 were 438.13 ± 26.77 and 167.27 ± 23.70 nmol/L, respectively. In the staining experiment of human brain slices of AD, both compounds a and b were found to bind to Aβ plaques, and their staining effect was stronger than positive control (0.1 % ThS). In the lipid-water partition coefficient experiment, compound a and compound b exhibited logP values of 0.84 and 1.85, respectively, indicating good lipid solubility. In the in vitro stability experiment, compound b exhibited a concentration of 74.79 %, 80.47 %, and 70.00 % after incubation in liver, brain homogenate, and blood, respectively. ConclusionWe have successfully designed and synthesized two probes labeled with fluoride that can be used for diagnosing AD. These probes have a high affinity for Aβ protein and good lipid solubility. As A contrast agent based on Aβ protein, compound b showed greater potential than compound a.

Mapping enhancer and chromatin accessibility landscapes charts the regulatory network of Alzheimer's disease

BackgroundEnhancers are regulatory elements that target and modulate gene expression and play a role in human health and disease. However, the roles of enhancer regulatory circuit abnormalities driven by epigenetic alterations in Alzheimer's disease (AD) are unclear. MethodsIn this study, a multiomic integrative analysis was performed to map enhancer and chromatin accessibility landscapes and identify regulatory network abnormalities in AD. We identified differentially methylated enhancers and constructed regulatory networks across brain regions using AD brain tissue samples. Through the integration of snATAC-seq and snRNA-seq datasets, we mapped enhancers with DNA methylation alterations (DMA) and chromatin accessibility landscapes. Core regulatory triplets that contributed to AD neuropathology in specific cell types were further prioritized. ResultsWe revealed widespread DNA methylation alterations (DMA) in the enhancers of AD patients across different brain regions. In addition, the genome-wide transcription factor (TF) binding profiles showed that enhancers with DMA are pervasively regulated by TFs. The TF-enhancer-gene regulatory network analysis identified core regulatory triplets that are associated with brain and immune cell proportions and play important roles in AD pathogenesis. Enhancer regulatory circuits with DMA exhibited distinct chromatin accessibility patterns, which were further characterized at single-cell resolutions. ConclusionsOur study comprehensively investigated DNA methylation-mediated regulatory circuit abnormalities and provided novel insights into the potential pathogenesis of AD.

Exploratory research on Neurogranin ratios for biomarkers of neurodegeneration

AbstractBackgroundNeurogranin (Ng) is a cerebrospinal fluid biomarker for synaptic dysfunction at early stage of Alzheimer’s disease (AD). It has been reported that some truncated forms from Ng is increased in AD brain tissue. However, the clinical significance of Ng forms in plasma has remained unclear. We have developed a simultaneous detection method of a plurality of Ng forms in plasma by using immunoprecipitation combined with matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (IP‐MALDI‐MS). In this study, we applied the IP‐MALDI‐MS technique to investigate blood‐based biomarkers for neurodegeneration.MethodPlasma samples were obtained from cognitively normal (CN), mild cognitive impairment (MCI), Alzheimer’s dementia (AD) and non‐Alzheimer’s dementia (non‐AD) individuals in National Center for Geriatrics and Gerontology. These subjects were classified as Aβ positive and negative based on PET with PIB tracer. Ng forms were immunoprecipitated from the samples spiked by stable isotope‐labeled (SIL) Ng peptides using magnetic beads coupled with anti‐Ng monoclonal antibody. After the IP was performed two times consecutively, Ng forms eluted from antibody beads were applied on the MALDI plate. SIL‐Ng peptides were used as internal standards for normalizing an intensity of each endogenous Ng form in plasma.ResultWe calculated intensity ratio of a Ng form‐to‐other Ng form (Ng ratio) in all combinations of peaks corresponding to mass of Ng forms. To investigate the possibility of Ng as a biomarker for neurodegeneration, we screened the Ng ratios with the difference between CN and Aβ positive AD or Aβ negative non‐AD. Eleven Ng ratios were significantly higher in the Aβ positive AD and Aβ negative non‐AD groups than the CN group. In ROC analyses, AUC values greater than 0.750 were found in discriminating the Aβ positive AD or Aβ negative non‐AD group from the CN group. Among the eleven Ng ratios, five were significantly correlated with MMSE (ρ←0.400).ConclusionSome Ng forms cleaved at or nearly the IQ domain have been found in the AD brain, which suggests the function loss of Ng in synaptic plasticity. Increasing Ng ratios in plasma may be biomarker candidates for synaptic dysfunction involved in the change of Ng forms in the brain.

Rapid, scalable assay of amylin‐β amyloid co‐aggregation in brain tissue and blood

AbstractBackgroundIslet amyloid polypeptide (amylin) secreted from the pancreas crosses the blood brain barrier into the brain parenchyma and interacts with Alzheimer’s disease (AD) associated cerebral β‐amyloid (Aβ) plaques, which are found in both sporadic and early‐onset familial AD. Immunoprecipitated amylin from human AD brain tissue analyzed by Western blot using an anti‐Aβ antibody detects Aβ immunoreactivity, which indicates that the amylin and Aβ peptides aggregate in the brains of humans with AD, forming amylin‐Aβ hetero‐oligomers that are sodium dodecyl sulfate (SDS)‐soluble. We report the development of an ELISA to detect amylin‐Aβ hetero‐oligomers in brain tissue and blood.MethodThe amylin‐Aβ ELISA relies on a monoclonal anti‐Aβ middomain antibody (detection) and a polyclonal anti‐amylin antibody (capture) designed to recognize an epitope that is distinct from the high affinity amylin‐Aβ binding sites. We screened temporal cortex homogenates obtained from 13 cognitively unaffected (CU) humans and 47 persons with sporadic AD (sAD) for amylin‐Aβ hetero‐oligomers. Individuals were of similar age (sAD, 85.65 ± 7.04 years vs. CU, 87.22 ± 7.30 years; p = 0.482). Brain tissue amylin and Aβ concentrations were measured using conventional ELISAs. Amylin‐Aβ oligomer levels were also measured in whole blood lysates from APP/PS1 rats and APP/PS1 rats expressing amyloid forming amylin in the pancreas (APP/PS1/HIP rats; all rats age‐matched, 16‐months).ResultThe brains of most AD patients had detectable amylin‐Aβ hetero‐oligomer concentrations, whereas about half of those CU had brain tissue amylin‐Aβ hetero‐oligomers below the detection limit (0.02 ng/mg total protein; one‐way ANOVA, P &lt; 0.0001). Amylin‐Aβ hetero‐oligomers were more common in brains with frequent neuritic plaques (CERAD C). Higher brain tissue amylin‐Aβ hetero‐oligomer levels were associated with greater brain tissue amylin concentrations (r = 0.38; P &lt; 0.05). There was no correlation between cerebral amylin‐Aβ hetero‐oligomer accumulation and age in the analysis of samples from sAD and CU groups (r = ‐0.22; P &gt; 0.05). Levels of amylin‐Aβ hetero‐oligomers in whole blood lysates obtained from APP/PS1/HIP rats were higher than those from APP/PS1 littermates (P &lt; 0.05).ConclusionStudies are needed to determine the extent to which amylin‐Aβ hetero‐oligomerization can be detected in human blood.

Rapid, scalable assay of amylin‐β amyloid co‐aggregation in brain tissue and blood

AbstractBackgroundIslet amyloid polypeptide (amylin) secreted from the pancreas crosses the blood brain barrier into the brain parenchyma and interacts with Alzheimer’s disease (AD) associated cerebral ß‐amyloid (Aß) plaques, which are found in both sporadic and early‐onset familial AD. Immunoprecipitated amylin from human AD brain tissue analyzed by Western blot using an anti‐Aß antibody detects Aß immunoreactivity, which indicates that the amylin and Aß peptides aggregate in the brains of humans with AD, forming amylin‐Aß hetero‐oligomers that are sodium dodecyl sulfate (SDS)‐soluble. We report the development of an ELISA to detect amylin‐Aß hetero‐oligomers in brain tissue and blood.MethodThe amylin‐Aß ELISA relies on a monoclonal anti‐Aß middomain antibody (detection) and a polyclonal anti‐amylin antibody (capture) designed to recognize an epitope that is distinct from the high affinity amylin‐Aß binding sites. We screened temporal cortex homogenates obtained from 13 cognitively unaffected (CU) humans and 47 persons with sporadic AD (sAD) for amylin‐Aß hetero‐oligomers. Individuals were of similar age (sAD, 85.65 ± 7.04 years vs. CU, 87.22 ± 7.30 years; p = 0.482). Brain tissue amylin and Aß concentrations were measured using conventional ELISAs. Amylin‐Aß oligomer levels were also measured in whole blood lysates from APP/PS1 rats and APP/PS1 rats expressing amyloid forming amylin in the pancreas (APP/PS1/HIP rats; all rats age‐matched, 16‐months).ResultThe brains of most AD patients had detectable amylin‐Aß hetero‐oligomer concentrations, whereas about half of those CU had brain tissue amylin‐Aß hetero‐oligomers below the detection limit (0.02 ng/mg total protein; one‐way ANOVA, P &lt; 0.0001). Amylin‐Aß hetero‐oligomers were more common in brains with frequent neuritic plaques (CERAD C). Higher brain tissue amylin‐Aß hetero‐oligomer levels were associated with greater brain tissue amylin concentrations (r = 0.38; P &lt; 0.05). There was no correlation between cerebral amylin‐Aß hetero‐oligomer accumulation and age in the analysis of samples from sAD and CU groups (r = ‐0.22; P &gt; 0.05). Levels of amylin‐Aß hetero‐oligomers in whole blood lysates obtained from APP/PS1/HIP rats were higher than those from APP/PS1 littermates (P &lt; 0.05).ConclusionStudies are needed to determine the extent to which amylin‐Aß hetero‐oligomerization can be detected in human blood.

ZCCHC17 Modulates Neuronal RNA Splicing and Supports Cognitive Resilience in Alzheimer's Disease.

ZCCHC17 is a putative master regulator of synaptic gene dysfunction in Alzheimer's disease (AD), and ZCCHC17 protein declines early in AD brain tissue, before significant gliosis or neuronal loss. Here, we investigate the function of ZCCHC17 and its role in AD pathogenesis using data from human autopsy tissue (consisting of males and females) and female human cell lines. Co-immunoprecipitation (co-IP) of ZCCHC17 followed by mass spectrometry analysis in human iPSC-derived neurons reveals that ZCCHC17's binding partners are enriched for RNA-splicing proteins. ZCCHC17 knockdown results in widespread RNA-splicing changes that significantly overlap with splicing changes found in AD brain tissue, with synaptic genes commonly affected. ZCCHC17 expression correlates with cognitive resilience in AD patients, and we uncover an APOE4-dependent negative correlation of ZCCHC17 expression with tangle burden. Furthermore, a majority of ZCCHC17 interactors also co-IP with known tau interactors, and we find a significant overlap between alternatively spliced genes in ZCCHC17 knockdown and tau overexpression neurons. These results demonstrate ZCCHC17's role in neuronal RNA processing and its interaction with pathology and cognitive resilience in AD, and suggest that the maintenance of ZCCHC17 function may be a therapeutic strategy for preserving cognitive function in the setting of AD pathology.